Marianne Leruez-Ville
email: marianne.leruez@aphp.fr

Sally Ren

Jean-François Magny

François Jacquemard

Sophie Couderc

Patricia Garcia

Anne-Marie Maillotte

Melinda Benard

Didier Pinquier

Philippe Minodier

Dominique Astruc

Hugues Patural

Melissa Ugolin

Sophie Parat

Bernard Guillois

Armelle Garenne

Marine Parodi

Laurence Bussières

Julien Stirnemann

Pascale 19

Sonigo

Anne Elodie Millischer

Yves Ville

Accuracy of prenatal ultrasound screening to identify fetuses infected by cytomegalovirus who will develop severe long-term sequelae

Keywords: cytomegalovirus, congenital infection, ultrasound, outcome

Objectives: To compare the ability to identify infected fetuses who will develop long-term sequelae, of routine ultrasound detailed examination performed without the knowledge of maternal serology and the fetal status to that of targeted prenatal imaging performed in prenatal diagnosis units in known cases of fetal infection.

Methods: All prenatal imaging reports were collected for 256 children with congenital CMV in a registered cohort between 2013 and 2017 (NCT01923636). All pregnancies underwent detailed fetal ultrasound examination at 20-24 and 30-34 weeks' as part of routine antenatal care. All cases of known fetal CMV infection underwent targeted prenatal ultrasound. Postnatal structured follow-up up until 48 months included audiology and neurological assessment including Brunet-Lezine scoring. All imaging reports were analyzed retrospectively with the knowledge of congenital CMV infection, searching for features that were or could have been related to fetal infection.

Results: 237 children with complete data were followed-up to a median 24 months, of whom 30% (71/237) and 70% (166/237) were diagnosed prenatally or within 3 weeks of life respectively. 73% (29/40) of children with any sequelae and 74% (14/19) with severe sequelae (bilateral hearing loss and/or neurologic sequelae) were not identified in the prenatal period. In those diagnosed prenatally, sensitivity of prenatal imaging for any and severe sequelae was 91 and 100%. In the group diagnosed postnatally non-specific infection-related features were reported in 48% and 64% with any and severe sequelae without raising suspicion.

Conclusions: Routine ultrasound screening in pregnancy is not an appropriate screening tool for congenital CMV infection leading to long-term sequelae, contrasting with the high performance of targeted prenatal imaging in known cases of fetal infections. Non-specific nature of ultrasound features and their evolution and lack of awareness of caregivers about cCMV are the main explanations. Sonologists' awareness and knowledge of maternal serological status in the first trimester seem key to the performance of routine prenatal ultrasound.

Contribution What are the novel findings of this work

Without CMV serology screening in pregnancy, routine ultrasound examination identified 26% of infected fetuses that developed long-term sequelae, although non-specific infection-related features were reported in 64% without raising suspicion. However targeted ultrasound of known infected fetuses had 91% sensitivity and 96% negative predictive value.

What are the clinical implications of this work

Routine ultrasound screening in pregnancy is not an appropriate screening tool for congenital CMV infection leading to long-term sequelae. Sonologists' awareness and the knowledge of maternal serological status in the first trimester are key to the performance of prenatal ultrasound.

Introduction

Congenital CMV infection (cCMV) affects 0.7% of all newborns worldwide 1 and around 15 to 20% of those suffer long-term sequelae 2 . In high-income countries half of infected newborns are infected following maternal primary infection in pregnancy and the other half following maternal non-primary infection (reactivation or reinfection) [3][START_REF] Puhakka | Primary versus non-primary maternal cytomegalovirus infection as a cause of symptomatic congenital infection -register-based study from Finland[END_REF] . All long-term sequelae develop in fetuses that have been infected after maternal infection occurring in the first trimester of pregnancy [START_REF] Foulon | Hearing Loss With Congenital Cytomegalovirus Infection[END_REF][6] .

Around 30 to 35% of newborns infected after maternal primary infection in the first trimester develop significant neurological sequelae 7 . Series of infected fetuses diagnosed prenatally following documented maternal primary infection report high sensitivity and negative predictive value of targeted prenatal ultrasound for the occurrence of symptoms at birth and of sequelae at follow-up [START_REF] Faure-Bardon | Refining the prognosis of fetuses infected with cytomegalovirus in the first trimester of pregnancy by serial prenatal assessment: A single center retrospective study[END_REF][START_REF] Leruez-Ville | Feasibility of predicting the outcome of fetal infection with cytomegalovirus at the time of prenatal diagnosis[END_REF][START_REF] Leyder | Primary maternal cytomegalovirus infections: accuracy of fetal ultrasound for predicting sequelae in offspring[END_REF][START_REF] Lipitz | Risk of cytomegalovirus-associated sequelae in relation to time of infection and findings on prenatal imaging[END_REF][12] . Following maternal prenatal serological screening and a positive amniocentesis, ultrasound performed by 23 weeks' identified up to 90% of cases with postnatal sequelae; and even more so by adding MRI examination at 28-32 weeks' [START_REF] Leruez-Ville | Feasibility of predicting the outcome of fetal infection with cytomegalovirus at the time of prenatal diagnosis[END_REF] . Normal ultrasound follow-up of fetuses known to be infected leaves a residual risk of isolated unilateral neurosensory hearing loss of 14% [START_REF] Faure-Bardon | Refining the prognosis of fetuses infected with cytomegalovirus in the first trimester of pregnancy by serial prenatal assessment: A single center retrospective study[END_REF] .

No country outside Germany and Italy, have yet implemented prenatal maternal serological screening for CMV [START_REF] Society | Diagnosis and antenatal management of congenital cytomegalovirus infection[END_REF] . Therefore, most maternal primary infections remain unknown and all maternal non-primary infections remain undiagnosed in the absence of any validated diagnostic tools. Consequently, sonologists are mainly unaware of the maternal status at the time of routine second or third trimester ultrasound examination and only 20% of symptomatic neonates could be identified in one study [START_REF] Guerra | Ultrasound prediction of symptomatic congenital cytomegalovirus infection[END_REF] . However, symptoms at birth are associated with long-term sequelae in hearing loss [START_REF] Fowler | Congenital cytomegalovirus infection[END_REF] . The value of routine detailed ultrasound examination of the fetal anatomy to identify fetuses who will become infants with long-term sequelae is unknown. The aim of this study was to assess the type and proportion of sequelae that are missed by detailed ultrasound examination in the second and in the third trimester, as part of routine antenatal care offered to all pregnant women in France.

Material and methods

Population

The Cymepedia study included infected neonate/mother pairs in 11 French perinatal centers between 2013 and 2017. Neonates included in the study were diagnosed with congenital CMV either prenatally because of a primary infection in the mother and/or compatible prenatal ultrasound features in the fetus or postnatally. The study was proposed to the parents of all children diagnosed with cCMV and only around 5% of those refused to be included. The cohort was composed of a total of 256 neonates.

The Cymepedia study is registered in clinicaltrial.gov website under NCT01923636. All parents gave written consent for their children to participate to the study. Ethics committee approved the study (2013-A00213-42).

Determination of the timing of maternal CMV infection

CMV serological assessment was centralized in Necker Reference laboratory. Among the 237 cases, maternal serology was screened at 12 weeks' in 78. In the remaining cases, maternal serology results were obtained retrospectively on stored sera. In France, serum samples are systematically collected in first and third trimesters of pregnancy for various serology testing. Moreover, women seronegative for toxoplasmosis (70% of the pregnant population) are tested monthly. All sera are stored for 1 year and can therefore easily be tested retrospectively.

Levels of CMV specific IgG and IgM antibodies were determined with LIAISON® CMV IgG II and LIAISON® CMV IgM on Liaison XL platform (Diasorin, Antony, France). IgG avidity was measured by the LIAISON® CMV IgG Avidity II and/or by VIDAS® CMV IgG avidity II (BioMerieux, Marcy L'Etoile, France) as described elsewhere 16 . The algorithm of CMV serology interpretation is shown in Figure S1.

Follow-up protocol

All infected children were followed with the same protocol including visits at 4, 12, 18, 24, 36 and 48 months of age. Clinical examination was standardized to assess motor, cognitive, speech and psychological development according to age as previously described 6 . Audiological assessment and the presence of otitis media were recorded at each visit. A Brunet-Lezine test which is an early childhood psychomotor development scale covering four areas of neurodevelopment: movement and posture, coordination, language, and sociability was done at M12 and M24 17 . Auditory Brainstem Responses (ABRs) or audiology tests were done depending on the child's age. Conductive SNHL (Sensorineural Hearing Loss) with presence of otitis media with effusion was considered non-interpretable and testing was repeated 4 to 6 months after otitis healing. Transtympanic drains were placed when indicated. Hearing was considered normal if the child could hear stimuli of between 0 and 20dB. SNHL was mild, moderate, severe and profound for detection of sound within 21 to 30 dB, 31 to 60 dB, 61 to 90 dB and ≥91 dB respectively. Vestibular functions were assessed in children with SNHL and/or delayed walking as previously reported 6 .

Sequelae were considered to be mild in cases with isolated unilateral hearing loss and/or vestibular disorders. Sequelae were considered to be severe in cases with bilateral hearing loss and/or neurologic sequelae.

Antenatal ultrasound results

Reports of all routine detailed ultrasound examinations performed in the second trimester (20 to 24 weeks) and in the third trimester (30 to 34 weeks) of pregnancy were identified from the women pregnancy files and collected retrospectively from the sonologists. They are working in different settings including teaching hospitals, regional hospital, private hospital or private clinics all over France. Since the aim of the study was to compare the performance of ultrasound examination in routine setting (maternal serology unknown, fetal infection unknown) to the performance of ultrasound when both maternal and fetal status were known, we deliberately not attempt to review the ultrasound images and only took into account the information obtained at the time of the examination and as written by the operator in the ultrasound report.

Prenatal detailed ultrasound examination, as part of the pregnancy care in France, includes a number of measurements and of features to be made and checked for using standardized ultrasound planes at recommended gestational ages of 20-24 and 30-34 weeks. This is offered to all pregnant women and the examinations are performed by registered sonologists 18 . The items checked for that could be relevant to fetal CMV infection include fetal biometry, size of the cerebral lateral ventricles, aspect of the cerebral midline, aspect and diameter of the cerebellum, size and position of the stomach bubble, aspect of the fetal bowel, placenta and the amount of amniotic fluid. Therefore, infection-related features that could be suspected include: small for gestational age (<10° percentile), microcephaly (< 2 DS), ventriculomegaly, abnormal cerebral midline, abnormal posterior fossa, abnormal cerebellum, hyperechogenic bowel, hepatosplenomegaly compressing or displacing the stomach bubble, placentomegaly, and oligo or polyhydramnios 18 .

When amniocentesis was positive for CMV, the women underwent a follow-up protocol with US scan every 2 to 3 weeks and systematic cerebral MRI between 32 and 34 weeks. Target ultrasound scans were realized by sonographers in fetal medicine Units.

Statistical analysis

The proportions between groups were compared by exact Fischer test. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), negative likelihood ratio (LR-) and positive likelihood ratio (LR+) were calculated. All analyses were conducted using R (R Foundation for Statistical Computing, Vienna, Austria).

Results

Description of the population (Figure 1) Antenatal ultrasound reports were recovered for 255 mothers of the 256 eligible children. One mother received very limited antenatal care. Among the 255 cases with antenatal ultrasound results available, 16 (6.2%) children were lost for follow-up before the age of one year. In addition, two children were excluded from the study because they were both diagnosed with a genetic disorder associated with neurologic symptoms and neurodevelopmental delay (Silver Russel and Galloway Mowat syndromes respectively). 71/237 (30%) of the cases were diagnosed prenatally with a positive CMV PCR in the amniotic fluid at amniocentesis. Amniocentesis was done because of a serology CMV screening with identification of a primary maternal infection in 89% (63/71) of these cases or because routine ultrasound screening picked up compatible symptom(s) in 11% (8/71). 166/237 (70%) cases were neither identified nor suspected in the antenatal period and were diagnosed only postnatally in the first 3 weeks of life. Among these 166 infected neonates, 38 were recruited through a universal

Performance of antenatal imaging in cases diagnosed prenatally (fetuses known as infected at the time of prenatal imaging)

The sensitivity, NPV and LR-of targeted prenatal imaging following a positive prenatal diagnosis were 91%, 96% and 0.21 to diagnose all cases with sequelae and 100%, 100% and 0 for cases with severe sequelae (Table 2). The PPV and the LR+ of imaging features were low when all features were considered. However, the specificity, PPV and LR+ of severe cerebral symptoms to predict a severe outcome reached 96%, 80% and 20 respectively (Table 3). Indeed, among children whom infection was diagnosed prenatally, 5 had severe sequelae and in 4 of the 5, severe cerebral abnormalities were identified by targeted ultrasound and MRI examinations.

The one child identified prenatally and who did not show severe cerebral abnormalities, developed bilateral hearing loss with no psychomotor delay.

Performance of antenatal imaging in cases diagnosed at birth (fetuses not known as infected at the time of prenatal imaging)

Based upon the review of ultrasound reports, the sensitivity, NPV and LR-of ultrasound screening could have been 48%, 87% and 0.70 to identify all cases with sequelae and 64%, 95% and 0.49 to identify cases with severe sequelae (Table 2). However, in all of these cases, although ultrasound features of infection were present the sonographer or the attending obstetrician did not raise the suspicion of CMV fetal infection. The sensitivity and NPV of ultrasound screening could have been 37% and 74% respectively to identify cases with severe sequelae in the group of children infected after a maternal primary infection in the first trimester (Table S1).

Comparison of imaging results between cases diagnosed prenatally and those diagnosed only postnatally

Table 4 reports the types and proportion of ultrasound features of infection: SGA, extra cerebral features, non-severe cerebral features and severe cerebral features, in the 2 groups of patients according to the presence of sequelae and to the type and date of maternal infection.

SGA was the most frequent US finding and was detected in similar proportions between the prenatal and the postnatal groups in cases with sequelae (36% Vs 45%) and in cases without sequelae irrespective of the trimester of maternal infection.

However, the presence of at least 1 extra-cerebral feature of infection was significantly more frequent in the prenatal group than in the postnatal one in cases with sequelae (82% Vs 3%, p<0.001) but also in the subgroup of cases with no sequelae following maternal infection in the first trimester (37% Vs 7%, p=0.002).

In the group of children diagnosed postnatally, no severe cerebral abnormalities were identified in the 29 children who developed sequelae (including the 14 with severe sequelae). None of these cases had access to fetal MRI but postnatal MRI was performed in 13/14 cases and showed severe white matter lesions in 10 of 13. Conversely, in the group diagnosed prenatally, 36% (4/11) of children with sequelae, and 80% (4/5) of those with severe sequelae showed severe white matter abnormalities identified by prenatal MRI (p=0.003 and p=0.001).

Discussion

In this cohort of children with congenital CMV infection, 73% (29/40) of any sequelae and 74% (14/19) of severe sequelae were not anticipated prenatally although prenatal care followed the national French guidelines including 2 standardized detailed ultrasound examinations of the fetal anatomy at between 20-24 and 30-34 weeks. In over half of the cases missed in utero who developed sequelae non-specific ultrasound features were present but was not raised by the sonologist nor by the attending clinician receiving the report. Even assuming that those ultrasound features would have been related to CMV infection, the low sensitivity of ultrasound screening compares poorly with the sensitivity of diagnostic ultrasound performed with the knowledge of fetal infection, that reached 91% in all cases and 100% in severe cases, thus confirming previous reports [START_REF] Leruez-Ville | Feasibility of predicting the outcome of fetal infection with cytomegalovirus at the time of prenatal diagnosis[END_REF][START_REF] Leyder | Primary maternal cytomegalovirus infections: accuracy of fetal ultrasound for predicting sequelae in offspring[END_REF][START_REF] Lipitz | Risk of cytomegalovirus-associated sequelae in relation to time of infection and findings on prenatal imaging[END_REF][12] . Therefore, a known infected fetus with a normal ultrasound and MRI was left with a residual risk of 10% of mild sequelae as previously reported [START_REF] Faure-Bardon | Refining the prognosis of fetuses infected with cytomegalovirus in the first trimester of pregnancy by serial prenatal assessment: A single center retrospective study[END_REF] .

The PPV of extra cerebral and non-severe cerebral features for sequelae were respectively 23% and 20% in our study, while PVV over 60% has been reported in most studies including both termination (TOP) cases and live children [START_REF] Leruez-Ville | Feasibility of predicting the outcome of fetal infection with cytomegalovirus at the time of prenatal diagnosis[END_REF][START_REF] Leyder | Primary maternal cytomegalovirus infections: accuracy of fetal ultrasound for predicting sequelae in offspring[END_REF][START_REF] Lipitz | Risk of cytomegalovirus-associated sequelae in relation to time of infection and findings on prenatal imaging[END_REF][12] . One explanation that might reflect a weakness of our study is that our population is a postnatal cohort and therefore TOP cases could not be identified and accounted for in the performance of antenatal assessment. Indeed, the specificity, PPV and LR+ of severe cerebral features, mostly on MRI, to predict severe sequelae were high (96, 80% and 20 respectively).

We reported that with a strategy based on CMV serology screening in the first trimester for maternal primary infection followed by prenatal diagnosis, the risks of neurologic sequelae and of hearing loss were of 12 and 27% respectively while 17% of affected cases were terminated for severe cerebral lesions 6 . Interestingly, the natural history of cCMV following maternal infection in the first trimester is associated with neurologic sequelae and hearing loss in 32% and 23% respectively 7 . Although no direct comparison can be made, the dramatic difference in neurological sequelae between these populations suggests that cCMV antenatal screening is likely to identify the most severe cases and give parents the alternative choice of terminating the pregnancy or anticipating the birth of a child with potential severe neurologic sequelae. The identification of cases following maternal non-primary infection is more difficult since serology is not helpful [19][20] . However, we show here that around 90% (9/10) of severe cases following maternal non-primary infection presented with at least one feature at ultrasound screening, mainly SGA.

Several hypotheses could be made to explain the poor performance of routine ultrasound examination even when this involves a detailed and structured examination of fetal biometry and anatomy: 1) Ultrasound screening had a poor sensitivity to detect subtle extra cerebral features of infection (hyperechogenic bowel, hepatosplenomegaly) that may be transient: in cases with sequelae they were only reported in 3% of those diagnosed postnatally while they were reported in 82% of those diagnosed prenatally (p< 0.001). Conversely, SGA, which was the most frequent warning sign, was reported in the same proportion in cases diagnosed postnatally and prenatally.

Ultrasound screening has therefore a good performance to detect SGA related to CMV. However this symptom is frequent and nonspecific and the awareness of the sonologists should therefore be raised regarding its link to the most frequent congenital infection affecting 0.37% of all births in France 3 . 2) The development of fetal lesions is a continuous and heterogeneous process that may not offer the possibility to be recognized at only two points in time in the pregnancy. Indeed, none of the severe cases in the group that did not benefit from a prenatal diagnosis had severe cerebral lesions picked-up by routine ultrasound examination, while in the group with prenatal diagnosis, 80% of the severe cases had severe cerebral anomalies identified by targeted imaging (ultrasound and/or MRI). Indeed, the knowledge of fetal infection will generate serial and targeted ultrasound follow-up and planned MRI in the third trimester; all more likely to identify even subtle significant lesions.

The prevalence of neonatal infection is 0.37% in France with around 2,960 infected neonates per year. One third of these, therefore 986 neonates, are infected following maternal infection in the first trimester and 315 (32% of 986) are therefore expected to bear long-term neurological sequelae. However, only around 60 cases of TOP in relation with severe cCMV infection are reported annually in France through the mandatory annual report to Health Authorities 21 . This is therefore compatible with our results and also suggests that only around 20% of all severe cases might be identified prenatally also at national level.

Strengths and Limitations

One weakness of this study is that the cases included in this pediatric cohort did not come for a systematic neonatal screening program. It is therefore impossible to estimate the true proportion of severe cases that were missed by routine ultrasound. Another limitation is that the population did not include cases of termination of pregnancy. However, this could also be seen as a strength since in prenatal cohorts, terminated cases are classified as severe cases based upon the lesions seen at autopsy although it remains difficult to preclude if those interrupted fetuses might have developed long-term sequelae or otherwise. Another limitation of the study is the length of follow-up with a median of only 24 months since late-onset hearing loss has been reported up to5 years of age. However, as extensively documented in the literature late onset hearing loss is rare after 2 years of age [START_REF] Lanzieri | Congenital Cytomegalovirus Longitudinal Study Group. Hearing Loss in Children With Asymptomatic Congenital Cytomegalovirus Infection[END_REF][START_REF] Townsend | Longterm outcomes of congenital cytomegalovirus infection in Sweden and the United Kingdom[END_REF] . Finally, this study is multicentre including 11 different paediatrics centres all over France and the women had their prenatal follow-up in various settings including teaching hospitals, district general hospitals, private hospitals and private clinics therefore reflecting the diversity of medical care.

Conclusion

Our study shows that even a well-organized systematic ultrasound screening in second and third trimester, as it is implemented in France, is not sensitive enough to identify most CMV cases with cerebral lesions that will lead to neurologic long-term sequelae. This calls for raising awareness of all antenatal care providers, and particularly sonologists; this should help identifying severe cases following both primary and non-primary maternal infection. Moreover, we believe that the results of this study should be taken into account in the evaluation of offering maternal serology testing in the first trimester.

Role of funding source

This work was funded by the French government (Direction de la recherche Clinique et Développement); Cymepedia Clinicaltrial.gov numbers, NCT01923636).

Figure legend

  neonatal screening program implemented in 2 centers between 2013 and 2015 3 ; 113 were diagnosed because of compatible symptoms (SGA, hearing loss, thrombocytopenia…) and 15 were diagnosed because of a known primary infection in pregnancy. 45/237 (19%) of the neonates were preterm: 21,19 and 5 were born between 36 and 37 weeks, 31 and 35 weeks and 26 and 31 weeks respectively. The median follow-up of the 237 children was 24 months (range: 12-48 months), only 22 children were lost for follow-up between 12 and 24 months. 17% (40/237) of the children had long-term sequelae, mild in 9% (21/237) and severe in 8% (19/237). The detailed prenatal ultrasound findings and the type of sequelae for the 40 children with sequelae are reported in

Figure 1 :

 1 Figure 1: Flow chart of the study

Table 1 .

 1 27% (11/40) of cases with any sequelae and 26% (5/19) of cases with severe sequelae were diagnosed prenatally, following maternal serology screening in 9 cases or because of evocative ultrasound features in 2 cases. 73% (29/40) of cases with any sequelae and 74% (14/19) of cases with severe sequelae were not identified in the prenatal period. There was a similar proportion of prematurity in children with sequelae than in those without: 15% (6/40) and 19% (39/197) respectively.

Table 1 :

 1 Description of cases with sequelae: type and date of maternal infection, type of sequelae and prenatal imaging (N=40)

		Type of maternal infection	Sequelae	Prenatal US features
		(date in weeks)		throughout the pregnancy
	Cases diagnosed prenatally (positive CMV PCR in amniotic fluid ) N=11
	001-001	PI (8)	Moderate uni HL	SGA, placentamegaly
				splenomegaly
	001-003	PI (7)	Profound uni HL	HEB, hepatomegaly
	001-073	PI (2)	Profound uni HL	SGA
	001-083	PI (3 )	Profound uni HL	SGA, HEB
	001-100	PI (10 )	Severe uni HL	Placentomegaly, HEB, hepatomegaly,
				splenomegaly
	001-117	PI (2)	Profound Bil HL	SGA, HEB
	001-130	NPI	Psychomotor delay	Splenomegaly, mild ventriculomegaly, sub
				ependymal cysts, LSV
	002-020	PI (11)	Moderate uni HL	0
	007-008	PI (8)	Moderate uni HL, spastic	HEB, sub ependymal cysts, LSV
			dysplegia
	016-005	PI (4)	Profound bil HL	HEB, hepatomegaly, splenomegaly,
				Sub ependymal cysts
	017-002	NPI	Psychomotor delay	Hepatomegaly, polyhydramnios,
				microcephaly
	Cases not diagnosed prenatally (amniocentesis not done) N=29
	001-025	PI (8)	Profound uni HL	0
	001-026	Pi (8 )	Profound uni HL	0
	001-048	NPI	Moderate uni HL vestibular	0

Table 2 :

 2 Sensitivity, specificity, PPV and NPV of prenatal ultrasound for long-term sequelae

	Se	Sp	PPV NPV LR+ LR-

Population diagnosed prenatally N=71/ severe sequelae n=5 100% 38% 11% 100% 1.61 0 Se= sensitivity; Sp=specificity; PPV= positive predictive value, NPV= negative predictive value; LR+= positive likelihood ratio; LR-= negative likelihood ratio

Table 3 :

 3 Sensitivity, specificity, PPV and NPV of specific imaging features for long-term sequelae in the subgroup diagnosed prenatally

	N=71 Number of sequelae =11 Number of severe sequelae=5 Se	Sp	PPV NPV LR+ LR-
	At least one extra cerebral symptom / all sequelae	81% 66% 23% 76% 2.38 0.28
	At least one non severe cerebral symptom/ all sequelae	18% 86% 20% 85% 1.28 0.95
	At least one severe cerebral symptom /all sequelae	36% 98% 80% 89% 18	0.65
	At least one severe cerebral symptom/severe sequelae	80% 96% 80% 98% 20	0.21

Se= sensitivity; Sp=specificity; PPV= positive predictive value, NPV= negative predictive value; LR+= positive likelihood ratio; LR-= negative likelihood ratio

Table 4 :

 4 Prenatal imaging according to the presence of sequelae and the time and type of maternal infection Maternal primary infection, NPI= Maternal non-primary infection, T1= First trimester of pregnancy, T2= Second trimester of pregnancy, T3= Third trimester of pregnancy, LSV= Vasculopathy of the lenticulo-striate vessels, Cysts= sub-ependymal cysts; VM= ventriculomegaly, mild ventriculomegaly (<15 mm), WMA: white matter abnormalities SGA= small for gestational age (< 10 th percentile); Mc= microcephaly; *= excluding SGA

	N=237	Prenatal	Prenatal	P	Prenatal	Prenatal	p	Prenatal	Prenatal	p	Prenatal	Prenatal	p
		diagnosis	diagnosis	OD	diagnosis	diagnosis		diagnosis	diagnosis		diagnosis	diagnosis
		positive	not done	CI95%	positive	not done		positive	not done		positive	not done
		PI and NPI	PI and NPI		PI T1	PI T1		PI T2 /T3	PI T2 /T3		NPI	NPI or type
		with	with		no	no		no	no		No	of infection
		sequelae	sequelae		sequelae	sequelae		sequelae	sequelae		sequelae	unknown
		N=11	N=29		N=30	N=42		N=29	N=69		N=1	no sequelae
												N=26
	No US	1/11	15/29	0.02	13/30	35/42	<0.001	11/29	50/69	0.004	1/1	16/26	NS
	features	(10%)	(52%)		(43%)	(83%)		(38%)	(72%)			(61%)
	SGA	4/11 (36%)	13/29	NS	3/30 (10%) 4/42 (9%)	NS	8/29	11/69	NS	0	8/26 (31%) NS
			(45%)					(27%)	(16%)
	1 extra	4/11 (36%)	1/29 (3%)	0.01	7/30 (23%) 3/42 (7%)	NS	5/29	10/69	NS	0	2/26 (7%) NS
	cerebral							(17%)	(14%)
	feature *
	2 extra	4/11 (36%)	0/29	0.003 3/30 (10%)	0/42	NS	2/29	5/69 (7%)	NS	0	0/26	NS
	cerebral		(0%)			(0%)		(7%)
	features *
	3 extra	2/11 (18%)	0/29	NS	1/30 (3%)	0/42	NS	3/29	0/69	0.02	1/1	1/26 (4%) NS
	cerebral		(0%)			(0%)		(10%)
	features *

PI=

Acknowledgments:

We thank all women and parents who participated in the trial. We also thank URC-CIC Paris Descartes (Laurence Lecomte, Guillaume Masson, Eric Dufour, Myriam Virlouvet, Cindy Parent) for the implementation, monitoring and data management of the study.

Conflict of interest


Jonathan Jumeau
email: jumeau.jonathan@gmail.com

Déborah Wolf

Léo Guthmann

Nicolas Gorlero

Françoise Burel

Yves Handrich

The use of military bunkers by the European badger and red fox in Western Europe

Keywords: refuges, artificial habitat, military fortifications, farmlands, burrows 27

Intensification of agriculture and land consolidation policy induced the removal of most 8 natural features from farmland, reducing the habitat of many wild species. Abandoned WWII 9 bunkers, unaffected by land consolidation, are a potential shelter for wildlife and could be 10 used as burrows by carnivores such as European badgers and red foxes.

11

We explored 182 abandoned bunkers in a farmland area in eastern France in summer 2016.

Bunkers were located in crops, forests, or groves. Over a three-month period, signs of 13 presence, the number and type of burrows inside the bunkers and in their immediate vicinity 14 were noted. Environmental variables were obtained using GIS, and binomial models were 15 created to reveal parameters influencing bunker use by badgers and foxes. The degree of 16 bunkers burrowing was used as a co-variable. Twenty-one groves without bunkers were also 17 studied in order to estimate the added value of bunkers in groves.

18

Badger and fox tracks were found on 34% of the sites, and burrows on 24% of them with similar rates for badgers and foxes. Animals used partially buried bunkers more than 20 underground or overground bunkers. The sizes of annual crops and urban areas around the 21 bunker were positively correlated with bunker use. The presence of a bunker in groves was 22 positively correlated with the presence of burrows in groves. intensive farming landscapes. We recommend the management of these ruins to facilitate the 25 sustainable installation of a variety of species.

Introduction

30

A huge number of military fortifications were built before and during the Second World 31 War, often located close to border areas like the Maginot Line or the Atlantic Wall. These 32 concrete bunkers (English), Blockhäuser (German) or casemates (French) are often located 33 underground, either by design or due to burying over time. They have a small number of 34 rooms, and the largest constructions have a well to collect water (van der Well & van Beveren 35 2016). Entrances to the larger defence bunkers are hard to reach, either due to ditches or 36 because they are completely underground. The entrances of bunkers used as storage units or 37 as garrisons (blockhouses) are larger and easier to reach. At the end of the war, these 38 structures were often abandoned. They are currently in a good state of conservation but are 39 less accessible. This is particularly true on farmland, where bunkers have been covered by 40 successive ploughing. As they were built in defensive or support lines, bunkers are widely 41 dispersed, covering large areas of land [START_REF] Mermy | Inventaire 468 départemental des cavités souterraines hors mines du Bas-Rhin[END_REF]. In Europe, they are found from 42 the Stalin Line in the East to the Atlantic Wall and the GHQ line in the West. There is no precise inventory of these structures, but the number present along the different defensive 44 lines represented in Figure 1 could be estimated between 120 000 and 200 000. Although the [START_REF] Tscharntke | Landscape 501 perspectives on agricultural intensification and biodiversity -Ecosystem service 502 management[END_REF], where pesticide use is common. The habitat quality and biodiversity 78 of these environments are usually low (Benton et al. 2003;[START_REF] Maxwell | The ravages of guns, nets and 464 bulldozers[END_REF]. During the 79 second half of the 20 th century, land consolidation policy led to the removal of natural 80 habitats such as wetlands or copses [START_REF] Bonfanti | Landscape analysis in areas affected by land 413 consolidation[END_REF][START_REF] Butet | Effects of agriculture development on vole dynamics and 415 conservation of Montagu's harrier in western French wetlands[END_REF]Robinson & 81 Sutherland 2002). This continuing agricultural intensification mainly has a negative impact on 82 biodiversity [START_REF] Poschlod | Changing land use and its impact on 475 biodiversity[END_REF][START_REF] Desjeux | Evaluating the impact of rural development measures on nature value indicators 430 at different spatial levels: Application to France and the Netherlands[END_REF]Benton et al., 2003). However, bunkers 83 found in these areas can play a role in preserving semi-natural elements in their vicinity such 84 as hedges, pathway verges and groves, which can all be used by a variety of species in 85 including mesopredators [START_REF] Dondina | How to manage hedgerows as effective 435 ecological corridors for mammals: A two-species approach[END_REF], or can even be used by animal species as 86 alternative habitats.

87

The red fox (Vulpes vulpes) and the European badger (Meles meles) are probably the most 88 common fossorial carnivores in Western European farmlands (Hoffmann & Sillero-Zubiri food stocked in warehouses and silos (König 2013). They also limit the dispersion of invasive 92 species, as illustrated by the reduction in the populations of American mink (Neovison vison) 93 by the red fox in Fennoscandia (Ritchie et al. 2012). Scavenging by foxes is considered as an 94 ecosystemic service by providing sanitary services [START_REF] Sekercioglu | Increasing awareness of avian ecological function[END_REF][START_REF] Devault | Mesopredators dominate 433 competition for carrion in an agricultural landscape[END_REF]. The

95

European badger provides services such as the dissemination of seeds and their resurfacing 96 from the soil seed bank, and abandons burrows that will then be used by other mammal 97 species (Kurek et al. 2013).

98

Burrow use is very different in these two species, which both use a number of burrows 99 that are spread across their territory. The red fox mainly has small burrows with one or two 100 entrances. The badger has a variety of burrows (called "setts") that vary in size and function.

101

The main sett is large with up to hundred entrances and can be very old, sometimes having 102 been used by several generations of badgers [START_REF] Roper | Internal structure and contents of three 490 badger ( Meles meles ) setts[END_REF]). There is one main sett per 103 group territory, where breeding occurs. Annexe and subsidiary setts have fewer entrances 104 and are located near the main burrow. Outlier setts are located on the edge of the territory 105 and have just one or two entrances [START_REF] Roper | The structure and function of badger setts[END_REF]. While the main sett is used for breeding, 106 thermoregulation and avoiding predators, the benefits provided by outlier burrows remain 107 unclear. They could play a role in regulating ectoparasite load, reducing the incidence of 108 breeding suppression and minimizing the costs of travelling through the territory (Davison et 109 al. 2008).

110

We hypothesized that bunkers are used as outlier burrows by both of these fossorial 111 species but that entrances located at an accessible height are necessary for this to occur.

122

with the majority located on farmland that is mainly composed of corn and wheat crops with 123 rare groves. In this study, a grove was defined as a small group of trees (>3 m high) with a size 124 between 0.01 and 1 ha, with or without an understorey. If the area exceeded 1ha, the 125 landscape type was considered to be forest. Six hundred and fourteen fortifications have 126 been found and geolocated in this region, but this list is non-exhaustive and the overall 127 number could be far higher [START_REF] Mermy | Inventaire 468 départemental des cavités souterraines hors mines du Bas-Rhin[END_REF]. Indeed, new bunkers are regularly found 128 through archaeological excavations.

129 Not all the bunkers were studied; those located within towns and cities were excluded 130 from the present study. The remaining bunkers (N=120) were randomly sampled to obtain a 131 representation of all the bunkers in the studied area. In addition to the 120 selected bunkers, 132 a further 62 bunkers were found during inspections. The analyses therefore concern a total of 133 182 bunkers (Figure 2). Twenty-one groves without bunkers and surrounded by crops were 134 also studied over the same period in order to compare their attractivity with that of groves the same environmental type.

163

• Overground: the bunker had at least one large human-made entrance that allowed easy 164 access to both carnivores and humans.

165

Landscape analysis

166

GIS was used to map landscape elements within an 800 m radius of each bunker. This 167 corresponds to an average badger home range in western Europe, and represents a smaller 168 home range than the red fox [START_REF] Meia | Home ranges and movements of red foxes in central 466 Europe: stability despite environmental changes[END_REF]Balestrieri et al. 2016). The distance to the 169 nearest landscape element of each type was computed, and the size of each landscape 170 element type was calculated within each 800 m radial buffer. Landscape elements types were 171 as follows: watercourses, ponds and lakes, groves, hardwood forests, scrublands, mixed considered to occur if any evidence of badger or fox presence was observed), (2) the occupied burrows (also called burrow in use) indicated by fresh evidence of their use, and (4) 181 the type of badger sett (central/outlier). These dependent variables were used for bunkers 182 and for groves without bunkers.

232

On eight sites (4.4%), bunkers were used by humans (house, wood/tractor storage, waste 233 disposal site). In two bunkers, several underfed amphibians were found with a number of dead animals of different species trapped in underground rooms that could only be reached 235 using a ladder.

236

Landscape analysis

237

Selection of collinear variables resulted in 14 collinear groups. The 14 main explanatory variables were extracted (Table 2). Data for both species were pooled to obtain 239 sufficient data for landscape analysis.

240

Partially buried Bunker

Resting area

Configuration 2: The bunker is small and partially buried. The inside can only be reached via a small opening no larger than a burrow hole. A resting area is present in the bunker. The bunker is considered as a burrow.

Configuration 3: A large underground bunker has an opening (i.e. a collapsed wall) where the debris and soil have formed a slope. Burrows are found in slopes inside the bunker.

Burrow

Bunker Configuration 1: The bunker creates a depression in the ground. Burrows are found in the slopes outside the bunker.

Burrow

Distance to:

Watercourses, Ponds and lakes, Scrublands, Vineyards Size of: Groves, watercourses, annual crops, ponds and lakes, scrublands, mixed forests, meadows, urban area, orchards, vineyards The use of bunkers and the use of groves without bunkers were both positively 242 correlated to the annual crop size in the surrounding buffers (least significant values: Z=2.831; 243 p<0.01). Both types of use were also negatively correlated to the distance to annual crops by 244 a significant negative correlation between the annual crop size and distance to annual crops 245 (ρ=-0.905; p<0.01).

246

The presence of burrows and the number of burrows in use were positively correlated 247 to annual crop size (least significant values: Z=3.044; p<0.01) and to the size of urban area

248

(least significant values: Z=3.148; p<0.01).

249

The effect of accessibility 250

The accessibility of bunkers had a significant effect on use (χ²=72.074, df=2, p<0.01), on 251 the presence of burrows (χ²=54.877, df=2, p<0.01) and on the number of burrows in use 252 (χ²=35.037, df=2, p<0.01) (Figure 4). that badgers and foxes have had to adapt to a man-made habitat due to the reduction in their 270 natural environment. These are not the only carnivorous species using bunkers; wildcats (Felis 271 silvestris) are also known to inhabit a large number of bunkers [START_REF] Crossland | From Wehrmacht to Wildcats: World War II Bunkers Turn 423 Into Wildlife Haven[END_REF].

272

Camera traps could be a more appropriate tool to study this type of discrete species in forest 273 bunkers (Anile et al. 2009;[START_REF] Velli | Non-invasive monitoring of the 504 European wildcat (Felis silvestris silvestris Schreber, 1777): comparative analysis of three 505 different monitoring techniques and evaluation of their integration[END_REF]. This technique could also allow a more precise 274 quantification of bunker use and an evaluation of the effect of human disturbance.

275

The abundance of badgers in the studied area may be lower than that of foxes, as

296

When burrows were found inside or in the vicinity of these highly accessible bunkers, it was 297 always in cases where the structures created a ground depression outside the bunker or 298 when a large amount of dirt was found inside. In both cases, a dirt slope was present, in which 299 burrows had been dug.

annual crops and greater urbanisation. The destruction of natural habitats for the benefit of 302 crops and urban area may force badgers and foxes to live in bunkers, which are preserved 303 from overly intense human action (Remonti et al. 2006). Bunkers may represent safe areas

304

where foxes and badgers could rest, feed and reproduce, i.e. a source of refuge on farmland.

305

The importance of refuge habitats has already been raised for other carnivores, such as the 306 polecat in the Mediterranean landscape [START_REF] Rondinini | Habitat use and preference by polecats ( Mustela 487 putorius L.) in a Mediterranean agricultural landscape[END_REF] Bunkers were located in crops, forests, or groves. Over a three-month period, signs of 13 presence, the number and type of burrows inside the bunkers and in their immediate vicinity 14 were noted. Environmental variables were obtained using GIS, and binomial models were 15 created to reveal parameters influencing bunker use by badgers and foxes. The degree of 16 bunkers burrowing was used as a co-variable. Twenty-one groves without bunkers were also 17 studied in order to estimate the added value of bunkers in groves.

18

Badger and fox tracks were found on 34% of the sites, and burrows on 24% of them with similar rates for badgers and foxes. Animals used partially buried bunkers more than habitats such as wetlands or copses [START_REF] Bonfanti | Landscape analysis in areas affected by land 413 consolidation[END_REF][START_REF] Butet | Effects of agriculture development on vole dynamics and 415 conservation of Montagu's harrier in western French wetlands[END_REF]Robinson & 81 Sutherland 2002). This continuing agricultural intensification mainly has a negative impact on 82 biodiversity [START_REF] Poschlod | Changing land use and its impact on 475 biodiversity[END_REF][START_REF] Desjeux | Evaluating the impact of rural development measures on nature value indicators 430 at different spatial levels: Application to France and the Netherlands[END_REF]Benton et al., 2003). However, bunkers

83

found in these areas can play a role in preserving semi-natural elements in their vicinity such 84 as hedges, pathway verges and groves, which can all be used by a variety of species in 85 including mesopredators [START_REF] Dondina | How to manage hedgerows as effective 435 ecological corridors for mammals: A two-species approach[END_REF], or can even be used by animal species as 86 alternative habitats.

87

The red fox (Vulpes vulpes) and the European badger (Meles meles) are probably the most 88 common fossorial carnivores in Western European farmlands (Hoffmann & Sillero-Zubiri 89 2016;Kranz et al. 2016). In stable ecosystems, these mesopredators regulate agricultural food stocked in warehouses and silos (König 2013). They also limit the dispersion of invasive 92 species, as illustrated by the reduction in the populations of American mink (Neovison vison)

93

by the red fox in Fennoscandia (Ritchie et al. 2012). Scavenging by foxes is considered as an 94 ecosystemic service by providing sanitary services [START_REF] Sekercioglu | Increasing awareness of avian ecological function[END_REF][START_REF] Devault | Mesopredators dominate 433 competition for carrion in an agricultural landscape[END_REF]). The

95

European badger provides services such as the dissemination of seeds and their resurfacing 96 from the soil seed bank, and abandons burrows that will then be used by other mammal 97 species (Kurek et al. 2013).

98

Burrow use is very different in these two species, which both use a number of burrows 99 that are spread across their territory. The red fox mainly has small burrows with one or two 100 entrances. The badger has a variety of burrows (called "setts") that vary in size and function.

101

The main sett is large with up to hundred entrances and can be very old, sometimes having 102 been used by several generations of badgers [START_REF] Roper | Internal structure and contents of three 490 badger ( Meles meles ) setts[END_REF]). There is one main sett per

110

We hypothesized that bunkers are used as outlier burrows by both of these fossorial 111 species but that entrances located at an accessible height are necessary for this to occur. with the majority located on farmland that is mainly composed of corn and wheat crops with 123 rare groves. In this study, a grove was defined as a small group of trees (>3 m high) with a size 124 between 0.01 and 1 ha, with or without an understorey. If the area exceeded 1ha, the 125 landscape type was considered to be forest. Six hundred and fourteen fortifications have 126 been found and geolocated in this region, but this list is non-exhaustive and the overall 127 number could be far higher [START_REF] Mermy | Inventaire 468 départemental des cavités souterraines hors mines du Bas-Rhin[END_REF]. Indeed, new bunkers are regularly found 128 through archaeological excavations.

129 Not all the bunkers were studied; those located within towns and cities were excluded

130
from the present study. The remaining bunkers (N=120) were randomly sampled to obtain a 131 representation of all the bunkers in the studied area. In addition to the 120 selected bunkers, 132 a further 62 bunkers were found during inspections. The analyses therefore concern a total of 133 182 bunkers (Figure 2). Twenty-one groves without bunkers and surrounded by crops were 134 also studied over the same period in order to compare their attractivity with that of groves the same environmental type.

186

• The percentage of each landscape element type in each 800m radial buffer;

187

• The nearest distance to each landscape element type;

188

• The accessibility of each bunker (underground, partially buried, overground).

189

The three first dependent variables were also used to compare groves with and without a 190 bunker. To explain their variations, GLMβ were also used with the following variables as co-

191

variables:

192

• The size of the grove;

193

• The absence/presence of a bunker in the grove;

194

• The species (fox, badger or both of these species). 2). Data for both species were pooled to obtain 239 sufficient data for landscape analysis.

240

Partially buried Bunker

Resting area

Configuration 2: The bunker is small and partially buried. The inside can only be reached via a small opening no larger than a burrow hole. A resting area is present in the bunker. The bunker is considered as a burrow. that badgers and foxes have had to adapt to a man-made habitat due to the reduction in their 270 natural environment. These are not the only carnivorous species using bunkers; wildcats (Felis 271 silvestris) are also known to inhabit a large number of bunkers [START_REF] Crossland | From Wehrmacht to Wildcats: World War II Bunkers Turn 423 Into Wildlife Haven[END_REF].

272

Camera traps could be a more appropriate tool to study this type of discrete species in forest 273 bunkers (Anile et al. 2009;[START_REF] Velli | Non-invasive monitoring of the 504 European wildcat (Felis silvestris silvestris Schreber, 1777): comparative analysis of three 505 different monitoring techniques and evaluation of their integration[END_REF]. This technique could also allow a more precise 274 quantification of bunker use and an evaluation of the effect of human disturbance.

275

The abundance of badgers in the studied area may be lower than that of foxes, as Balestrieri, A., Cardarelli, E., Pandini, M., Remonti, L., Saino, N. & Prigioni, C. (2016)

Partially buried Bunker

Resting area

Configuration 2: The bunker is small and partially buried. The inside can only be reached via a small opening no larger than a burrow hole. A resting area is present in the bunker. The bunker is considered as a burrow.

  European badgers and by red foxes, (2) to identify how these species use them, and (3) to 114 understand the factors associated with this use. studied are located on the German defensive line of Strasbourg and 119 at the eastern extremity of the Maginot Line, located in the Bas-Rhin (Alsace, Grand Est, 120 France). The annual average temperature of Bas-Rhin is 10.4 °C. Annual precipitation is about 121 700 mm/y with an average altitude of 150 m. Bunkers are scattered throughout the territory,

Figure 2 :•

 2 Figure 2: Location of the 182 bunkers and the 21 groves without bunkers

•••••

  models (GLMβ) were used to explain the variations of dependent 185 variables of bunkers, using the following variables as co-variables:186 The percentage of each landscape element type in each 800m radial buffer;187 The nearest distance to each landscape element type;188 The accessibility of each bunker (underground, partially buried, overground).189The three first dependent variables were also used to compare groves with and without a 190 bunker. To explain their variations, GLMβ were also used with the following variables as co-The absence/presence of a bunker in the grove;194 The species (fox, badger or both of these species).

Figure 3 :

 3 Figure 3: The three different configurations of burrows observed in this study.

276

  badgers were less observed than foxes in non-exhaustive naturalist local surveys (Data 2008-4 755 km² of the studied area;Odonat, 2017). If these figures are correct, the observation of 279 equal use of bunkers by each species in this study thus suggests that badgers are particularly 280 attracted by these remaining habitats in highly changed landscapes. These animals also have a 281 greater network of burrows than foxes, with setts that are composed of different types of 282 chamber. Most of the badger burrows found were outlier setts, characterized by few 283 entrances and usually a nest chamber. A similar configuration was found in partially buried 284 bunkers. They were regularly used by badgers and foxes. As nesting sites were found inside 285 these partially buried bunkers, we considered the latter to be burrows. used by the studied species, indicating that visible human-made openings are needed to 288 make them accessible to these animals. This observation suggests that badgers and foxes do 289 not dig to reach the underground rooms, even when the bunker roof is visible. Partially buried 290 bunkers could be habitat opportunities requiring an initial physical effort to gain access, yet 291 they are not particularly sought after by these species. Foxes and badgers can also use 292 bunkers that are highly accessible and can be easily entered by humans, but the quality of 293 these shelters seems lower than that provided by partially buried bunkers. This difference in 294 quality could be explained by the larger size of inner rooms, the luminosity and human 295 disturbances, all of which are greater when bunkers are not buried (Aaris-Sørensen 1987).

20

  underground or overground bunkers. The sizes of annual crops and urban areas around the 21 bunker were positively correlated with bunker use. The presence of a bunker in groves was 22 positively correlated with the presence of burrows in groves. intensive farming landscapes. We recommend the management of these ruins to facilitate the 25 sustainable installation of a variety of species. 26 Keywords: refuges, artificial habitat, military fortifications, farmlands, burrows Introduction 30 A huge number of military fortifications were built before and during the Second World 31 War, often located close to border areas like the Maginot Line or the Atlantic Wall. These 32 concrete bunkers (English), Blockhäuser (German) or casemates (French) are often located 33 underground, either by design or due to burying over time. They have a small number of 34 rooms, and the largest constructions have a well to collect water (van der Well & van Beveren 35 2016). Entrances to the larger defence bunkers are hard to reach, either due to ditches or 36 because they are completely underground. The entrances of bunkers used as storage units or 37 as garrisons (blockhouses) are larger and easier to reach. At the end of the war, these 38 structures were often abandoned. They are currently in a good state of conservation but are 39 less accessible. This is particularly true on farmland, where bunkers have been covered by 40 successive ploughing. As they were built in defensive or support lines, bunkers are widely 41 dispersed, covering large areas of land[START_REF] Mermy | Inventaire 468 départemental des cavités souterraines hors mines du Bas-Rhin[END_REF]. In Europe, they are found from 42 the Stalin Line in the East to the Atlantic Wall and the GHQ line in the West. There is no precise inventory of these structures, but the number present along the different defensive 44 lines represented in Figure1could be estimated between 120 000 and 200 000. Although the majority of bunkers were constructed before and during WW2, some were built after the end of these environments are usually low(Benton et al. 2003;[START_REF] Maxwell | The ravages of guns, nets and 464 bulldozers[END_REF]. During the 79 second half of the 20 th century, land consolidation policy led to the removal of natural 80

103

  group territory, where breeding occurs. Annexe and subsidiary setts have fewer entrances 104 and are located near the main burrow. Outlier setts are located on the edge of the territory 105 and have just one or two entrances[START_REF] Roper | The structure and function of badger setts[END_REF]. While the main sett is used for breeding, 106 thermoregulation and avoiding predators, the benefits provided by outlier burrows remain 107 unclear. They could play a role in regulating ectoparasite load, reducing the incidence of 108 breeding suppression and minimizing the costs of travelling through the territory(Davison et 109 al. 2008).

  European badgers and by red foxes, (2) to identify how these species use them, and (3) to 114 understand the factors associated with this use. studied are located on the German defensive line of Strasbourg and 119 at the eastern extremity of the Maginot Line, located in the Bas-Rhin (Alsace, Grand Est, 120 France). The annual average temperature of Bas-Rhin is 10.4 °C. Annual precipitation is about 121 700 mm/y with an average altitude of 150 m. Bunkers are scattered throughout the territory, 122

Figure 2 :•

 2 Figure 2: Location of the 182 bunkers and the 21 groves without bunkers

221Figure 3 :

 3 Figure 3: The three different configurations of burrows observed in this study.

Configuration 3 :

 3 A large underground bunker has an opening (i.e. a collapsed wall) where the debris and soil have formed a slope. Burrows are found in slopes inside the bunker. Burrow Bunker Configuration 1: The bunker creates a depression in the ground. Burrows are found in the slopes outside the bunker. Burrow p<0.01). Both types of use were also negatively correlated to the distance to annual crops by 244 a significant negative correlation between the annual crop size and distance to annual crops 245 (ρ=-0.905; p<0.01).246The presence of burrows and the number of burrows in use were positively correlated 247 to annual crop size (least significant values: Z=3.044; p<0.01) and to the size of urban area248(least significant values: Z=3.148; p<0.01).249The effect of accessibility 250The accessibility of bunkers had a significant effect on use (χ²=72.074, df=2, p<0.01), on 251 the presence of burrows (χ²=54.877, df=2, p<0.01) and on the number of burrows in use 252 (χ²=35.037, df=2, p<0.01) (Figure4).

  Finally, 360 certain interesting bunkers located close to urban areas could be video-equipped as an 361 awareness-raising project to educate people about wildlife. 362 Conclusion 363 Bunkers are widely used as shelters by red foxes and European badgers. However, a 364 high proportion of these military structures, especially on farmland, are in poor condition for 365 their use as a habitat. The present study provides the necessary information to significantly 366 improve the habitat potentiality of bunkers for the two most representative fossorial 367 carnivore species living on Western European farmlands. Knowing the high number of 368 bunkers concerned and their relatively equal distribution in the studied area, their 369 management could help to slow the inexorable deterioration and homogenisation of the 370 agricultural landscape. for their comments on the previous version of this manuscript. We also thank Joanna Lignot 374 and Lana Petrod for the English editing. carried out data analysis; JJ, YH and FB organised the writing of the manuscript. All authors 381 played an active part in the drafting of the manuscript and gave their final approval for conducted though the CERISE project research and was funded by the 385 French Minister of Ecology (DREAL Alsace) and the Departmental Council of the Bas-Rhin 386 (CD67). They had no role in the study design, writing, collection, analysis and interpretation of 387 data. They agree to the publication of this study.

Figure 1 :

 1 Figure 1: Location of defensive lines in Europe (according to Kaufmann & Jurga 1999).

Figure 2 :Figure 3 :

 23 Figure 2: Location of the 182 bunkers and the 21 groves without bunkers

  Figure 4: Effects of the accessibility of bunkers on their use by animals (global use shown in

Table 1 :

 1 Number of studied bunkers per accessibility type and main surrounding

	197	Variance Inflation Factor (VIF), associated with ACP and Spearman tests. Models were
	198	selected using AIC backward elimination/forward selection. Modalities of variables were
	199	compared with a Tuckey-HSD post-hoc test. Statistics were performed with R software
	200	(v3.3.0) and GIS analysis using QGIS (v2.18.3). The significance threshold was set to p<0.05.
	201	Results
	202	Characteristics of bunkers
	203	Nineteen bunkers were located in groves (10.44%), 57 in forests (31.32%) and 106 on
	204	crops (58.24%). Accessibility type was more balanced, with 39 underground bunkers
	205	(21.43%), 60 partially buried bunkers (32.97%) and 83 overground bunkers (45.60%)(Table 1).
	206	Only five underground bunkers were located in groves and in forests. The small defensive
	207	lines in the studied area were composed of an average 9.4 bunkers (±2.3 SE, N=15), with an
	208	average inter-distance of 389 metres (±64.64 SE).
	209
	210	landscape.
		Surroundings Underground Partially buried Overground Total
		Crop	34	26	46	106
		Grove	2	11	6	19
		Forest	3	23	31	57
		Total	39	60	83	182
	211
	212	Use of bunkers
	213	Evidence of badger or fox presence was found in 61 of the 182 studied bunkers
	214	(33.52%). Each species used a significantly similar number of bunkers (W = 21214, p=0.357).
		Burrows were found on 43 sites (23.63%), 30 (69.77%) of which were still occupied at the

  Use of groves was not correlated to the presence of a bunker (χ²=0.523, df=1, p=0.469).

	257
	258
	259	Comparison of groves with and without a bunker.

Figure 4: Effects of the accessibility of bunkers on their use by animals (global use shown in 255 white, presence of burrows in grey, and presence of burrows in use in black), for each of the 256 three accessibility categories. Error bars are standard errors. 260 There were more burrows (χ²=10.354, df=1, p<0.01) and more burrows in use (χ²=5.107, df=1, 261 p=0.02) when a bunker was present. The area of the groves had no effect on grove use, on 262 the presence of burrows and on the number of burrows in use (most significant values: Z=-263 0.361; p=0.718). Among the eleven species-determined burrows found in groves, seven were 264 badger setts and two were fox burrows. 265 Discussion 266 Bunkers as habitat 267 Bunkers form a network of habitats which provide resources for wildlife. The presence of 268 burrows in over one third of the studied bunkers and a relatively high occupation rate suggest 269

  Indeed, even when entirely surrounded by scrubland, bunkers with large human-made 350 openings are attractive to people who sometimes put these spaces to surprising use, such as their comments on the previous version of this manuscript. We also thank Joanna Lignot 374 and Lana Petrod for the English editing.the aspects of the work that they conducted, and will ensure that any questions related to the 378 accuracy or integrity of any part of their work are appropriately investigated and resolved. JJ 379 conceived the idea and design methodology; JJ, DW, NG and LG collected the data; JJ and LG 380 carried out data analysis; JJ, YH and FB organised the writing of the manuscript. All authors 381 played an active part in the drafting of the manuscript and gave their final approval forBalestrieri, A., Cardarelli, E., Pandini, M., Remonti, L., Saino, N. & Prigioni, C. (2016). Spatial 400 organisation of European badger (Meles meles) in northern Italy as assessed by camera-401 trapping. Eur. J.Wildl. Res., 62,[219][220][221][222][223][224][225][226]

	1
	2 3	326
	4 5	327 351 402	As bunkers were built around strategic points in small defensive lines, they form a
	6 7 8	328 352 403 404	network of potential shelters for wildlife. The distance between the successive bunkers of one the exhibition of thousands of hubcaps we found in one bunker. The surroundings of bunkers Benton, T.G., Vickery, J.A. & Wilson, J.D. (2003). Farmland biodiversity: is habitat heterogeneity the key? Trends Ecol. Evol., 18, 182-188.
	9 10 11	329 353 486 405 406 487	small defensive line allows badgers to have three or four outlier burrows in their territory, located on cropland should be included on the land registry plan to ensure their Billeter, R., Liira, J., Bailey, D., Bugter, R., Arens, P., Augenstein, I., Aviron, S., Baudry, J., in Great Britain. J. Appl. Ecol., 39, 157-176. Bukacek, R., Burel, F., Cerny, M., De Blust, G., De Cock, R., Diekötter, T., Dietz, H., Rondinini, C., Ercoli, V. & Boitani, L. (2006). Habitat use and preference by polecats ( Mustela
	12 13 14 15 16	330 407 488 354 408 489 409 355 410 490	whilst foxes have even more. Towards an ecological management of bunkers management. A ground depression should be created around underground bunkers to Dirksen, J., Dormann, C., Durka, W., Frenzel, M., Hamersky, R., Hendrickx, F., Herzog, F., putorius L.) in a Mediterranean agricultural landscape. J. Zool., 269, 213-219. excavate them and create slopes that can be used by wildlife to create burrows and settle Klotz, S., Koolstra, B., Lausch, A., Le Coeur, D., Maelfait, J.P., Opdam, P., Roubalova, M., Schermann, A., Schermann, N., Schmidt, T., Schweiger, O., Smulders, M.J.M., Speelmans, Roper, T.J. (1992). The structure and function of badger setts. J. Zool., 227, 691-694. M., Simova, P., Verboom, J., Van Wingerden, W.K.R.E., Zobel, M. & Edwards, P.J. (2008). Roper, T.J., Tait, A.I., Fee, D. & Christian, S.F. (1991). Internal structure and contents of three
	17 18 19	491 411 492	(Macdonald et al. 2004). Burrows were found inside or in the vicinity of 35 of the 99 bunkers Indicators for biodiversity in agricultural landscapes: A pan-European study. J. Appl. Ecol., badger ( Meles meles ) setts. J. Zool., 225, 115-124. 45, 141-150. Sachanowicz, K. & Zub, K. (2002). Numbers of hibernating Barbastella barbastellus (Schreber,
	20 21 22	493 494	1774) (Chiroptera, Vespertilionidae) and thermal conditions in military bunkers. Mamm. Biol. -Zeitschrift für Säugetierkd., 67, 179-184.
	23 24 25	495 382	Sekercioglu, C.H. (2006). Increasing awareness of avian ecological function. Trends Ecol. Evol., 21, 464-71.
	26 27		publication.
	28
	29
	30
	31
	32
	33
	34
	35
	36
	37
	38
	39
	40 41 42	506 507	61, 657-668.
	43 44 45	508 366 429 509	Warchałowski, M., Łupicki, D., Cichocki, J., Pietraszko, M., Rusek, A., Zawadzka, A. & Nikodem, M. (2013). Zimowanie nietoperzy Chiroptera w wybranych obiektach wolno stojących
	46 47 48 49 50 51 52 53 54 55 56 57 58	343 344 345 430 510 367 368 369 370 431 471 511 432 512 472 433 434 473 474 513 514 435 436 475	was insufficient. These results show that bunkers are currently used by wildlife, but some have lost their habitat potentiality. This situation can be changed and improved by the management of (2014). Evaluating the impact of rural development measures on nature value indicators Frontu Fortecznego Łuku Odry i Warty. Kulon, 18, 139-147. improve the habitat potentiality of bunkers for the two most representative fossorial carnivore species living on Western European farmlands. Knowing the high number of bunkers concerned and their relatively equal distribution in the studied area, their management could help to slow the inexorable deterioration and homogenisation of the at different spatial levels: Application to France and the Netherlands. Ecol. Indic., 59, 41-61. van der Well, L. & van Beveren, A. (2016). Bunkersite.com [WWW Document]. URL DeVault, T.L., Olson, Z.H., Beasley, J.C. & Rhodes, O.E. (2011). Mesopredators dominate Odonat : Office des données naturalistes du Grand-Est. (2017). Atlas des espèces d'Alsace http://bunkersite.com/index.php competition for carrion in an agricultural landscape. Basic Appl. Ecol., 12, 268-274. [WWW Document]. www.faune-alsace.org. URL http://www.faune-alsace.org/index.php?m_id=620 Wretenberg, J., Pärt, T. & Berg, Å. (2010). Changes in local species richness of farmland birds in relation to land-use changes and landscape structure. Biol. Conserv., 143, 375-381. Dondina, O., Kataoka, L., Orioli, V. & Bani, L. (2016). How to manage hedgerows as effective ecological corridors for mammals: A two-species approach. Agric. Ecosyst. Environ., 231, Poschlod, P., Bakker, J.P. & Kahmen, S. (2005). Changing land use and its impact on Zduniak, P., Czechowski, P. & Jedro, G. (2011). The effect of nesting habitat on reproductive biodiversity. Basic Appl. Ecol., 6, 93-98. 283-290. agricultural landscape.
	59
	60
	61
	62
	63
	64

. They also provide resources 307 for other species such as birds, plants, spiders, insects and other mammals

(Sheperd & 308 Swihart 1995;[START_REF] Zollner | Comparing the landscape level perceptual abilities of forest sciurids in[END_REF] Benton et al. 2003;[START_REF] Duelli | Regional biodiversity in an agricultural landscape: the Hoffmann[END_REF] Grashof-Bokdam & van 309 Langevelde 2005; Billeter et al. 2008;[START_REF] Wretenberg | Changes in local species richness of farmland birds 513 in relation to land-use changes and landscape structure[END_REF][START_REF] Redon (de) | Road 477 network in an agrarian landscape: Potential habitat, corridor or barrier for small Ritchie[END_REF]

.

310

Wildlife obtains different services from bunkers and groves without bunkers when 311 they use them as refuge habitats. Indeed, the presence of a bunker in a grove increased the 312 probability of finding a burrow and a burrow in use there. A major difference between these 313 two landscape elements was the presence of slopes created by ground depression around 314 bunkers.

315

Distribution of bunkers in the studied area 316 Few bunkers were found in groves. These were mainly small bunkers used for storage and 317 were hidden from the enemy when constructed in wooded areas. However, as other storage 318 bunkers were found on crops, we presume that land consolidation strategy has ensured the 319 maintenance of groves. Administratively speaking, if the presence of a bunker made a piece 320 of land "uncultivable" on the land registry plan, the piece of land remained registered as such 321 and the bunker and grove were both preserved in their natural state. If not, the piece of land 322 became cultivable and farmers were free to plant crops around the bunker. Moreover, the 323 use of land for crops has led to the burial of bunkers, as shown by the high number of these structures have lost their habitat potentiality.

331

Although bunkers are widely used by carnivores, they are often unhealthy structures, 332 with stagnant water containing high numbers of mosquitoes and animal carcasses. Hygienic 333 conditions are usually poor, and it is unlikely that animals can live in these places for long 334 periods of time. The frequent presence of frogs trapped in bunker chambers that are only 335 reachable by a ladder merits attention and should be notified to the competent authorities to 336 ensure that the concerned bunkers are made safe, especially when they represent a danger 337 for children in urban areas.

338

Some trees were found close to the bunkers, even in middle of large crops fields. They 339 did not provide enough cover to form a grove but provided roosting areas for avian 340 mesopredators. Furthermore, preserved groves located on farmland can be used by these 341 species, including corvids, hawks, buzzards and owls. There was no evidence of badgers or 342 foxes around bunkers that did not have visible human-made openings, suggesting that access 346 bunkers which would be advantageous not only for badger and foxes but also for a variety of 347 other species. First, the accessibility of bunkers with large apertures should be reduced by 356 that had a slope (35.4%). This means that if all the studied bunkers (182) were managed with 357 a slope, 64 potential burrows could be expected, and hundreds could be observed if all the 358 bunkers in the study area (at least 614) managed in the same way. The planting of trees 359 would create small groves around the bunkers to provide benefits for avian predators. Finally, 360 certain interesting bunkers located close to urban areas could be video-equipped as an 361 awareness-raising project to educate people about wildlife. 362 Conclusion 363 Bunkers are widely used as shelters by red foxes and European badgers. However, a 364 high proportion of these military structures, especially on farmland, are in poor condition for 365 their use as a habitat. The present study provides the necessary information to significantly for 383 Role of the funding source 384 This study was conducted though the CERISE project research and was funded by the 385 French Minister of Ecology (DREAL Alsace) and the Departmental Council of the Bas-Rhin 386 (CD67). They had no role in the study design, writing, collection, analysis and interpretation of 387 data. They agree to the publication of this study. 412 Bonfanti, P., Fregonese, A. & Sigura, M. (1997). Landscape analysis in areas affected by land 413 consolidation. Landsc. Urban Plan., 37, 91-98. 414 Butet, A. & Leroux, A.B.A. (2001). Effects of agriculture development on vole dynamics and 415 conservation of Montagu's harrier in western French wetlands. Biol. Conserv., 100, 289-416 295. 417 Ciechanowski, M. & Sachanowicz, K. (2014). Fat Dormouse Glis glis (Rodentia: Gliridae) in 418 Albania: Synopsis of Distributional Records with Notes on Habitat Use. Acta Zool. Bulg., 419 66, 39-42. 420 CIGAL. (2013). Données faune Alsace [WWW Document]. Coopération pour l'information 421 géographique en Alsace. URL https://www.cigalsace.org/portail/ 422 Crossland, D. & Schöne, S. (2007). From Wehrmacht to Wildcats: World War II Bunkers Turn 423 Into Wildlife Haven [WWW Document]. SPIEGEL ONLINE. URL 424 http://www.spiegel.de/international/germany/from-wehrmacht-to-wildcats-world-war-425 ii-bunkers-turn-into-wildlife-haven-a-507880.html 426 Davison, J., Huck, M., Delahay, R.J. & Roper, T.J. (2008). Urban badger setts: characteristics, 427 patterns of use and management implications. J. Zool., 275, 190-200. 428 Desjeux, Y., Dupraz, P., Kuhlman, T., Paracchini, M.L., Michels, R., Maign??, E. & Reinhard, S. 437 Duelli, P. & Obrist, M.K. (2003). Regional biodiversity in an agricultural landscape: the (Vulpes vulpes) changes soil conditions and vegetation in a European temperate forest. 456 Ecol. Res., 29, 1-11. 457 Macdonald, D.W., Newman, C., Dean, J., Buesching, C.D. & Johnson, P.J. (2004). The 458 distribution of Eurasian badger, Meles meles , setts in a high-density area: field 459 observations contradict the sett dispersion hypothesis. Oikos, 106, 295-307. 460 Marton, M., Markolt, F., Szabo, L., Kozak, L., Lanszki, J., Patko, L. & Heltai, M. (2016). Den site 461 selection of the European badger, Meles meles and the red fox, Vulpes vulpes in 462 Hungary. FOLIA Zool., 65, 72-79. 463 Maxwell, S.L., Fuller, R.A., Brooks, T.M. & Watson, J.E.M. (2016). The ravages of guns, nets and 464 bulldozers. Nature, 536, 146-145. 465 Meia, J.-S. & Weber, J.-M. (1995). Home ranges and movements of red foxes in central 466 Europe: stability despite environmental changes. Can. J. Zool., 73, 1960-1966. 467 Mermy, D.C., Guignat, H.S., Zammit, R., Heckenmeyer, E. & Lizée, M. (2011). Inventaire 468 départemental des cavités souterraines hors mines du Bas-Rhin. 469 Nowicki, F., Dadu, L., Carsignol, J., Bretaud, J.-F. & Bielsa, S. (2008). Rapport bibliographique : 470 Routes et chiroptères Etat des connaissances. Les Rapp., 1-253. 476 Redon (de), L., Le Viol, I., Jiguet, F., Machon, N., Scher, O. & Kerbiriou, C. (2015). Road 477 network in an agrarian landscape: Potential habitat, corridor or barrier for small 496 Sheperd, B.F. & Swihart, R.K. (1995). Spatial dynamics of fox squirrels ( Sciurus niger ) in 497 fragmented landscapes. Can. J. Zool., 73, 2098-2105. 498 Théou, P. & Bego, F. (2013). Etude des populations de chiroptères de l'île de Sazani. Note 499 naturaliste Initiative PIM. 500 Tscharntke, T., Klein, A.M., Kruess, A., Steffan-Dewenter, I. & Thies, C. (2005). Landscape 501 perspectives on agricultural intensification and biodiversity -Ecosystem service 502 management. Ecol. Lett., 8, 857-874. 503 Velli, E., Bologna, M.A., Silvia, C., Ragni, B. & Randi, E. (2015). Non-invasive monitoring of the 504 European wildcat (Felis silvestris silvestris Schreber, 1777): comparative analysis of three 505 different monitoring techniques and evaluation of their integration. Eur. J. Wildl. Res., 515 output of the Barn Swallow (Hirundo rustica). A comparative study of populations from 516 atypical and typical nesting habitats in western Poland. BELGIAN J.

Zool., 141, 38-43. 517 Zollner, P.A. (2000)

. Comparing the landscape level perceptual abilities of forest sciurids in 518 fragmented agricultural landscapes. Landsc.

Ecol., 15,[523][524][525][526][527][528][529][530][531][532][533]

Table 1 :

 1 Variance Inflation Factor (VIF), associated with ACP and Spearman tests. Models were Number of studied bunkers per accessibility type and main surrounding Burrows were found on 43 sites (23.63%), 30 (69.77%) of which were still occupied at the were outlier burrows and seven (26%) were central burrows. Five of these seven central 218 burrows were found outside the bunkers. "Resting areas" composed of hay and badger/fox 219 fur were found inside nine small partially buried bunkers. The latter were considered as 220 outlier burrows in the analyses but with no assigned species. We identified three different

	197
	198	selected using AIC backward elimination/forward selection. Modalities of variables were
	199	compared with a Tuckey-HSD post-hoc test. Statistics were performed with R software
	200	(v3.3.0) and GIS analysis using QGIS (v2.18.3). The significance threshold was set to p<0.05.
	201	Results
	202	Characteristics of bunkers
	203	Nineteen bunkers were located in groves (10.44%), 57 in forests (31.32%) and 106 on
	204	crops (58.24%). Accessibility type was more balanced, with 39 underground bunkers
	205	(21.43%), 60 partially buried bunkers (32.97%) and 83 overground bunkers (45.60%)(Table 1).
	206	Only five underground bunkers were located in groves and in forests. The small defensive
	207	lines in the studied area were composed of an average 9.4 bunkers (±2.3 SE, N=15), with an
	208	average inter-distance of 389 metres (±64.64 SE).
	209
	210	landscape.
		Surroundings Underground Partially buried Overground Total
		Crop	34	26	46	106
		Grove	2	11	6	19
		Forest	3	23	31	57
		Total	39	60	83	182
	211
	212	Use of bunkers
	213	Evidence of badger or fox presence was found in 61 of the 182 studied bunkers
	214	(33.52%). Each species used a significantly similar number of bunkers (W = 21214, p=0.357).

  white, presence of burrows in grey, and presence of burrows in use in black), for each of the

	256
	257	three accessibility categories. Error bars are standard errors.
	258
	259	Comparison of groves with and without a bunker.
	260	Use of groves was not correlated to the presence of a bunker (χ²=0.523, df=1, p=0.469).
	261	There were more burrows (χ²=10.354, df=1, p<0.01) and more burrows in use (χ²=5.107, df=1,
	262	p=0.02) when a bunker was present. The area of the groves had no effect on grove use, on
	263	the presence of burrows and on the number of burrows in use (most significant values: Z=-
	264	0.361; p=0.718). Among the eleven species-determined burrows found in groves, seven were
	265	badger setts and two were fox burrows.
	266	Discussion
	267	Bunkers as habitat
	268	Bunkers form a network of habitats which provide resources for wildlife. The presence of
	269	burrows in over one third of the studied bunkers and a relatively high occupation rate suggest

  [START_REF] Macdonald | The 458 distribution of Eurasian badger, Meles meles , setts in a high-density area: field 459 observations contradict the sett dispersion hypothesis[END_REF]). Burrows were found inside or in the vicinity of 35 of the 99 bunkers 356 that had a slope (35.4%). This means that if all the studied bunkers (182) were managed with 357 a slope, 64 potential burrows could be expected, and hundreds could be observed if all the bunkers in the study area (at least 614) managed in the same way. The planting of trees 359 would create small groves around the bunkers to provide benefits for avian predators.

	302 326	annual crops and greater urbanisation. The destruction of natural habitats for the benefit of these structures have lost their habitat potentiality.
	303 327 351	crops and urban area may force badgers and foxes to live in bunkers, which are preserved As bunkers were built around strategic points in small defensive lines, they form a openings are attractive to people who sometimes put these spaces to surprising use, such as
	328 352	from overly intense human action (Remonti et al. 2006). Bunkers may represent safe areas network of potential shelters for wildlife. The distance between the successive bunkers of one the exhibition of thousands of hubcaps we found in one bunker. The surroundings of bunkers
	329 353	small defensive line allows badgers to have three or four outlier burrows in their territory, located on cropland should be included on the land registry plan to ensure their
	306 330 354	The importance of refuge habitats has already been raised for other carnivores, such as the whilst foxes have even more. management. A ground depression should be created around underground bunkers to
	307 331 355	polecat in the Mediterranean landscape (Rondinini et al. 2006). They also provide resources Towards an ecological management of bunkers excavate them and create slopes that can be used by wildlife to create burrows and settle
	332	for other species such as birds, plants, spiders, insects and other mammals (Sheperd & Although bunkers are widely used by carnivores, they are often unhealthy structures, (
	333	with stagnant water containing high numbers of mosquitoes and animal carcasses. Hygienic
	334	conditions are usually poor, and it is unlikely that animals can live in these places for long
	335	periods of time. The frequent presence of frogs trapped in bunker chambers that are only
	336	reachable by a ladder merits attention and should be notified to the competent authorities to
	337	ensure that the concerned bunkers are made safe, especially when they represent a danger
	314 338	two landscape elements was the presence of slopes created by ground depression around for children in urban areas.
	315 339	bunkers. Some trees were found close to the bunkers, even in middle of large crops fields. They
	316 340	Distribution of bunkers in the studied area did not provide enough cover to form a grove but provided roosting areas for avian
	294 317 341	Few bunkers were found in groves. These were mainly small bunkers used for storage and mesopredators. Furthermore, preserved groves located on farmland can be used by these
	295 318 342	quality could be explained by the larger size of inner rooms, the luminosity and human were hidden from the enemy when constructed in wooded areas. However, as other storage species, including corvids, hawks, buzzards and owls. There was no evidence of badgers or
	296 319 343	disturbances, all of which are greater when bunkers are not buried (Aaris-Sørensen 1987). bunkers were found on crops, we presume that land consolidation strategy has ensured the foxes around bunkers that did not have visible human-made openings, suggesting that access
	297 320 344	When burrows were found inside or in the vicinity of these highly accessible bunkers, it was maintenance of groves. Administratively speaking, if the presence of a bunker made a piece was insufficient.
	298 321 345	always in cases where the structures created a ground depression outside the bunker or of land "uncultivable" on the land registry plan, the piece of land remained registered as such These results show that bunkers are currently used by wildlife, but some have lost
	299 322 346	when a large amount of dirt was found inside. In both cases, a dirt slope was present, in which and the bunker and grove were both preserved in their natural state. If not, the piece of land their habitat potentiality. This situation can be changed and improved by the management of
	323 347	burrows had been dug. became cultivable and farmers were free to plant crops around the bunker. Moreover, the bunkers which would be advantageous not only for badger and foxes but also for a variety of
		use of land for crops has led to the burial of bunkers, as shown by the high number of other species. First, the accessibility of bunkers with large apertures should be reduced by

276

badgers were less observed than foxes in non-exhaustive naturalist local surveys (Data 2008-used by the studied species, indicating that visible human-made openings are needed to 288 make them accessible to these animals. This observation suggests that badgers and foxes do 289 not dig to reach the underground rooms, even when the bunker roof is visible. Partially buried 290 bunkers could be habitat opportunities requiring an initial physical effort to gain access, yet 291 they are not particularly sought after by these species. Foxes and badgers can also use 292 bunkers that are highly accessible and can be easily entered by humans, but the quality of 293 these shelters seems lower than that provided by partially buried bunkers. This difference in

Table 1 :

 1 . Spatial 400 organisation of European badger (Meles meles) in northern Italy as assessed by camera-401 trapping. Eur. J. Wildl. Res., 62, 219-226. 402 Benton, T.G., Vickery, J.A. & Wilson, J.D. (2003). Farmland biodiversity: is habitat 403 heterogeneity the key? Trends Ecol. Evol., 18, 182-188. 404 Billeter, R., Liira, J., Bailey, D., Bugter, R., Arens, P., Augenstein, I., Aviron, S., Baudry, J., 405 Bukacek, R., Burel, F., Cerny, M., De Blust, G., De Cock, R., Diekötter, T., Dietz, H., 406 Dirksen, J., Dormann, C., Durka, W., Frenzel, M., Hamersky, R., Hendrickx, F., Herzog, F., 407 Klotz, S., Koolstra, B., Lausch, A., Le Coeur, D., Maelfait, J.P., Opdam, P., Roubalova, M., 408 Schermann, A., Schermann, N., Schmidt, T., Schweiger, O., Smulders, M.J.M., Speelmans, 409 M., Simova, P., Verboom, J., Van Wingerden, W.K.R.E., Zobel, M. & Edwards, P.J. (2008). 410 Indicators for biodiversity in agricultural landscapes: A pan-European study. J. Appl. Ecol., contribution of seminatural habitat islands. Basic Appl. Ecol., 4, 129-138. Number of studied bunkers per accessibility type and main surrounding landscape.

	1
	2
	3
	4
	5
	6
	7
	8
	9
	10
	11
	12
	13
	14
	15
	16
	17
	18
	19
	20
	21
	22
	23
	24
	25
	26
	27
	28
	29
	30
	31
	32
	33
	34
	35
	36
	37
	38
	39
	40
	41
	42
	43
	44
	45
	46
	47
	48
	49
	50
	51
	52
	53
	54
	55
	56
	57
	58
	59
	60
	61
	62
	63
	64
	65

Table 2 :

 2 List of used environmental variablesDistance to:Watercourses, Ponds and lakes, Scrublands, Vineyards Size of: Groves, watercourses, annual crops, ponds and lakes, scrublands, mixed forests, meadows, urban area, orchards, vineyards

agriculture landscapes are characterized by large monospecific farm plots(Foley et al. 2005;

Backtrack D-Tour). Freshness of evidence and the actual use of burrows were confirmed with

annual crops(CIGAL© data (CIGAL 2013), accuracy 1/10 000). The average distance between

bunkers of a same small defensive line was measured, as well as the size of all the groves.

presence of a burrow inside a bunker or in its immediate vicinity, (3) the presence of currently

time of the study. We found 18 (41.86%) burrows inside the bunkers and 25 (58.14%) outside.

contribution of seminatural habitat islands. Basic Appl.Ecol., 4,[129][130][131][132][133][134][135][136][137][138]

Foley, J.A., Defries, R., Asner, G.P.,Barford, C., Bonan, G., Carpenter, S.R., Chapin, F.S., Coe,

mammals? Acta Oecologica, 62, 58-65.

location in agricultural ecosystems ofNW Italy. Folia Zool., 55.1,[19][20][21][22][23][24][25][26][27]

Ritchie, E.G., Elmhagen, B., Glen, A.S., Letnic, M.,[START_REF] Redon (de) | Road 477 network in an agrarian landscape: Potential habitat, corridor or barrier for small Ritchie[END_REF].

[START_REF] Robinson | Post-war changes in arable farming and biodiversity 485 in Great Britain[END_REF]. Post-war changes in arable farming and biodiversity

ⱡ Corresponding author. Tel.: +33 06 07 62 33 63

E-mail address: jumeau.jonathan@gmail.com

(Sturnus vulgaris ; Evans, Ardia, & Flux, 2009) or the barn swallow (Hirundo rustica ; Zduniak,

[START_REF] Zduniak | The effect of nesting habitat on reproductive 515 output of the Barn Swallow (Hirundo rustica). A comparative study of populations from 516 atypical and typical nesting habitats in western Poland[END_REF]. However, bunkers do not generally appear to be an optimal

habitat. This is illustrated by an Albanian island where chiropterans were rare despite an

755 km² of the studied area;Odonat, 2017). If these figures are correct, the observation of

Indeed, even when entirely surrounded by scrubland, bunkers with large human-made

Acknowledgements Acknowledgements

Method 177

Four dependent variables were used: (1) the use or non-use of the bunker/grove (use was Author Contributions

376

All authors approve this version of the manuscript. They agree to be accountable for consequences of land use. Science (80-. )., 309, 570-4.

58

Few studies have explored the impact of bunkers on biodiversity. The reduced variations 59 of temperatures in the wells of bunkers make them an ideal hibernation site for the 60 Barbastelle bat (Barbastella barbastellus) [START_REF] Sachanowicz | Numbers of hibernating Barbastella barbastellus (Schreber, 492 1774) (Chiroptera, Vespertilionidae) and thermal conditions in military bunkers[END_REF]. In Germany, the one 61 hundred bunkers and 32 kilometres of galleries making up the Festungsfront Oder-Warthe-

62

Bogen bunker network are colonized by 37 000 bats from ten different species, possibly 63 making it the artificial structure hosting the highest number of individuals in Europe 64 [START_REF] Warchałowski | Zimowanie nietoperzy Chiroptera w wybranych obiektach wolno stojących 509 Frontu Fortecznego Łuku Odry i Warty[END_REF]Jan Cichocki in AFP 2014). Small terrestrial mammals like the 65 edible dormouse (Glis glis) also colonize bunkers [START_REF] Ciechanowski | Fat Dormouse Glis glis (Rodentia: Gliridae) in 418 Albania: Synopsis of Distributional Records with Notes on Habitat Use[END_REF].

66

Study area abundance of bunkers; however, the rarity of this species on the island may also be explained Watercourses, Ponds and lakes, Scrublands, Vineyards Size of: Groves, watercourses, annual crops, ponds and lakes, scrublands, mixed forests, meadows, urban area, orchards, vineyards The use of bunkers and the use of groves without bunkers were both positively 242 correlated to the annual crop size in the surrounding buffers (least significant values: Z=2.831; equal use of bunkers by each species in this study thus suggests that badgers are particularly 280 attracted by these remaining habitats in highly changed landscapes. These animals also have a 281 greater network of burrows than foxes, with setts that are composed of different types of 282 chamber. Most of the badger burrows found were outlier setts, characterized by few 283 entrances and usually a nest chamber. A similar configuration was found in partially buried 284 bunkers. They were regularly used by badgers and foxes. As nesting sites were found inside 285 these partially buried bunkers, we considered the latter to be burrows.

where foxes and badgers could rest, feed and reproduce, i.e. a source of refuge on farmland. [START_REF] Sheperd | Spatial dynamics of fox squirrels ( Sciurus niger ) in 497 fragmented landscapes[END_REF][START_REF] Zollner | Comparing the landscape level perceptual abilities of forest sciurids in[END_REF]Benton et al. 2003;[START_REF] Duelli | Regional biodiversity in an agricultural landscape: the Hoffmann[END_REF]Grashof-Bokdam & van 309 Langevelde 2005;Billeter et al. 2008;[START_REF] Wretenberg | Changes in local species richness of farmland birds 513 in relation to land-use changes and landscape structure[END_REF][START_REF] Redon (de) | Road 477 network in an agrarian landscape: Potential habitat, corridor or barrier for small Ritchie[END_REF].

310

Wildlife obtains different services from bunkers and groves without bunkers when 311 they use them as refuge habitats. Indeed, the presence of a bunker in a grove increased the 312 probability of finding a burrow and a burrow in use there. A major difference between these Author Contributions

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All authors approve this version of the manuscript. They agree to be accountable for 377 the aspects of the work that they conducted, and will ensure that any questions related to the accuracy or integrity of any part of their work are appropriately investigated and resolved. JJ 379 conceived the idea and design methodology; JJ, DW, NG and LG collected the data; JJ and LG consequences of land use. Science (80-. )., 309, 570-4. Landsc. Ecol.,20,15,[523][524][525][526][527][528][529][530][531][532][533]