Karim Tararbit
email: karim.tararbit@inserm.fr

MD, MS Nathalie Lelong

MS Anne-Claire Thieulin

MS Lucile Houyel

MD Damien Bonnet

PhD François Goffinet

PhD Babak Khoshnood

The risk for four specific congenital heart defects associated with assisted reproductive techniques: a population-based evaluation

Keywords: Reproductive techniques, assisted Intracytoplasmic sperm injection Heart defects, congenital Tetralogy of Fallot Epidemiology

Study question. Are the risks of hypoplastic left heart syndrome, transposition of great arteries, tetralogy of Fallot (TOF) and coarctation of the aorta increased in infants conceived by different assisted reproductive techniques (ART)?

Study answer. ART, and particularly intracytoplasmic sperm injection (ICSI), are specifically associated with a higher risk of TOF.

What is already known. ART are associated with an increase in the overall risk of birth defects.

The risk for congenital heart defects (CHD) associated with ART has been evaluated as a whole but there is limited information on the risks for specific CHD.

Study design, material and methods. We conducted a case-control study using populationbased data from the Paris registry of congenital malformations for the period 1987-2009 and a cohort study of CHD (EPICARD) on 1583 cases of CHD and 4104 malformed controls with no known associations with ART. ART included ovulation induction only, IVF and ICSI.

Results. Exposure to ART was significantly higher for TOF than controls (6•6 vs. 3•5%, P=0•002); this was not the case for the other three CHD. ART (all methods combined) were associated with a 2•4-fold higher odds of TOF after adjustment for maternal characteristics, paternal age and year of birth (Adjusted OR 2•4, 95% CI 1•5-3•7) with the highest risk associated with ICSI (Adjusted OR 3•0, 95%CI 1•0-8•9). No statistically significant associations were found for the other CHD.

Limitations. Our study cannot disentangle to what extent the observed associations between risk of TOF and ART are due to causal effects of ART and/or the underlying infertility problems of couples who conceive following ART.

Implications. The developmental basis of the specific association between the risk of TOF and ART and in particular the possible role of neural crest cells in this association need to be further investigated.

Introduction

Assisted reproductive techniques (ART) are known to be associated with a modest increase in the overall risk of congenital anomalies (Wennerholm et al., 2000;[START_REF] Hansen | The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization[END_REF][START_REF] Koivurova | Neonatal outcome and congenital malformations in children born after in-vitro fertilization[END_REF][START_REF] Hansen | Assisted reproductive technologies and the risk of birth defects--a systematic review[END_REF][START_REF] Klemetti | Children born after assisted fertilization have an increased rate of major congenital anomalies[END_REF][START_REF] Olson | In vitro fertilization is associated with an increase in major birth defects[END_REF][START_REF] Schieve | Risk of birth defects among children conceived with assisted reproductive technology: providing an epidemiologic context to the data[END_REF]. Relatively little specific information exist on the risk of congenital heart defects (CHD) for foetuses conceived following ART [START_REF] Anthony | Congenital malformations in 4224 children conceived after IVF[END_REF][START_REF] Hansen | The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization[END_REF][START_REF] Katalinic | Pregnancy course and outcome after intracytoplasmic sperm injection: a controlled, prospective cohort study[END_REF][START_REF] Lie | Birth defects in children conceived by ICSI compared with children conceived by other IVFmethods; a meta-analysis[END_REF]Zhu et al., 2006;[START_REF] Reefhuis | Assisted reproductive technology and major structural birth defects in the United States[END_REF][START_REF] Tararbit | Risk of congenital heart defects associated with assisted reproductive technologies: a population-based evaluation[END_REF]. Available evidence suggest an overall risk for CHD in relation to ART that is comparable to that found for all congenital anomalies combined (OR~1•4-1•5) [START_REF] Hansen | Assisted reproductive technologies and the risk of birth defects--a systematic review[END_REF][START_REF] Tararbit | Risk of congenital heart defects associated with assisted reproductive technologies: a population-based evaluation[END_REF]. Specific associations between different methods of ART and categories of CHD have also been reported by our group [START_REF] Tararbit | Risk of congenital heart defects associated with assisted reproductive technologies: a population-based evaluation[END_REF] using a subset of the data used in the present study.

However, previous studies have mostly examined the risk of CHD in relation to ART for all CHD combined or for broad categories of CHD rather than for specific CHD. Moreover, the associations between different methods of ART and specific CHD have not been examined.

Assessment of such specific associations is important as known teratogens are generally associated with the risk of one or a few specific malformations. Furthermore, specific associations between types of CHD and ART may provide clues about the underlying mechanism of the higher risk of congenital malformations in foetuses conceived following ART.

Using population-based data from the Paris registry of congenital malformations and a cohort study of children with CHD (the EPICARD study), we estimated the risks for four major specific CHD: hypoplastic left heart syndrome (HLHS), transposition of great arteries (TGA), tetralogy of Fallot (TOF), and coarctation of the aorta (CoA) in relation to different methods of ART.

Material and methods

Data sources

Two sources of data were used for this study: 1) the Paris registry of congenital malformations and 2) the EPICARD study (Epidemiological study on the outcomes for congenital heart diseases). These two sources of data are briefly described below.

The Paris registry of congenital malformations

Since 1981, the Paris registry of congenital malformations registers all cases of birth defects and chromosomal anomalies among live-births, still-births (≥ 22 weeks of gestation), and pregnancy terminations. The registry covers the population of women who live in the Greater Paris area (Paris and its surrounding suburb) and deliver or have a termination of pregnancy for foetal anomaly in a Parisian maternity unit. The annual number of deliveries in our population is about 38,000.

The Paris registry is a member of the European network of registries of congenital malformations (European Surveillance of Congenital Anomalies, EUROCAT) and of the International clearinghouse for birth defects surveillance and research (Eurocat special report, 2009;[START_REF] Cocchi | International trends of Down syndrome 1993-2004: Births in relation to maternal age and terminations of pregnancies[END_REF][START_REF] Greenlees | Paper 6: EUROCAT member registries: organization and activities[END_REF][START_REF] Khoshnood | Paper 2: EUROCAT public health indicators for congenital anomalies in Europe[END_REF]. The registry follows the EUROCAT methodology and quality of data is routinely monitored by both EUROCAT and the French National Committee of Registries. Review of procedures regarding confidentiality of data is overseen by both the National Committee of Registries and the National Committee of Informatics and Freedom (CNIL). Data are based on medical records and are collected from several sources including maternity units, neonatology wards, cytogenetic, and pathology services.

In the present study, data from the registry corresponded to the period January 1st 1987 to December 31st 2009 as the first case of a malformation with exposure to in vitro fertilization occurred in 1987 and 2009 was the last year for which data were available at the time of the study.

EPICARD

The EPICARD study is an on-going prospective cohort study of all children with a CHD [START_REF] Khoshnood | Prevalence, timing of diagnosis and mortality of newborns with congenital heart defects: a population-based study[END_REF] and iv) identify the factors associated with their health outcomes, especially the role of events during the neonatal period and of the initial medical and surgical management. All cases (live births, pregnancy terminations, foetal deaths) diagnosed in the prenatal period or up to one year of age in the birth cohorts between May 1 st 2005 and April 30 th 2008 were eligible for inclusion.

The total number of cases of CHD included in the study was 2867, including 2348 live births (82%), 466 pregnancy terminations (16•2%) and 53 foetal deaths (1•8%). Diagnoses were confirmed in specialized paediatric cardiology departments and for the majority of pregnancy terminations and foetal deaths by a foetopathologist examination. For others in which a pathology exam could not be done, the diagnoses were confirmed by consensus by a paediatric cardiologist and a specialist in echocardiography in the study group based on results of prenatal echocardiography examination.

Methods

A case-control study with malformed controls was performed. Cases were foetuses/children with hypoplastic left heart syndrome (HLHS), transposition of great arteries (TGA), tetralogy of Fallot (TOF) and coarctation of the aorta (CoA). Cases included in both the Paris registry and the EPICARD study were counted once. Malformed controls were isolated congenital defects other than CHD for which no evidence of an association with ART was found in literature. As recommended by [START_REF] Hook | Normal or affected controls in case-control studies of congenital malformations and other birth defects: reporting bias issues[END_REF], we selected a wide spectrum of heterogeneous birth defects as controls in order to decrease the risk of selection bias due to shared etiologic factors between cases and controls [START_REF] Swan | Reporting and selection bias in case-control studies of congenital malformations[END_REF][START_REF] Lieff | Selection bias and the use of controls with malformations in case-control studies of birth defects[END_REF]. The malformations in the control group comprised cases of club-foot, angioma, skin abnormality, polydactyly, syndactyly and congenital hip dislocation in the Paris registry.

The risk (odds) of each CHD in relation to ART was the main outcome measure. Data on exposure to ART were obtained from medical records. The same procedure for data collection and coding was used for information on ART in the two datasets (Paris registry and EPICARD) used in this study. Exposure to ART included the following categories: ovulation induction (OI) only, IVF, and ICSI. Exposure to ART was assessed as: i) a binary variable (ART yes/no) , ii) a variable in four categories (no ART, OI, IVF, ICSI) and iii) a variable combining IVF and ICSI (IVF + ICSI) in a single category.

Potential confounding factors considered were maternal characteristics (age, occupation and geographic origin), paternal age, and year of birth (or pregnancy termination). Although their exact relations to the risk for specific CHD are not well known, these factors are associated with both exposure to ART and prevalence of birth defects in general [START_REF] Vrijheid | Socioeconomic inequalities in risk of congenital anomaly[END_REF].Maternal

Statistical analysis

The odds of each of the four specific CHD vs. controls in relation to ART was estimated using logistic regression models, after taking into account year of birth, maternal characteristics (age, occupation and geographic origin), and paternal age. Paternal age was missing for 20•6% of the study population. We used multiple imputation [START_REF] Little | Statistical analysis with missing data[END_REF] for missing data on paternal age. Paternal age was imputed in twenty sets of data for each CHD separately using the case/control status, exposure to ART, maternal age, and year of birth/termination. The pooled (over the 20 datasets) adjusted ORs for the association between ART and risk of each specific CHD were estimated using the method described by [START_REF] Little | Statistical analysis with missing data[END_REF]. In order to explore the possible role of multiple pregnancies in the association between ART and CHD, we also conducted analyses with further adjustment for multiple pregnancies and tested for any interactions effect between multiple pregnancies and ART.

The statistical significance level was set at α = 0.05 and all tests were two-sided.

Analyses were done with Stata 11 software (Statacorp, Texas, USA).

Ethics approval

No specific ethical approval was needed for this particular analysis. The French National Committee of Informatics and Freedom (CNIL) has authorised the surveillance and research activities of the registry using anonymous data and has approved the EPICARD study.

Results

Study population

After excluding cases with missing data on ART (3% of cases), the study population comprised 353 cases of HLHS, 444 cases of TGA, 395 cases of TOF and 391 cases of CoA. Approximately 14% of cases of HLHS, 3% of TGA, 20% of TOF and 10% of CoA were associated with chromosomal anomalies. The study population included 4104 malformed controls with complete information on ART, which comprised 1436 with congenital hip dislocation, 824 with club-foot, 782 with polydactyly, 517 with angioma, 381 with skin abnormality, and 164 with syndactyly with complete information on ART; 3% of controls had missing data on ART.

Table 1 summarises the results of the comparison of the maternal, paternal and pregnancy characteristics of cases of CHD (all four specific CHD combined) and controls. Overall, mothers of cases of CHD were older, more likely to be from North Africa and in the occupational category "none" as compared with mothers of controls. Still births and terminations of pregnancy for foetal anomaly were more frequent for cases of CHD than controls.

When comparisons of the characteristics of cases and controls were done for the four defects separately (detailed results not shownavailable from authors), for CHD other than TOF, the characteristics of cases and controls were for the most part comparable, except that mothers of cases of CoA were more likely to be from North Africa than controls. Most sociodemographic characteristics were different between cases of TOF and controls. Mothers of cases of TOF were significantly older and more likely to be from North Africa than controls. Mothers of cases of TOF were also more likely to be in the occupational category "none" than controls (data not shown).

Risk of CHD associated with ART

All cases

Exposure to ART (all methods combined, Table 2) was significantly higher for cases of TOF than controls (6•6% vs. 3•5%, p=0•002). Exposure to the different methods of ART (data not shown) was also significantly different between cases of TOF and controls, in particular 2•5% of TOF were born following IVF vs. 1•3% of controls and 1•3% of TOF were born following ICSI vs. 0•3% of controls (p=0•004). Exposure to ART was not associated with a significantly higher risk of other CHD.

Exposure to ART was associated with a 2•4-fold increase in the maternal characteristics and year of birth-adjusted odds of TOF (Adjusted OR= 2•4, 95%CI 1•5 -3•7) (Table 3). The odds of TOF associated with ART remained similar after further adjustment for paternal age. In contrast, ART were not associated with statistically significant increases in the risks of HLHS, TGA or CoA and the ORs were generally close to the null value (Table 3). All three methods of ART were associated with significantly higher odds of TOF (Table 4). In particular, ICSI was associated with a three-fold higher odds of TOF after adjustment for maternal characteristics and year of birth (Adjusted OR= 3•0, 95%CI 1•0-8•9). There was no evidence that IVF was associated with a higher odds of TOF as compared with OI (for IVF: Adjusted OR=2•0, 95%CI 1•0 -4•2; for OI: Adjusted OR= 2•5, 95%CI 1•3 -4•8). For the other three specific CHD, no statistically significant associations were observed. Further adjustment for paternal age using the multiple imputation estimates did not modify appreciably the above estimates (data not shown).

Cases without associated chromosomal anomalies

Tables 3 and5 show the results of the analyses for the associations between the risks of the four CHD and ART (all methods combined, Table 3) and separately for different methods of ART (Table 5) for the subset of cases without associated chromosomal anomalies. All estimates were essentially the same as those found for all cases combined (i.e. when cases of each specific CHD with and without associated chromosomal anomalies were analysed together).

Results of the analyses, which included further adjustment for multiple pregnancies were essentially the same as those found without adjustment for multiple pregnancies (data not shown). We found no statistically significant interaction effects between ART and multiple pregnancies for any of the four CHD (data not shown).

Discussion

Using population-based data on nearly 1600 cases of specific congenital heart defects (CHD), we assessed the risk of four specific CHD in relation to assisted reproductive techniques (ART).

We found that ART (all methods combined) were associated with a 2•4-fold increased risk of tetralogy of Fallot (TOF), after taking into account maternal age, occupation, geographic origin, paternal age, and year of birth. In particular, ICSI was associated with a three-fold higher adjusted odds of TOF. In contrast, we did not find any statistically significant increases in the risk of CHD in relation to ART for the other CHD in our study, i.e., hypoplastic left heart syndrome (HLHS), transposition of the great arteries (TGA), and coarctation of the aorta (CoA).

Risk estimates were comparable when cases with chromosomal anomalies were excluded, suggesting that the associations between ART and TOF are not due to the association of the latter with chromosomal anomalies. Further adjustement for multiple pregnancies did not substantially modify our results.

On the basis of our findings, we calculated attributable risk fractions, which would represent the proportion of cases of TOF that may be caused by ART, or equivalently, the proportion of cases of TOF that would be avoided were the exposure to ART removed ceteris paribus, "if" the association we found between the risk of TOF and ART can be assumed to represent a causal relation (this may of course not be the case in part for reasons that are discussed further below).

The attributable risk fraction estimates suggested in particular that around 6.5% of the TOF may have been caused by ART (all methods combined) and 2% by ICSI.

Our study has certain limitations. We had limited power to detect OR lower than two in the association between ART (for all methods combined) and specific CHD and three in case of the different methods of ART. Therefore, our study may have had insufficient power to detect statistically significant associations for other CHD.

The models used to estimate the odds ratios for the different defects in relation to ART were not nested (i.e., were separate models) and we did not formally test the statistical significance of differences in the odds ratios for one defect vs. another. The associations were not statistically significant for any of the defects except for TOF, whereas the numbers of cases for the other CHD were comparable to those of TOF.

A potential source of bias in our study is related to the use of malformed controls [START_REF] Swan | Reporting and selection bias in case-control studies of congenital malformations[END_REF][START_REF] Lieff | Selection bias and the use of controls with malformations in case-control studies of birth defects[END_REF]. The main advantage of using malformed controls is to reduce the risk of recall or other sources of information bias. But malformed controls may also be a source of selection bias if malformations included as controls are either directly or indirectly associated with ART. Risks could be under (over)-estimated if malformations included in the control group occur more (less) frequently in foetuses conceived following ART. By selecting a heterogeneous group of malformations with no known association with ART, as recommended by [START_REF] Hook | Normal or affected controls in case-control studies of congenital malformations and other birth defects: reporting bias issues[END_REF], we aimed to minimize such bias. However, the possibility of residual bias due to shared aetiologies between cases and malformed controls cannot be excluded.

A differential misclassification bias for exposure assessment cannot be excluded if exposure to ART is ascertained in a different way for cases and controls. However, we have no reason to believe that ART may have been ascertained differentially for cases of TOF vs. the other CHD examined in our study.

We had a relatively high proportion of missing data on paternal age. The latter is known to be associated with ART and more specifically with ICSI. Estimates for ICSI could therefore be biased if distribution of paternal age was different for subjects with missing data. We used multiple imputations for imputing missing paternal age using case/control status, exposure to ART, maternal age and year of birth and adjustment for paternal age did not appreciably change our results. However, residual bias due to other paternal characteristics cannot be excluded.

The question of multiple pregnancies and its association with both ART and the risk of congenital anomalies is an important issue to consider. There is evidence suggesting that multiple pregnancies may be associated with a higher risk of congenital anomalies [START_REF] Mastroiacovo | Congenital malformations in twins: an international study[END_REF][START_REF] Glinianaia | Congenital anomalies in twins: a register-based study[END_REF]. This may specifically be the case for CHD, although relatively little, and at times contradictory, information exist on the associations between multiple pregnancies and CHD [START_REF] Manning | A study to determine the incidence of structural congenital heart disease in monochorionic twins[END_REF][START_REF] Bahtiyar | Prevalence of congenital heart defects in monochorionic/diamniotic twin gestations: a systematic literature review[END_REF][START_REF] Campbell | Congenital heart defects in twin gestations[END_REF]. Moreover, it is not clear to what extent any association between multiple pregnancies and CHD may in fact be due to ART. Our results remained similar after further adjustment for multiple pregnancies and we did not find any statistically significant interaction effects between ART and multiple pregnancies for any of the CHD, although this may have been due to limited power of our study for detecting interaction effects. In any case, none of the above precludes the possibility that multiple pregnancies may be on the causal pathway between ART and CHD. It is worth noting however that the public health impact of ART on the risk for birth defects, including that of TOF found in our study, includes all (singleton and multiple) pregnancies.

Specific associations between ART and certain categories of CHD, particularly the so-called conotruncal defects, which include TOF, have been reported [START_REF] Reefhuis | Assisted reproductive technology and major structural birth defects in the United States[END_REF]Tararbit et al., 2001). In a recent study [START_REF] Tararbit | Risk of congenital heart defects associated with assisted reproductive technologies: a population-based evaluation[END_REF], the risk of CHD associated with ART was also shown to vary more generally for different methods of ART and categories of CHD defined based on anatomic and clinical criteria [START_REF] Houyel | Population-based evaluation of a suggested anatomic and clinical classification of congenital heart defects based on the International Paediatric and Congenital Cardiac Code[END_REF]. In particular, the authors found a stronger association between ICSI and the category "Malformations of the outflow tracts and ventriculoarterial connections" that comprised, among other CHD, the conotruncal defects.

The developmental origins of TOF are complex and not fully understood but they may involve abnormal development of neural crest cells. None of the other three CHD studied is known to be of cardiac neural crest origin. In particular TGA which is a defect of the outflow tract does not belong to the group of the conotruncal defects [START_REF] Houyel | Population-based evaluation of a suggested anatomic and clinical classification of congenital heart defects based on the International Paediatric and Congenital Cardiac Code[END_REF] and migration/proliferation of neural crest cell appear to be normal in this condition [START_REF] Bajolle | Rotation of the myocardial wall of the outflow tract is implicated in the normal positioning of the great arteries[END_REF]. In order to further investigate, the hypothesis of the involvement of neural crest cells in the association between TOF and ART, we assessed the risk for other, rarer CHD thought to be of neural crest origin (TOF with pulmonary atresia, TOF with absent pulmonary valve, and common arterial trunk).

We found an increased overall risk associated with ART (data not shown) but the confidence intervals were wide due to small sample sizes.

Given the uncertainties about both the developmental origins of cardiac defects and possible effects of ART on foetal development, the hypothesis of a potential implication of neural crest cells in the association between ART and TOF must be regarded as very tentative and no more than a reasonable speculation. Future observational and experimental studies using other designs (e.g., animal studies, genetic studies, fundamental research in biology of reproduction / ART as well as additional epidemiological studies) are needed to both further assess our observations and in order to understand the possible underlying mechanisms of the association between the risk of TOF and ART.

In conclusion, we found that cases of TOF were more likely to have been conceived following ART when compared with controls. ART were associated with a 2•4-fold higher risk of TOF after adjustment for maternal age, occupation, geographic origin, paternal age and year of birth; ICSI was specifically associated with a three-fold higher risk of TOF. In contrast, we did not find statistically significant associations between ART and HLHS, TGA or CoA and most ORs were close to the null value. Our study cannot disentangle to what extent the observed associations between risk of TOF and ART may be due to any causal effects of ART and/or the underlying infertility problems of couples who conceive following ART. Nevertheless, the developmental basis of the specific association between risk of TOF and ART, particularly ICSI, and the potential implication of neural crest cells in this association, need to be further investigated.

  born to women living in the Greater Paris area (Paris and its surrounding suburbs) between 2005 and 2008 regardless of the place of delivery (N = 317,538 births). The principal objectives of the study are to use population-based data from a large cohort of patients with CHD to: i) estimate the total and live birth prevalence, ii) examine timing of diagnosis and assess medical and surgical management of children with CHD, iii) evaluate neonatal mortality and morbidity and neuro-developmental outcomes of children with CHD;

  occupation was coded in five categories (professional, intermediate, administrative/public service, other, and none) following the French National Institute of Statistics and Economic Studies (INSEE) classification. Geographic origin was coded in four categories: French, North African, Sub-Saharan African, and other countries.

Table 1 . Associations between predictor variables and case/control status.

 1

	n	% §	n	% §

† Co pariso of the proportio of hildre /fetuses o ei ed after ART et ee the spe ifi CHD a d the alfor ed o trols.

Table 2 .

 2 Numbers of cases and controls and proportions of fetuses conceived after Assisted Reproductive Technologies (ART).

	Controls *
	All cases
	Cases without chromosomal anomalies

5 -2.6 †Adjusted for ater al age, geographi origi , o upatio a d year of irth.

Table 4 .

 4 Logistic regression analyses of the associations between the different methods of assisted reproductive technologies (ART) and four specific congenital heart defects (CHD). Odds ratios (OR) represent the odds of a birth (including live births, stillbirths and pregnancy terminations) with congenital heart defect (cases) relative to the odds of a birth with one of the malformed controls (club-foot, angioma, skin abnormality, polydactyly, syndactyly and congenital hip dislocation).

	Maternal
	Adjusted † OR*

* 6 -2.9 †Adjusted for ater al age, geographi origi , o upatio a d year of irth.

Table 5 .

 5 Logistic regression analyses of the associations between assisted reproductive technologies (ART) and four specific congenital heart defects (CHD) without associated chromosomal anomalies.Maternal Adjusted † OR* * Odds ratios (OR) represent the odds of a birth (including live births, stillbirths and pregnancy terminations) with congenital heart defect (cases) relative to the odds of a birth with one of the malformed controls (club-foot, angioma, skin abnormality,polydactyly, syndactyly and congenital hip dislocation)

Funding. This work was supported by grants from the Agence de Biomédecine (Saint-Denis La Plaine, France) (to B.K.). The Paris Registry of Congenital Malformations received financial support from INSERM (Paris, France) and the Institut de Veille Sanitaire (Saint-Maurice, France). The EPICARD study was supported by three grants from the Ministry of Health (PHRC 2004(PHRC , 2008(PHRC and 2011)).Additional funding for the EPICARD study was provided by the AREMCAR Association (Association pour la Recherche et l"Etude des Maladies Cardiovasculaires).

Sources of funding

This work was supported by grants from the Agence de Biomédecine (Saint-Denis La Plaine, France) (to B.K.). The Paris Registry of Congenital Malformations received financial support from INSERM (Paris, France) and the Institut de Veille Sanitaire (Saint-Maurice, France). The EPICARD study was supported by three grants from the Ministry of Health (PHRC 2004(PHRC , 2008(PHRC and 2011)). Additional funding for the EPICARD study was provided by the AREMCAR Association (Association pour la Recherche et l"Etude des Maladies Cardiovasculaires).

Conflict of interest


Julien Cucherousset

Stéphanie Boulêtreau

Frédéric Azémar

Arthur Compin

Mathieu Guillaume

Frédéric Santoul
email: frederic.santoul@univ-tlse3.fr

1 Freshwater Killer Whales": Beaching Behavior of an Alien Fish to Hunt Land Birds

The behavioral strategies developed by predators to capture and kill their prey are fascinating, notably for predators that forage for prey at, or beyond, the boundaries of their ecosystem. We report here the occurrence of a beaching behavior used by an alien and large-bodied freshwater predatory fish (Si/urus glanis) to capture birds on land (i.e. pigeons, Columbia livia). a ュ ッ ョ セ @ a total of 45 beaching behaviors observed and filmed, 28% were successful in bird capture. Stable isotope analyses (3 1 C and B 15 N) of predators and their putative prey revealed a highly variable dietary contribution of land birds among individuals. Since this extreme behavior has not been reported in the native range ofthe species, our results suggest that sorne individuals in introduced predator populations may adapt their behavior to forage on novel prey in new environments, leading to behavioral and trophic specialization to actively cross the water-land interface.

Introduction

To capture, handle, kill and/or swallow their prey, predators have developed numerous strategies, including pack hunting, venin, cooperation, trapping webs and the use of tools. For instance, savannah chirnpanzees have been reported to construct wooden spear-like tools to hunt their preys [START_REF] Pruetz | Savanna Chimpanzees, Pan troglodytes verus, Hunt with Tools[END_REF] while New Caledonian crows can use stick tools to capture highly energetic wood-boring beede larvae [START_REF] Rutz | The Ecological Significance of Tool Use in New Caledonian Crows[END_REF]. However, perhaps the most astonishing strategies are when the preys targeted by the predator are located outside of the predator's ecosystem boundaries. These strategies can be grouped into three broad categories. First, the predators can passively lie in ambush until the preys cross the water-land interface (voluntarily or accidendy) and capture them. This is notably the case of crocodiles that capture migrating wildebeest crossing rivers and of terrestrial predators such as birds, spiders and bats that capture emerging aquatic insects [START_REF] Nakano | Reciprocal subsidies: Dynamic interdependence between terrestrial and aquatic food webs[END_REF][START_REF] Baxter | Tangled webs: reciprocal flows ofinvertebrate prey link streams and riparian zones[END_REF]. Second, sorne predators may develop strategies that force the prey to enter their ecosystem. For instance, archerfish (Toxotes jaculatrix Pallas) have developed complex optical and morphological adaptations to 'shoot clown' insects located on trees by expelling droplets on the insects that will subsequendy fall in the water [START_REF] Scbuster | Archer flsb learn to compensate for complex optical distortions to determine the absolute size of their aerial prey[END_REF]. Third, and most spectacularly, predators can actively cross the water-land interface to capture the prey. Sorne marine predators such as killer whales (Orcinus orca L.) and botdenose dolphins (Tursiops sp.) display intentional 'beaching' behavior to catch prey on beaches [START_REF] Guinet | Development of intentional stranding hunting techniques in killer whale ( Orcinus orca) calves at Crozet Archipelago[END_REF][START_REF] Sargeant | Specialization and development of beach hunting, a rare foraging behavior, by wild bottlenose dolphins (Tursiops sp.)[END_REF]. ln many predators, these extreme hunting behaviors represent a form of ecological specialization [START_REF] Bolnick | The ecology of individuals: incidence and implications of individual specialization[END_REF][START_REF] Araujo | The ecological causes ofindividual specialisation[END_REF] that is displayed only by a subset of individuals in the populations [START_REF] Sargeant | Specialization and development of beach hunting, a rare foraging behavior, by wild bottlenose dolphins (Tursiops sp.)[END_REF].

Here, we report the occurrence of a hunting behavior, analogous to the intentional beaching of marine mammals, in an alien freshwater fish species (European catfish Silurus glo:nis L., the world's third largest and Europe's largest freshwater fish) [START_REF] Stone | The Last of the Leviathans[END_REF]11] to capture land birds. Additionally, we demonstrate the existence of trophic niche variability within the population with only sorne individuals foraging on land birds.

Materials and Methods

(a) Behavioral Monitoring

European catfish originates from Europe (east of Rhine River) and has been introduced in many ecosystems of Western Europe, including Spain, ltaly and Southwestern France [11]. We conducted the present study in the Tarn River (Southwestern France) within the historical city center of Albi, a UNESCO World Heritage Centre. European catfish were introduced in the Tarn River in 1983 and have since established self-sustained populations [12]. Behavioral monitoring was performed from a bridge above a gravel island where pigeons (Columbia livia Gmelin) regroup for drinking and cleaning (43° 55' 51.77" N, 2° 08' 41.83" E). At the studied stretch, the Tarn River is approxirnately 100 rn wide with a mean depth of 3 rn (maximum depth 5.4 rn) and belongs to a protected area where angling is prohibited. ln total, 24 surveys (approx. 3-hour long on average, total observation and filming rime of approx. 72 h) were performed from June 30th to October l9th 20 ll in the morning or in the afternoon. The number and success rate of beaching behavior were determined by filming the predatory fish nearby the gravel island. Throughout the survey, river discharge was 1ow and the water was clear, allowing full observation of ali disp1ayed behavior (Figure 1 and Movie Sl).

(b) Stable Isotope Analyses

Tissue samples of European catfuh and their potential ー イ セ @ were collected in the observation site from 18th September to 14 October 2011 to quantify the contribution of land birds to the catfiSh diet using stable isotope analyses. Sampling was allowed by the pennit "Arrêté Préfectoral no. 2011--Q18". Stable isotope analyses (principally o 13 C and o 15 N) are now commonly used by eco1ogists to provide reliab1e estimates of long-tenn dietary patterns of predators [13, 14], including trophic specialization [15,16) and the ecological impacts of non-native fish species [14,17]. A total of 14 European catfJSh 1ocated within the study area (estinJated body size ranging from 900 to 2000 mm) were sampled. Fin clips were collected by angling from 5 individuals while muscle samp1es were collected on the flank of 9 additional

• • • • • 13 • • • • • • • • • z 11 • +• !!l 10 • • Q セ @ ' 1 \ • 9 1 <Il •1 • • 7 • 5 • 29 -28 -27 •26 • 25 • 24 -23 •22 ôtlC f ャ ァ オ セ @ 2. s エ ャ ャ セ ャ ・ @ Isotope values of European 」 。 エ ヲ ャ ウ セ @ 。 ョ セ @

{c) Mixing Models

A Bayesian mixing model (Stable Isotope Analysis in R, SIAR) [START_REF] Parnell | Source Partitioning Using Stable Isotnpes: Coping with Too Mucb Variation[END_REF] was used to estimate the contribution of each prey (ftsh, crayfish and pigeon) to the diet of each individual European catftsh (function siarsoltJmancv4, 500000 iterations) since it accounts for potential variability in the stable isotope values of consumers, prey and trophic fractionation [START_REF] Parnell | Source Partitioning Using Stable Isotnpes: Coping with Too Mucb Variation[END_REF]. Since no specifie trophic fractionarion values exist for European catftSh, we followed a conservative approach [18] that consisted in using commonly reported fractionation factors with errer estimates (1 %o (± 1 SD) and 3.4 %o (:t 1 SD) for ô 13 C and ô 15 N, respectively) [16,[START_REF] Post | Using stable isotnpes to estimate trophic position: models, methods, and assumptions[END_REF]. For each individual, the lower and upper 50, 75 and 95% Bayesian credibility intervals of the contribution of each prey were predicted by the mixing models. Potential effects of individual body size, sampling date and sampled tissue on the mean predicted contribution of pigeon to the diet of each individual were tested using Spearman correlations and Kruskal-Wallis test, respectively. Ali statistical analyses were performed using R [START_REF] Develnpment | R: A language and environment for statistical computing[END_REF].

Results

(a) Behavioral Observations

During the 24 surveys conducted in an urbanized stretch of the Tarn River (France), between 1 and 9 European catfish (mean 3.9±2.1 SD, estimated body size range: 900--1500 mm) were observed swinuning nearby a small island where pigeons regrouped for drinking and cleaning (Figure 1). Fifty-four beaching behaviors with partial and mosdy complete stranding were observed and filmed (Movie S1), among which 28% (n = 15) were ruccessful, i.e. the land birds were captured on land, returned to the river and swallowed (Figure 1 and Movie SI). On one occasion, complete stranding was observed but the attack was unsuccessful in capturing the pigeon. In approximately 40% of ail observations, European catfish bad more than half of their body outside of the water. The beaching behavior was quick, lasting from less than one second ta no more than 4 seconds. The attacks were systematically triggered by active pigeons. lndeed, motionless pigeons, even very closed to the European catfish, were never attacked. Before the attack, European catfish were observed to exhibit erected upper jaw barbels on the upper jaw when they approach pigeons, suggesting that water vibrations, rather than visual eues, were used to detect and attack the prey.

(b) Stable Isotope Analyses

Stable isotope analyses of European catfish revealed a high level of trophic niche variability among individuals, notably for 6 13 C that ranged from -26.6 %a to -22.8 %o while the variability in ô 1 'N was somewhat 1ower (12.1 %o to 14.7 %o, Figure 2). As ftsh and crayfish were 13 C-depleted compared to pigeons (Figure 2 and Table SI), this indicated varying importance of aquatic (i.e. ftsh and crayfish) and terrestrial (i.e. pigeons) prey in the diet of the sampled individuals. Specifically, mixing models predicted a lùghly variable dietary contribution of pigeons among individuals, with 95% Bayesian credibility intervals ranging from 0--51% to 12-78% (Figure 3). While the dietary contribution of crayfish was relatively similar among individuals, the dietary contribution of pigeoDll increased as the dietary contribution of fish decreased (Figure 3). No significant effects of sampling date and sampled tissue on the mean predicted dietary contribution of pigeons were observed (Speannan correlation, P = 0.203 and Kruskal-Wallis test, P = 0.317, n = 14, respectively). However, the estimated body size range of European catfuh observed hunting for pigeons (range: 900--1500 mm) tended to somewhat smaller that the estimated body size range of the individuals sampled in the study arca (range: 900--2000 mm), suggesting a potential effect of individual body size on the display of this novel beaching behavior. This was partially confinned by the existence of a negative but marginally significant relationship between individual body size and the mean predicted dietary contribution of pigeons (Speannan correlation, r = -0.60, P = 0.065, n = 14).

Discussion

Although the consumption of terrestrial prey by aquatic predators is a ubiquitous phenomenon [START_REF] Baxter | Tangled webs: reciprocal flows ofinvertebrate prey link streams and riparian zones[END_REF][START_REF] Bartels | Reciprocal subsidies between freshwater and terrestrial ecosystems structure consumerresource dynamics[END_REF], we document here a novel behavior displayed by an alien freshwater predator that was not, to the best of our knowledge, reported in its native range. This behavior allowed the capture of birds on land through intentional crossing of the water-land interface (beaching). lntroduced species can display ecological and evolutionary adaptations in their new environment, and the occurrence of new behaviors can increase invasive species success [START_REF] Holway | Animal behavior: an essential component of invasion biology[END_REF]. Here, this new hunting strategy leads to a high level of trophic variability among individuals. Theories predict that behavioral and trophic specialization can have strong ecological and evolutionary consequences on intraspecific competition and individual fitness [START_REF] Bolnick | The ecology of individuals: incidence and implications of individual specialization[END_REF], which might subsequendy affect the invasive success of the population. However, since European catfish were not monitored at the individual level in the present study, the potential correlation between success rate of attacks, dietary contribution of pigeons and individuals fitness were not tested. Therefore, it would be of great interest to determine the individual ecological consequences of this hunting behavior in an introduced population.

Understanding the ecological causes triggering the occurrence and maintenance of this unusual predation behavior is important, but it remains unknown at this stage. The emergence of trophic specialization in wild populations can be triggered by an increased intraspecific competition caused by an increased population density and/or a decreased in prey availability [START_REF] Stone | The Last of the Leviathans[END_REF][START_REF] Tin | Food limitation leads to behavioral diversi6cation and dietary specialization in sea otters[END_REF]. European catfish have recendy widely expanded its non-native distributional range through multiple introductions and colonization [11,[START_REF] Poulet | Time trends in flsh populations in metropolitan France: insights from national monitoring data[END_REF], and although temporal patterns of the fish community in the study area are unknown, a potential increased density of European catfish and/ or a potential decreased in prey fish might have caused this behavior to occur. This hypothesis might be explained by the existence of a negative and marginally significant relationship between individual body size and the dietary contribution of pigeons, assuming that smaller individuals are Jess competitive than larger individuals to prey upon prey fish. Alternatively, the risk of being stranded on the riverbank and the energy cost of attacking a pigeon on land rnight be lower for smaller individuals than for large-bodied specimen (i.e. > 1500 mm). ln this case, the costs associated with displaying this new beaching behavior (e.g. learning, risk ofbeing stranded) rnight be counterbalanced by high energy returns provided by the consumption of the new prey, as observed elsewhere [START_REF] Baxter | Tangled webs: reciprocal flows ofinvertebrate prey link streams and riparian zones[END_REF]. Therefore, land birds certainly represent a new ecological opportunity [1 0] that increases the diversity of trophic resources available to the introduced predator and that could drive individual specialization, as observed in other populations ofpredators foraging on allochtonous prey (e.g. [START_REF] Darimont | Landscape heterogeneity and marine subsidy generate extensive intrapopulation niche diversity in a large terrestrial vertebrale[END_REF]). ln conclusion, these fmdings suggest that this new predation behavior might represent an extreme example of the ability of introduced species to adapt to a new environment that could have unexpected implications for consumer-resources dynamics and ecosystem functioning [27 ,28] that deserve further investigations.

Supporting Information

Table SI Stable isotope values of the three potential prey sources (fish, crayfish and pigeon) used in the mixing models. Reported values are the number of sampled individuals (n) and mean (± SD) li 13 C and li 15 N (in o/oo).

(DOC)

Movie SI Movie showing European catfish displaying beaching behavior to capture land birds, with two successful and two unsuccessful attacks. (WMV)

Figure 1 .

 1 Figure 1. e オ イ ッ ー セ セ 。 ョ @

  the putative prey.li 13 C and li 15 N values(%.) of eac:h individual (n= 14) and the putative aquat1c: (fish, n = 9 and c:rayfish, n = 3) and terrestr1al (pigeon, n = 6) prey are displayed. The large symbols for eac:h prey represent the mean value (::!: SD).individuals by scuba divers using a spear gun equipped with a 40mm length and 27-mm diameter stainless steel biopsy tip. Fin and muscle samples were pooled in the analyses since their stable isotope values do not differ significandy in this spec:ies [12]. No lipid correction was performed since samples have low and homogeneous C:N ratios (3.2±0.2 SD) [18]. Muscle samples of the putative aquatic and terrestrial prey present in the study area were al.so collected. These included crayfuh (OrconecfM linwsus Rafinesque n = 3) and Cyprinids prey fish (Blicca i!joerkna L. n"' 3, Cyprinus carpio L. n = 3, and RuiiJm rutiJus L. n = 3, pooled for analyses) which represent the most abundant aquatic prey. Additionally, muscle samples of pigeons were collected on in 2010 (n = 3) and 2011 (n = 3) from local hunting society within 2 km of the study site where pigeons forage. No other potential terrestrial prey sources were observed in the study area. Samples for SIA were oven dried (60°C for 48 h) and analyzed at the Comell Isotope Laboratory (COIL, lthaca, NY).

Figure 3 .

 3 Figure 3. Predicted contribution of putative prey to the diet of each Europan catfish. Putative prey are (a) fish (in red), (b} crayfish (in blue) and (cl pigeons (in green). Reported values are the lower and upper 50, 75 and 95% Bayesian credibility intervals predicted by the mixing models.

Acknowledgments

We thankJ.D. Olden,J.R. Britton, S. Blanchet, E. Danchin,J. Syvliranta and anonymous reviewers for comrnents on the manuscript, P. Marty, S. Planes, B. Lenclos and C. Musseau for field assistance and the ONEMA and FDAAPPMA 81. Sampling was conducted under the permit "Arrêté Préfectoral 2011--018" and we are grateful to local angling and hunting societies for providing sorne samples.

Author Contributions

Conceived and designed the experiments: JC AC FA FS. Performed the experiments: FA AC MG FS. Analyzed the data: JC SB FS. Contributed reagents/materials/analysis tools: FS. Wrote the paper: JC SB FS.


Tony Ducrocq
email: tony.ducrocq@inria.fr

Michaël Hauspie
email: michael.hauspie@univ-lille1.fr

Nathalie Mitton
email: nathalie.mitton@inria.fr

Geographic Routing with Partial Position Information *

Keywords: Wireless Sensor Networks, Geographic Routing Algorithm, Position Based Routing

Geographic routing protocols show good properties for Wireless Sensor Networks (WSN). They are stateless, local and scalable. However they require that each node of the network is aware of its own position. While it may be possible to equip each node with GPS receiver, even if it is costly, there are some issues and receiving a usable GPS signal may be difficult in some situations. For these reasons, we propose a geographic routing algorithm, called HGA, able to take advantages of position informations of nodes when available but also able to continue the routing in a more traditional way if position information is not available. We show with simulations that our algorithm offers an alternative solution to classical routing algorithm (non-geographic) and offers better performances for network with a density above 25 and more than 5% of nodes are aware of their position.

Introduction

Wireless sensor networks consist of sets of mobile wireless nodes without the support of any fixed infrastructure. Such wireless sensor networks offer great application perspectives. Sensors are tiny devices with hardware constraints (low memory storage, low computational resources) that rely on battery. Sensor networks thus require energy-efficient algorithms to make them work properly in a way that suits their hardware features and application requirements.

Low power sensor nodes have limited transmission power, thus they can communicate only to a limited number of nodes. This set of nodes is called the neighborhood of the node. In order to send messages at longer range, nodes are using multi-hop communication. Multi-hop communication means that data will need to be routed from source to destination by other nodes. An efficient way to route messages is to use nodes position information. A node uses a metric based on its own position, its neighbors positions and the destination position in order to choose the next hop for the route. To use such a technique it is possible to equip each node of the network with a GPS receiver or to configure them at setup with their position if they are static.

On the other hand there exist non geographic routing algorithms. They do not require node position to route data but only neighborhood knowledge.

In the Smart Cities context, it is likely that the network is heterogeneous [START_REF] Al-Hader | Smart City Components Architecture[END_REF]. Some nodes may be aware of their position while some other are not. The reason to not equip nodes with GPS receiver may be to save money or energy. It is also not possible to setup position of mobile nodes as it will change over time. Furthermore, even if all nodes are equipped with a GPS receiver, it is possible that some of them do not receive the signal because of environmental factors. For instance, two districts of a city may be connected through a tunnel, in which GPS signal is not received.

In such contexts, the availability of a routing algorithm taking advantages of nodes positions when possible but also functioning when the position is not available is interesting. Thus, we introduce the HGA algorithm (Hybrid-Greedy-AODV). The algorithm works like the Greedy geographic routing while position information allows it, and if position information is not available, a route request message is sent in the node neighborhood in order to find a path toward the destination.

The rest of the paper is organized as follows: Section 2 presents related work concerning geographic and pseudo geographic routing algorithms. We introduce some background works on which our algorithm relies in Section 3. Section 4 describes the HGA algorithm operation in details and Section 5 present simulation and results of its performances. Finally we conclude in Section 6.

Related Works

To the best of our knowledge, there is no existing routing solution that considers that only a subset of nodes is aware of its position. Classical routing algorithms and those using virtual coordinates are the closest approaches to the one we propose. In this section we describe related works as well as works on which our proposal relies.

The main idea of geographic routing protocols is to route data closer to the destination at each step of the routing. The Greedy algorithm, on which part of our work relies, chooses the closest node to the destination in a node's neighborhood as the next forwarder of the data packet. The Greedy algorithm and some variants from literature are shown on Figure 1. Some algorithms allow the deduction of position for nodes by using their neighbors ( Čapkun et al., 2002), [START_REF] Ermel | On Selecting Nodes to Improve Estimated Positions[END_REF]. Nevertheless, these solutions exhibit poor performances due to the difficulty to determine distance between two nodes without specialized hardware [START_REF] Benkic | Using RSSI value for distance estimation in wireless sensor networks based on ZigBee[END_REF]. Moreover, Seada et al. [START_REF] Seada | On the effect of localization errors on geographic face routing in sensor networks[END_REF] showed that a small error on position can generate several losses of packets in geographic routing algorithms.

Routing algorithms over virtual coordinates allow the benefits of geographic routing algorithms without the need of real geographic position of nodes. The main drawback of these solutions resides in the setup of the virtual coordinates system. Indeed, it is needed to flood all the network several times at the beginning of the network lifetime.

The VCap solution [START_REF] Caruso | GPS free coordinate assignment and routing in wireless sensor networks[END_REF] relies on virtual coordinates and a greedy routing. Some particular nodes are chosen for their interesting properties to act as starting points for the different coordinates of the system. These nodes are named anchors. The coordinate system relies on the distance in number of hops between a node and the different anchors. A predefined sink node is used to initiate anchors election (or any other node). This node, by broadcasting a message in the whole network allows to elect a first anchor. At its turn, this anchor broadcast a message in the whole network to elect a second one and so on. Anchors are chosen to be close to the network boundaries a far away to each other to limit duplicates in coordinates. In VCap the network is flooded at least 5 times. Otherwise, because uniqueness of coordinates is not guaranteed, the delivery can not be guaranteed.

The cost over progress concept idea [START_REF] Stojmenovic | Localized network layer protocols in wireless sensor networks based on optimizing cost over progress ratio[END_REF] is to optimize the ratio between the cost of a transmission by the progress made by the transmission. Vcost [START_REF] Elhafsi | Cost over Progress Based Energy Efficient Routing over Virtual Coordinates in Wireless Sensor Networks[END_REF] relies on this idea and on VCap coordinate system to reduce energy needs for the routing.

The LTP algorithm [START_REF] Chávez | Routing in Wireless Networks with Position Trees[END_REF] uses a root and a tree construction as the only coordinate system instead of anchors. Thanks to the tree, this routing algorithm guarantees delivery but the chosen routes may be long. Above all, the tree construction is costly and hard to maintain.

HECTOR [START_REF] Mitton | Towards a hybrid energy efficient multi-tree-based optimized routing protocol for wireless networks[END_REF] combines both coordinates systems of LTP and VCap. Its chooses the best node in the VCap coordinate system at the same level or that allow a progress in LTP coordinate system. HECTOR is then able to guarantee delivery of packets and to reduce the energy used for the routing.

Other virtual coordinates solutions have been proposed, some are without guaranteed delivery [START_REF] Benbadis | Jumps: Enhancing hop-count positioning in sensor networks using multiple coordinates[END_REF], [START_REF] Fang | GLIDER: gradient landmark-based distributed routing for sensor networks[END_REF], [START_REF] Niculescu | Ad hoc positioning system (APS)[END_REF], some other with guaranteed delivery [START_REF] Chávez | Routing in Wireless Networks with Position Trees[END_REF], [START_REF] Liu | Stateless and guaranteed geometric routing on virtual coordinate systems[END_REF]. However, algorithms using virtual coordinates have the drawback to not support node mobility. Furthermore, it is needed to flood the whole network at the beginning, introducing high latency at startup when the network is big.

Background

Our proposal relies on route requests and route replies similar to those used in AODV [START_REF] Perkins | Ad-hoc on-demand distance vector routing[END_REF]. We introduce their behavior in the following. Figure 2 shows a route search in AODV. AODV is a non-geographic routing, totally reactive. Indeed, routes are established only on-demand, when they are needed. To route a data packet toward the destination, a node broadcasts a RREQ (route request) in its neighborhood. Each node receiving the RREQ keeps track of it until expiration delay expires (defined to 3000 ms in AODV). Keeping track of RREQ allows the creation of the reverse path as shown in Figure 2(a) that will be used when destination is found. If a node S does not know a route towards the destination D, S is not the destination and it has not received this RREQ earlier, it broadcasts a RREQ at its turn in its neighborhood. When a node knows a route toward the destination, or is itself the destination, it sends a RREP packet (route reply) to the node that has sent the RREQ. The RREP packet then follows the reverse path and each node on the route saves the source of the RREP in its neighborhood table in order to create the reverse path.

Proposal

In this paper, we propose HGA, a Hybrid Greedy-AODV algorithm. The main idea of HGA is to apply a greedy geographic routing whenever it is possible. When a greedy routing is not possible, a RREQ is sent in the k-neighborhood of the node. When a node needs to route a data packet and none of its neighbors allows a progress toward the destination, a route is searched in AODV fashion [START_REF] Perkins | Ad-hoc on-demand distance vector routing[END_REF].

We first describe the notations and the model used and then the algorithm details.

Notations and Model

The k parameter is the maximum depth for searching a route and defines the variant of HGA : HGA-k. The set of nodes aware of their position is P, then if u knows its position u ∈ P. R defines the routing table of a node, then R(u) is the routing table of node u. RREQ T IMEOUT is the delay until a RREQ is ignored if no RREP has been received. The Euclidean distance between nodes u and v is noted |uv|. N(u) is the set of neighbors in communication range of u. RREQs in the "waiting" state belong to the set W .

Algorithm Description

The header of a data packet contains the destination position and the last known position where the packet passed by.

To route a data packet, a node u starts by verifying if one of its neighbors allows a progress toward the destination. If u knows its own position, the progress depends of the distance between u and the destination. If u is not aware of its position, the progress depends on the last known position contained in the data packet if available, otherwise, any node aware of its position will be considered as making a progress. If no neighbor of u allows a progress, R(u) is sought to find a route allowing a progress in an AODV fashion. If there is no such a route, a RREQ is sent N(u) and a new research in routing table is made after the delay RREQ T IMEOUT . Algorithm 1 sums up route search on a node u.

When u receives a RREQ, it can either transmit it, answer with a RREP or discard it. Node u starts by analyzing R(u) to verify, by comparing the sequence number and the source node of RREQ to those in the routing table entries, whether u has already received this RREQ. If so, the RREQ is ignored and nothing happens, otherwise u search in N(u) if a node is closer to the destination than the source of the RREQ. If a route is found among neighbors of u, a RREP is sent to the nodes that transmitted the RREQ to u. The RREP contains the source id and sequence number from the RREQ, it also contains position of the closest neighbor of u to destination. If no route is found, the RREQ is forwarded if the maximum depth k for RREQ (MAX HOP RREQ) has not been reached. When a RREQ is transmitted, a new entry in the routing table is created with a 'waiting" flag allowing to know that a RREQ has been sent but no route is known yet. Algorithm 2 describes operations made at RREQ reception.

When a RREQ is received by a node u, the corresponding entry with "waiting" flag in the routing table is updated. The flag is removed and the neighbor allowing the destination to be reached is written is the table (this information is known thanks to the RREQ). Likewise, position of the destination is updated, indeed, the RREP may point to a position allowing to approach Algorithm 1: Data packet reception at node u towards D with l being last node with known position. HGA does not guarantee delivery, indeed if no node in the k-neighborhood of a node is aware of its position, the node will not be able to route packets. However, the route search mechanism allows to circumvent dead ends by searching new routes in the k-hops limit. Figure 3 shows an example of routing from node S, which is not aware of its position, to D, aware of its position. Figure 3(a) highlights a route request with a maximum depth of 3. Nodes receiving the RREQ and knowing their position answer with a RREP and S chooses the one which allow the maximum progression to destination. Once the packet has been routed to the intermediate destination, a new RREQ is initiated, the packet continues its progression then it is routed using classical geographic routing technique as intermediate destination knows a neighbor allowing a progression through destination 3(b). Finally, on Figure 3(c) a last RREQ is performed to allow the destination to be reached.

1 if u = D then 2 exit /* success */ ; 3 next ← -1 ; 4 if u ∈ P then 5 dist ← |uD| ; 6 l ← u ; 7 else if l = -1 then 8 dist ← |lD| ; 9 else 10 dist ← +∞ ; 11 for v ∈ N(u) do 12 if |vD| < dist then 13 next ← v ; 14 dist ← |vD| ; 15 if next < 0 then 16 /* no route found in direct neighborhood */ 17 broadcast(RREQ) ; 18 R ← R ∪ RREQ ; 19 W ← W ∪ RREQ ; 20 wait(RREQ TIMEOUT) ; 21 if ¬(∃r, r ∈ R ∧ |rD| < dist) ∧ r / ∈ W then 22 R ← R \ RREQ ; 23 /*
We choose to compare HGA to VCap, a routing algorithm using a similar greedy routing technique but based on virtual coordinates. We also simulate a greedy routing algorithm, slightly adapted to operate with some nodes without position awareness. In this variant, nodes without position knowledge do not send HELLO messages, then they do not participate in routing but can emit data packets. When a node needs to send a data packet (because it is the source or it needs to transfer it), it chooses in its neighbors with position the closest to the destination allowing a progress. If the node sending the data packet is not aware of its position, the notion a progress does not exist, then the closest position aware neighbor to destination is chosen.

We simulated HGA with route search depth of 1, 2, 3 and 5 hops. VCap has been simulated with 3 and 5 anchors.

Simulations have been performed using WS-NET [START_REF] Fraboulet | Worldsens: Development and Prototyping Tools for Application Specific Wireless Sensors Networks[END_REF] simulator. Each simulation lasts for 10 minutes. We generated random connected topologies of 500, 750, 1000, 1500, 2000, 2500 and 3000 nodes in a field of 500 m × 500 m to achieve average density of 10, 15, 20, 30, 40, 50 et 60 nodes per communication range. Nodes have a communication range of 40 m. Each combination (algorithm, average density) is launch 50 times on different topologies. The same topology is used for a combination of average density and iteration number for each different algorithm. For instance the first simulation of HGA-1 uses the same topology as the simulation of VCap-3, Greedy, etc.

To simulate a data traffic, every 10 seconds, each node chooses randomly a destination node aware of its position. The source node sends the data packet in respect to the algorithm it executes. Nodes are desynchronized in order to distribute emissions in time.

We measure delivery ratio, control message number and memory used. The delivery ratio is the number of messages received divided by the number of messages sent. Control messages are HELLO, RREQ, RREP and messages to initiate the coordinate system. Finally, the memory used is the average number of neighbors saved by a node plus the number of known routes for each node. Simulation parameters are summed up in Table 1.

For the sake of clarity, in the following we refer to HGA with a depth search of 1 hop by HGA-1 (k = 1). Moreover, HGA-2 (resp. HGA-3 and HGA-5) refer to HGA algorithm with depth search of 2 (resp 3 and 5) hops. Finally VCap-3 and VCap-5 refer to the VCap algorithm with 3 and 5 anchors. VCap results curves have been duplicated on figures with 1%, 5% and 10% located nodes in order to ease readings.

Delivery ratio

Figure 4 shows delivery ratio for the four variants of HGA, the two variants of VCap and the Greedy algorithm. We can see on Figure 4(a) that whatever the average density of the network, with 1% of nodes aware of their position, HGA-5 has a delivery ratio higher that the two variants of VCap and Greedy. We also observe than from an average density of 40, HGA-5 reaches almost 100% delivery ratio. As we expected, HGA-1 offers lower performances than HGA-2, which offers lower performances than HGA-3 while HGA-5 gets the best performances. As well, VCap is better with 5 anchors than with 3 as expected since it reduces the This hierarchy is also observed with 5% and 10% of nodes aware of their position as seen on Figures 4(b) and 4(c).

We can observe that HGA-1 performs much better than Greedy. HGA-1 has a delivery ratio 5 times higher for a density of 10 and almost 15 times higher for a density of 40 although only 1% of nodes are aware of their position. It shows that with only a search depth of 1 hop (k = 1), we can achieve really interesting performances. We will see how it impact the cost in messages in the next section.

One notices that HGA algorithms take a better advantage for higher densities of nodes. Indeed, for 1% of nodes aware of their position (Fig. 4(a)), the delivery ratio increases for densities from 10 to 20. It is similar for all variants of HGA and VCap. Besides, from a density of 20, the increase is stronger for HGA than for VCap. It allows HGA-2 to outperform VCap-3 from a density of 25 and HGA-3 is over VCap-5 from a density of 35.

With 5% of nodes aware of their position (Fig. 4(b)), performances of HGA are improved. This behavior is logical as a RREQ is more likely to find a node aware of its position and then a node in the direc- Figure 4: Delivery ratio tion of the destination. It is interesting to highlight that the delivery ratio increase is higher that with 5% of nodes aware of their position. It allows all variants of HGA to outperform both VCap variant from a density of 25. We can observe that this behavior is even more true with a position knowledge of 10% (Fig. 4(c)). These results show that HGA benefits more of a higher density of nodes than VCap. Indeed with VCap, the number of nodes with the same coordinates do not necessary decrease with the increase of node density while RREQs of HGA have a higher probability to reach a node in the direction of the destination as explained.

Control Messages Cost

Figure 5 shows the average number of control messages sent by nodes. For 1% of nodes aware of their position (Fig. 5(a)), we observe for HGA that, the deeper is the search, the more there is control messages. Likewise, the higher is density, the more messages are sent. It can be explained by the fact that more nodes receive the RREQs when the density is higher, each node receiving a RREQ potentially re-emits it, explaining the exponential increase of the number of messages emitted with the average density. It also explains why the increase is stronger when the route search if deeper. Figures show that the number of control messages sent by Greedy is very low because only nodes aware of their position emit control messages. For Greedy and VCap, the number of control messages sent is constant as it does not depend on density. For a low density, the control messages number is close for all algorithms. The gap increases with the density. For low densities, it is interesting to prefer HGA-5 which offers high delivery ratio, compared to other solutions, while adding a reasonable amount of control messages. When the average density increases, VCap is more suitable as it offers a delivery ratio close to, if not better than some variants of HGA while generating less control messages.

When the number of nodes aware of their posi-tion increases to 5%, the number of control messages strongly decreases for HGA, especially with deep search. This behavior is explained because the probability of finding a route during a RREQ increases when the number of nodes aware of their position increases. This probability also increases when the density of nodes increases (there is more nodes in the neighborhood to satisfy the request). This explains the inversion of curves for HGA for 5% and 10% of nodes aware of their position (Fig. 5(b) and 5(c)). However, there is no need to use deep variants for 10% of nodes aware of their position as the delivery ratio rapidly reaches 100% in that case.

Memory Cost

Figure 6 shows memory usage for the different algorithms. We can see that memory needs are much higher for HGA than VCap when 1% of nodes are aware of their position (Fig. 6(a)). With 5% (Fig 6(b)), the gaps for memory usage are lower and even really close to VCap when average density increase. On Figure 6(c), with 10% of nodes with position knowledge, memory needs are much lower for HGA and even lower than VCap when density is over 40. The memory size for Greedy is only the number of neighbors aware of their position for each node. The high amount of memory needed by HGA is explained because in simulations, every route is kept until the end of the simulation. It would be possible to lower the memory needs by "forgetting" old routes with the counterpart of having to send more control messages. We can highlight that experimental conditions are demanding concerning memory needs. Indeed, sources and destinations of data packet are chosen randomly at a high frequency. In real applications, the number of destinations of data packets are often limited if not unique in case of data collection. In case of actuators, one base station sends messages to different sources. In that case the source is unique, thus the number of routes to memorize for each node is reduced.

Conclusion

In this article we propose a novel solution for geographic routing, that allow to use nodes position when available but also able to deal with unavailable positions. To the best of our knowledge, there is no solution in the literature which relies on the same assumptions. Indeed, we can find geographic routing solutions for networks with all nodes aware of their positions as well as solutions using virtual coordinates where no node is aware of its position. We propose an hybrid solution, taking advantages of real node position when available but able to operate if not always available. The geographic and the reactive methods illustrated in this paper could be replaced by some other methods from literature or new ones. Both part should be chosen regarding application needs. The underlying topology of the application will favor some geographic routing techniques while the traffic pattern will favor the classical routing part. For low data rates, the classical routing part could be proactive or even hybrid as proposed in ZRP [START_REF] Haas | The Zone Routing Protocol (ZRP) for Ad Hoc Networks[END_REF].

Our solution, HGA offers much better performances than Greedy geographic routing if part of the nodes are not aware of their position. We also compare HGA with a routing solution based on virtual coordinates, VCap, and show that for different scenarios, our solution offer better performances with a limited or no overhead.

For future works, we consider to validate our works with realistic physical and MAC layers. The use of real platform such as FIT or SmartSantander [START_REF] Sanchez | Smartsantander: The meeting point between future internet research and experimentation and the smart cities[END_REF] would allow us to validate these aspects while testing realistic environments.

Studying node mobility is another interesting aspect, we could characterize the needed timeout before routes expires with regards to nodes speed. Comparison with routing algorithm using virtual coordinates would also bring some arguments for HGA since these algorithms need to frequently rebuild coordinates system, and then, increase number of control messages exchanged in the network.

Finally it could be interesting to compare our solution with a variant in which each node keeps a k-hop neighborhood table instead of using k-hop RREQ. The study of control messages number would allow us to find threshold to know which variant is better in some cases.

Figure 1 :

 1 Figure 1: Comparison of geographic algorithms. Node a is chosen by MFR algorithm, node b by Greedy, node c by NFP and node e by Compass.

  Forward path setup.

Figure 2 :

 2 Route search example from S to D in AODV.

  Figure 2(b) shows the creation of the forward path and expiration of reverse paths.

Algorithm 2 :

 2 Reception of a RREQ initiated by S with sequence number seqnum on node u for destination D. 1 if (S, seqnum) ∈ R then 2 exit /* RREQ already sent */;

Figure 3 :

 3 Routing example in HGA.

Figure

Figure 6 :

 6 Average size in memory.

Table 1 :

 1 Simulation parameters

	Parameter	Value
	Duration (m)	10
	MAC layer	idealmac
	Interferences	none
	Data size (bytes)	10
	Header size (bytes)	88
	Field size	500 m × 500 m
	Communication range	40 m
	Number of nodes	500, 750, 1000, 1500, 2000, 2500, 3000
	Density	10, 15, 20, 30, 40, 50, 60
	Iterations	50

* This work is partially supported by CPER Nord-Pasde-Calais/FEDER Campus Intelligence Ambiante and ANR ECOTECH BinThatThinks.