Genetic identification of bat species for pathogen surveillance across France

Youssef Arnaout; Zouheira Djelouadji; Emmanuelle Robardet; Julien Cappelle; Florence Cliquet; Frédéric Touzalin; Giacomo Jimenez; Suzel Hurstel; Christophe Borel; Evelyne Picard-Meyer

doi:10.1371/journal.pone.0261344

. 2022 Jan 4;17(1):e0261344. doi: 10.1371/journal.pone.0261344

Genetic identification of bat species for pathogen surveillance across France

Youssef Arnaout ^1,², Zouheira Djelouadji ², Emmanuelle Robardet ¹, Julien Cappelle ^3,⁴, Florence Cliquet ¹, Frédéric Touzalin ⁵, Giacomo Jimenez ⁶, Suzel Hurstel ^7,⁸, Christophe Borel ⁶, Evelyne Picard-Meyer ^1,^*

Editor: Daniel Becker⁹

¹ANSES-Nancy Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies Serology, Malzéville, France

²VetAgro Sup Lyon Laboratory for Leptospira, Marcy l’Etoile, France

³UMR ASTRE, CIRAD, INRAE, Université de Montpellier, Montpellier, France

⁴UMR EPIA, INRAE, VetAgro Sup, Theix, France

⁵School of Biology and Environmental Science, Science Centre West, University College Dublin, Dublin, Ireland

⁶CPEPESC Lorraine, Neuves-Maisons, France

⁷GEPMA, Strasbourg, France

⁸LPO Alsace, Rosenwiller, France

⁹University of Oklahoma Norman Campus: The University of Oklahoma, UNITED STATES

Competing Interests: No authors have competing interests.

^✉

* E-mail: evelyne.picard-meyer@anses.fr

Roles

Youssef Arnaout: Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Zouheira Djelouadji: Conceptualization, Formal analysis, Investigation, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing

Emmanuelle Robardet: Formal analysis, Validation, Writing – original draft, Writing – review & editing

Julien Cappelle: Formal analysis, Validation, Writing – original draft, Writing – review & editing

Florence Cliquet: Validation, Writing – original draft, Writing – review & editing

Frédéric Touzalin: Validation, Writing – original draft, Writing – review & editing

Giacomo Jimenez: Validation, Writing – original draft, Writing – review & editing

Suzel Hurstel: Validation, Writing – original draft, Writing – review & editing

Christophe Borel: Validation, Writing – original draft, Writing – review & editing

Evelyne Picard-Meyer: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing

Daniel Becker: Editor

PMCID: PMC8726466 PMID: 34982782

Abstract

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.

Introduction

All bat species and their roosts are legally protected in France and in Europe by national and international legislation due to the significant decrease in their populations over the last few decades. Bats belong to order Chiroptera, the second largest order of mammals after rodents. With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Bats are divided into two sub-orders: Yangochiroptera including 12 microbat families, and Yinpterochiroptera including four microbat families in Rhinolophidea plus Old World fruit bats [1, 2]. Today, 51 bat species have been identified in Europe of which 35 occur in France, including one new cryptic species (Myotis crypticus) recently identified in France [3, 4]. All bats in Europe are insectivorous except Rousettus aegyptiacus, which is a frugivorous bat commonly reported in Africa, but also in Cyprus in Europe [5, 6]. Insectivorous bats in Europe are divided into four different families: Rhinolophidae, Vespertilionidae, Molossidae and Miniopteridae [7, 8]. Some studies have suggested a possible association between some bat species and micro-organisms, including more than 200 viruses, bacteria, parasites and pathogenic fungi. For example, Myotis daubentonii, found throughout Ireland and Europe and as far as Japan and Korea, has been shown to be infected with alphacoronaviruses, astroviruses, paramyxoviruses [9, 10], lyssaviruses [11] and Bartonella bacteria [12]. Myotis myotis, common in France, has been reported with the presence of alphacoronaviruses [13], herpesviruses and with Yersinia bacteria [14].

The role of bats in the transmission of zoonotic pathogens to both humans and animals is not clear. Their possible specific association with infectious micro-organisms highlights the importance of identifying bat species precisely to thoroughly investigate the link between the potential presence of pathogens and bat species. Species identification of individuals is generally performed by bat specialists using morphological keys [15]. The morphological determination of bats is usually based on geometric morphometrics since the 1980s [16–18]. For example, since 1989, to differentiate between M. myotis and M. blythii populations, each individual has been identified with external measurements such as left and right forearm lengths, third- and fifth-digit lengths, ear width, ear length, weight, ear surface area, tragus form, number of ear folds, calcar tip form, fur colour. This set of techniques has become popular and has been used for determining bats across a large number of taxa. In some cases, morphological determination can lead to misidentification of some bat species, especially from sites harbouring multiple species belonging to the same family [19]. Moreover, it is extremely difficult to determine some species precisely based on carcasses, for example Pipistrellus pipistrellus, P. kuhlii, P. pygmaeus and P. nathusii, or to distinguish between young P. pipistrellus and P. pygmaeus due to their similar morphological features. Finally, differences may be less obvious between these species when carcasses are in an advanced state of decomposition.

Many studies have shown the importance of genetic determination by amplification of mitochondrial DNA (mtDNA) for accurate identification of bat species [20–22]. Animal mtDNA is generally a small, circular molecule containing 37 genes that are essential for normal mitochondrion function [23]. Species identification and DNA barcoding have been shown to be a useful tool to better understand the relationship between the presence of pathogens and bat species. For instance, a study showed the role of bat species involved in the circulation of lyssaviruses across Canada [19, 20]. Genetic identification can be undertaken by testing for the partial cytochrome b gene (cyt b) [24], cytochrome c oxidase subunit 1 (cox1) gene [20] or a portion of the hypervariable domain II of the mtDNA control D-loop region that can differentiate mitochondrial haplotypes and diversity [25, 26].

Interestingly, new universal cyt b primers allow species identification of 63 animal species belonging to 38 families from 14 orders and 5 classes (Mammalia, Aves, Reptilia, Actinopterygii and Malacostraca) from putrefied samples [27]. Primer design is based on an alignment of referenced cyt b gene sequences (-1140 nt) from 751 Mammalia species, including bats. Primers have been used for the identification of different animal species belonging to 38 families, except bats. Many types of sample have been tested, including muscle, brain, lung or spleen tissue, blood, oral swabs, and others [20, 27]. However, the drawbacks of collecting these types of samples involve the need to capture and restrain the animals combined with the difficulty of handling them. To avoid sampling live animals, using a non-invasive sampling technique such as faeces sampling can be an alternative solution to the capture of bats. Faecal samples represent a simple and easy method to collect samples from living bats without disturbing them using capture/release methods [28, 29].

One study has demonstrated the possibility of genetically identifying bat species from guano samples and other non-invasive samples based on the amplification of a segment of the mitochondrial gene cox1 [21]. Despite the fact that some studies have shown disadvantages of studying faeces samples, due to the presence of PCR inhibitors, fragmented DNA and the poor quality of extracted nucleic acids [30], other studies have demonstrated the efficacy and success of studying bat guano [9, 21].

The aim of this study was 1) to optimize the rapid PCR method previously described in Lopez-Oceja et al. (2016) with the new universal cyt b primers to identify autochthonous bat species from different types of bat sample, namely guano and wing punches tested for the first time; 2) to genetically determine bats in France and 3) to compare the morphological and genetic species identification of bat carcasses submitted for rabies diagnosis in 2018 and 2019.

Materials and methods

Bat specimens

The specimens used in this study were selected from a frozen and archived collection of bat carcasses submitted to the ANSES-Nancy Laboratory for Rabies and Wildlife for rabies diagnosis between 2018 and 2019. Wing punches (each ~8 mm, ~ 0.02 mg) were sampled from bat carcasses diagnosed negative for rabies and stored at -20°C. All bats were previously identified using a morphological identification key by bat specialists [15]. The choice of bat samples was based on the following essential criteria: bat species and the geographic zone of collection. A total of 200 bat carcasses belonging to one of three families, Rhinolophidae, Vespertilionidae and Miniopteridae, representing 22 species were included in the study. Of the 200 bat wing punches tested, 37 were included in the development of the PCR and 163 were used in the PCR amplification of the partial cyt b gene followed by sequencing of amplified products and sequence analysis. Tables 1 and 2 gives the characteristics of the 200 bat specimens used in this study.

Table 1. Characteristics of the 163 bat carcasses included for genetic identification.

Family	Bat species^*	Number wing punches tested		Total
Family	Bat species^*	2018	2019	2018–2019
Vespertilionidae	Barbastella barbastellus	1		1
	Eptesicus serotinus	1	7	8
	Eptesicus nilssonii	1		1
	Myotis bechsteinii	1		1
	Myotis brandtii		1	1
	Myotis daubentonii		3	3
	Myotis emarginatus	1		1
	Myotis myotis		11	11
	Myotis mystacinus	1	4	5
	Myotis nattereri		3	3
	Nyctalus leisleri	3	10	13
	Nyctalus noctula	2	8	10
	Pipistrellus kuhlii	3	4	7
	Pipistrellus nathusii	2	15	17
	Pipistrellus pipistrellus	1	24	25
	Pipistrellus pygmaeus	2	2	4
	Plecotus		1	1
	Plecotus auritus	2	5	7
	Plecotus austriacus	3	6	9
	Vespertilio murinus		2	2
	Pipistrellus sp.	3	5	8
	n.d.		18	18
Rhinolophidae	Rhinolophus ferrumequinum		2	2
Rhinolophidae	Rhinolophus hipposideros		4	4
Miniopteridae	Miniopterus schreibersii		1	1
	Total of samples tested	27	136	163

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n.d.: not determined.

*: identification based on morphological criteria.

Table 2. Characteristics of the 37 bat carcasses included for optimization.

Family	Bat species^*	Number wing punches tested		Total
Family	Bat species^*	2018	2019	2018–2019
Vespertilionidae	Barbastella barbastellus		1	1
	Eptesicus serotinus	1	1	2
	Eptesicus nilssonii		1	1
	Myotis daubentonii		1	1
	Myotis emarginatus	1		1
	Myotis myotis	2		2
	Myotis mystacinus		2	2
	Myotis nattereri		1	1
	Nyctalus leisleri	1		1
	Nyctalus noctula	1		1
	Pipistrellus kuhlii	1	2	3
	Pipistrellus nathusii		1	1
	Pipistrellus pipistrellus		7	7
	Pipistrellus pygmaeus		1	1
	Plecotus auritus	1		1
	Plecotus austriacus	2	1	3
	Vespertilio murinus		2	2
Rhinolophidae	Rhinolophus ferrumequinum		1	1
Rhinolophidae	Rhinolophus hipposideros		2	2
Miniopteridae	Miniopterus schreibersii		3	3
	Total of samples tested			37

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*: identification based on morphological criteria.

In addition, bat guano (one faecal pellet ~50 mm²; ~ 0.02 mg) was also collected by bat specialists from the French Bird Protection League (LPO) Alsace as part of authorized bat studies. Faecal pellets were collected directly on the ground under the bat colony in three different sites in the Grand Est region in France. Bat species were determined in each selected area by inspected hanging individuals in the colony. A total of 31 bat faecal samples representing 7 species belonging to the families Rhinolophidae and Vespertilionidae were included in the genetic identification study (Table 3). Samples were collected in individual bags, stored at -20°C and then at -80°C before analysis.

Table 3. Characteristics of the 31 bat faecal specimens included in the study.

Family	Bat species^*	Number bat faecal specimens
Family	Bat species^*	2019
Vespertilionidae	Eptesicus serotinus	1
	Myotis emarginatus	2
	Myotis myotis	22
	Pipistrellus pipistrellus	1
	Plecotus austriacus and/or Plecotus auritus	2
Rhinolophidae	Rhinolophus hipposideros	3
	Total of samples tested	31

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*: identification based on morphological criteria.

Ethics statement

Bats are protected species in Europe and in France. All biological samples employed in this study had been submitted for rabies diagnosis by ANSES-Nancy Laboratory for Rabies and Wildlife in accordance with the formal authorization by the French Ministry of the Environment [31]. In France and within the European Union, the legal frame- work for using under experimentation purposes is governed by Regulation 2010/63/EU of the European parliament and of the council of 22 September 2010 (applicable and translated in French in 2013) and handling of wildlife animal in the field does not require any prior specific ethical approval.

DNA extraction

DNA extraction was performed using 1 punch per animal or 1 faecal pellet per site or per bat. Wing punches were directly used for DNA extraction, whereas a pre-extraction step was carried out to prepare bat faeces. Each faecal pellet was ground with 120 μL of 1X PBS buffer (phosphate buffered Saline, Sigma-Aldrich, Saint Quentin-Fallavier, France) then centrifuged for 5 min at 30,000 x g. For DNA extraction, 20 μL of supernatant was used and the extraction was performed using the Nucleospin Tissue Kit (Macherey Nagel, Hoerdt, France), following the manufacturer’s recommendations. DNA samples were quantified using a Qubit fluorometer (Invitrogen, Marseille, France) and stored at -20°C before use.

PCR: Amplification of the partial cytochrome b gene

Five μL of extracted DNA diluted to 1 ng/μL was used as template for amplification of a portion of the mitochondrial cyt b gene. We used the universal primers previously described in Lopez-Oceja et al. (2016) (forward primer L15601: 5’-TACGCAATCCTACGATCAATTCC-3’ and reverse primer H15748: 5’-GGTTGTCCTCCAATTCATGTTAG-3’) to amplify a 148 bp fragment of cyt b [27].

PCR amplification was performed in a 25 μL reaction volume containing 5 μL of DNA (1 ng/μL), 2.5 μL of 10X PCR Buffer without MgCl₂ (Invitrogen, Marseille, France), 1 μL of 50 mM MgCl₂, 1 μL of dNTPs (10 mM each) and 0.5 μL of Taq DNA polymerase (5 U) (Invitrogen, Marseille, France) and 1 μL of each primer (0.4 μM). The PCR was performed with the following conditions: 3 min at 95°C, 45 cycles of 30 s at 95°C, 30 s at 48°C and 45 s at 72°C and following with a final step of extension of 5 min at 72°C.

With each run, negative and positive PCR controls were performed for PCR validation.

PCR: Amplification of partial D-loop

Five μL of extracted DNA was used for PCR amplification of the hypervariable domain II of the mtDNA control D-loop region producing PCR amplicons of 424 bp. The PCR amplifications were performed in 25 μL reaction volumes using validated primers described in Moussy et al. (2015). PCR consisted of an initial denaturation step at 95°C for 3 min, followed by 45 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 45 sec, and a final extension step of 72°C for 5 min. The 25 μL reaction mixture consisted of 25 μL of DNA template diluted to 1:10, 0.5 μL of enzyme mix in 2.5 μL 10X reaction buffer, 1.0 μL of 50 mM MgCl₂, 1 μL of 10 mM dNTP mixture, and 1 μM of each forward and reverse primer. The D-loop primers used for PCR amplification were L-strand D-loop (5′-CTACCTCCGTGAAACCAGCAAC-3′) and H-strand D-loop (5′-CGTACACGTATTCGTATGTATGTCCT-3). With each run, negative and positive PCR controls were performed for PCR validation. The D-loop PCR was performed on serotine bats (n = 5), only. The specificity of the PCR products was confirmed by direct sequencing of the amplified amplicons.

Sequencing and phylogenetic analysis

Amplicons were analysed using 2% agarose gels stained with the intercalant SYBR Safe (Thermo Fisher Scientific, IIIkirch, France) then visualized using Bioimager (Bio-Rad, Roanne, France).

Sanger sequencing of PCR products was carried out by a service provider (Eurofins, Ebersberg, Germany) with the reverse and forward primers used in the PCR. All nucleotide sequences were assembled using Vector NTI software (version 11.5.3) (Invitrogen, France). Sequence alignments and determination of the percentages of identities and similarities were carried out with BioEdit Software (version 7.2.5) and MEGA X.

Genetic identification was determined using BLAST (Basic Local Alignment Search Tool) and by constructing a phylogenetic tree with MEGA-X using the maximum likelihood algorithm and the Tamura-Nei model between the 25 sequences from this study (representing 2 families and 15 species) and 52 representatives of bat species (3 families, 29 species) (Table 4). The bootstrap probabilities of each node were calculated using 500 replicates to assess the robustness of the maximum likelihood method. Bootstrap values over 70% were regarded as significant for phylogenetic analysis.

Table 4. Characteristics of the partial cytochrome b gene reference sequences retrieved from GenBank and other sequences amplified from wing punches and bat guano from this study.

No.	Country	Species	Year	GenBank Accession no.	Source
1	Japan	Rhinolophus ferrumequinum	2003	AB085730	[32]
2	Japan	Rhinolophus ferrumequinum	2003	AB085731	[32]
3	Japan	Plecotus auritus	2003	AB085734	[32]
4	Japan	Myotis daubentoni	2003	AB106589	[33]
5	Japan	Myotis nattereri	2003	AB106606	[33]
6	Japan	Vespertilio murinus	2010	AB287358	[34]
7	Swiss	Nyctalus leisleri	2001	AF376832	[35]
8	Swiss	Eptesicus nilssoni	2001	AF376836	[35]
9	Swiss	Myotis blythii	2001	AF376842	[35]
10	Swiss	Myotis bechsteinii	2001	AF376843	[35]
11	Swiss	Myotis brandtii	2001	AF376844	[35]
12	Swiss	Myotis capaccinii	2001	AF376845	[35]
13	Swiss	Myotis dasycneme	2001	AF376846	[35]
14	Swiss	Myotis emarginatus	2001	AF376849	[35]
15	Cyprus	Pipistrellus pygmaeus	2004	AJ504442	[36]
16	Greece	Pipistrellus pipistrellus	2004	AJ504443	[36]
17	Macedonia	Pipistrellus kuhli	2004	AJ504444	[36]
18	Swiss	Pipistrellus nathusii	2004	AJ504446	[36]
19	Swiss	Hypsugo savii	2004	AJ504450	[36]
20	Swiss	Myotis alcathoe	2004	AJ841955	[37]
21	Swiss	Nyctalus noctula	2004	AJ841967	[37]
22	Spain	Myotis myotis	2007	AM261883	[38]
23	China	Myotis blythii	2006	AM284170	[39]
24	Japan	Myotis daubentoni	2012	AY665137	[40]
25	Japan	Myotis brandtii	2012	AY665139	[40]
26	Japan	Plecotus auritus	2012	AY665169	[41]
27	China	Miniopterus schreibersii	2004	EF530339	[41]
28	China	Miniopterus schreibersii	2004	EF530342	[33]
29	China	Plecotus auritus	2015	EF570882	[42]
30	Spain	Rhinolophus euryale	2009	EU436671	[43]
31	Spain	Rhinolophus mehelyi	2009	EU436672	[43]
32	Azerbaijan	Eptesicus serotinus	2009	EU751000	[44]
33	Russia	Eptesicus nilssoni	2009	GQ272582	[45]
34	Russia	Eptesicus serotinus	2009	GQ272585	[45]
35	Russia	Eptesicus serotinus	2009	GQ272586	[45]
36	Armenia	Myotis myotis	2009	GU817388	[46]
37	France	Myotis escalerai	2012	JF412390	[47]
38	France	Myotis escalerai	2012	JF412391	[47]
39	France	Myotis nattereri	2012	JF412411	[47]
40	Portugal	Barbastella barbastellus	2012	JQ683211	[48]
41	Swiss	Nyctalus leisleri	2012	JX570901	[49]
42	Greece	Nyctalus noctula	2012	JX570902	[49]
43	France	Rhinolophus hipposideros	2013	KC978712	[50]
44	Spain	Rhinolophus mehelyi	2014	KF031265	[51]
45	Spain	Rhinolophus mehelyi	2014	KF031266	[51]
46	France	Rhinolophus euryale	2014	KF031267	[51]
47	France	Rhinolophus euryale	2014	KF031268	[51]
48	Greece	Myotis blythii	2013	KF312501	[52]
49	Iran	Pipistrellus pipistrellus	2013	KF874519	[53]
50	Caucasus region	Myotis mystacinus	2016	KU060256	[54]
51	Caucasus region	Myotis mystacinus	2016	KU060257	[54]
52	Caucasus region	Myotis alcathoe	2016	KU060271	[54]
53	France	Barbastella barbastellus_132883	2018	MZ066766	This Study
54	France	Eptesicus serotinus_133164	2019	MZ066767	This Study
55	France	Nyctalus noctula_132681	2018	MZ066769	This Study
56	France	Myotis mystacinus_133119	2019	MZ066772	This Study
57	France	Myotis mystacinus_133333	2019	MZ066774	This Study
58	France	Pipistresllus pipistrellus_133323	2019	MZ066788	This Study
59	France	Myotis nattereri_133147	2019	MZ066775	This Study
60	France	Nyctalus leisleri_132631	2018	MZ066776	This Study
61	France	Nyctalus noctule_132624	2018	MZ066777	This Study
62	France	Pipistrellus kuhli_133328	2019	MZ066781	This Study
63	France	Plecotus auratus_132673	2018	MZ066778	This Study
64	France	Plecotus austriacus_133165	2019	MZ066779	This Study
65	France	Pipistrellus nathusius_133149	2019	MZ066782	This Study
66	France	Pipistrellus pipistrellus_133225	2019	MZ066773	This Study
67	France	Pipistrellus pipistrellus_133120	2019	MZ066783	This Study
68	France	Pipistrellus pipistrellus_133152	2019	MZ066784	This Study
69	France	Pipistrellus pipistrellus_133522	2019	MZ066785	This Study
70	France	Pipistrellus pipistrellus_133330	2019	MZ066786	This Study
71	France	Pipistrellus pipistrellus_133331	2019	MZ066787	This Study
72	France	Myotis myotis_132714	2018	MZ066770	This Study
73	France	Myotis emarginatus_Hoerdt	2019	MZ066768	This Study
74	France	Plecotus austriacus_Weiler	2019	MZ066780	This Study
74	France	Rhinolophus ferrumequinum_133127	2019	MZ066789	This Study
76	France	Myotis myotis_GM-5-CB	2019	MZ066771	This Study
77	France	Rhinolophus hipposideros_133128	2019	MZ066790	This Study

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The nucleotide sequences were identified using BLASTN with the following parameters: standard nucleotide database and standard algorithm parameters by default (threshold of 0.05 and mismatch scores of 1,-2). In each case, the top BLAST hit was retained if the BLAST alignment covered more than 95% of the query length and the BLAST high-scoring segment pair identity was greater than ~90%.

Results

Genetic identification of bat carcasses and bat faeces

Bat carcasses

Of 163 bat wing punches tested using cyt b PCR, 152 were genetically identified by BLAST analysis and/or phylogeny. The 152 genetically identified samples represented the 3 families currently distributed throughout France with bat species belonging to the families Miniopteridae (n = 1), Rhinolophidae (n = 2) and Vespertilionidae (n = 19), respectively (Table 5). Twenty species out of the 35 bat species reported to date in France were genetically determined with an over representation of Pipistrelle bats in the sampling (37% = 61/163*100). BLAST analysis allowed the identification of 2 bat species belonging in the Rhinolophidae family with ~96% of nucleotide similarity with the GenBank sequences KU531352 (R. hipposideros) and MH029812 (R. ferrumequinum) and the identification of M. schreibersii from the Miniopteridae family with 93% of nucleotide similarity with the MK737740 sequence. Within, the Vespertilionidae family, 16 bat species were genetically identified by BLAST with a % nucleotide identity ranging from 87% to 100% (S1 Table).

Table 5. Results of PCR on the partial cytochrome b gene and species misidentification of bat wing punch samples compared with morphological species identification.

Family	Bat species ^*	Bat wing punches			Species
Family	Bat species ^*	2018–2019	Morphological misidentification of bat species	Clarifications	Species
Vespertilionidae	Barbastella barbastellus	1	0/1
	Eptesicus serotinus	8	0/8
	Eptesicus nilssonii	1	0/1
	Myotis bechsteinii	1	0/1
	Myotis brandtii	1	1/1
	Myotis daubentonii	3	1/3
	Myotis emarginatus	1	0/1
	Myotis myotis	11	2/11
	Myotis mystacinus	5	2/5
	Myotis nattereri	3	0/3
	Nyctalus leisleri	13	0/13
	Nyctalus noctula	10	1/10
	Pipistrellus kuhlii	7	2/7
	Pipistrellus nathusii	17	1/17
	Pipistrellus pipistrellus	25	3/25
	Pipistrellus pygmaeus	4	4/4
	Plecotus	1	0/1
	Plecotus auritus	7	2/7
	Plecotus austriacus	9	1/9
	Vespertilio murinus	2	0/2
	Pipistrellus sp.	8	0/8	8/8	Pp (n = 4), Pk (n = 4)
	n.d.	18	-	18/18	Pp (n = 6), Pk (n = 7), Rh (n = 1), Md (n = 1), Hs (n = 2), Pg/Pp (n = 1)
Rhinolophidae	Rhinolophus ferrumequinum	2	0/2
Rhinolophidae	Rhinolophus hipposideros	4	0/4
Miniopteridae	Miniopterus schreibersii	1	0/1
	Total of samples tested	163	21/163

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*: identification based on morphological criteria.

Abbreviations: Pipistrellus pipistrellus (Pp), Pipistrellus kuhlii (Pk), Rhinolophus hipposideros (Rh), Myotis daubentonii (Md), Hypsugo savii (Hs), Pipistrellus pygmaeus or Pipistrellus pipistrellus (Pg/Pp).

Twenty out of the 156 samples belonging in the Vespertilionidae family could not be identified by BLAST sequence analysis of the cyt b amplicons. These samples had previously been morphologically determined as E. serotinus (n = 6), V. murinus (n = 2), E. nilssonii (n = 1), and Plecotus sp (n = 11). Interestingly, the phylogeny allowed the genetic determination of two species, Plecotus austriacus and Plecotus auritus for 9 samples analysed with a boostrap of 99 (Fig 1).

Fig 1 — **B.barb**: *Barbastella barbastellus*, **E.nils**: *Eptesicus nilssonii*, **E.ser**: *Eptesicus serotinus*, **H.s**: *Homo sapiens*, **H.savi**: *Hypsugo savii*, **M.al**: *Myotis alcathoe*, **M.bech**: *Myotis bechsteinii*, **M.bly**: *Myotis blythii*, **M.br**: *Myotis brandtii*, **M.c**: *Myotis capaccinii*, **M.daub**: *Myotis daubentonii*, **M.das**: *Myotis dasycneme*, **M.em**: *Myotis emarginatus*, **M.esc**: *Myotis escalerai*, **M.myo**: Myotis *Myotis*, **M.mys**: *Myotis mystacinus*, **M. nat**: *Myotis nattereri*, **M.schr**: *Myotis schreibersii*, **N.leis**: *Nyctalus leisleri*, **N.noct**: *Nyctalus noctula*, **P.aur**: *Plecotus auritus*, **P.aust**: *Plecotus austriacus*, **P.kuh**: *Pipistrellus kuhlii*, **P.pip**: *Pipistrellus pipistrellus*, **P.pyg**: *Pipistrellus pygmaeus*, **P.nath**: *Pipistrellus nathusii*, **R.fer**: *Rhinolophus ferrumequinum*, **R.hip**: *Rhinolophus hipposideros*, **R.meh**: *Rhinolophus mehelyi*, **S.a**: *Sorex araneus*, **V.mur**: *Vespertilio murinus*.

The partial D-loop amplification (424-bp) of five bats morphologically identified as E. serotinus showed 100% of nucleotide similarity with E. serotinus (GenBank no. accession MF187797.1).

Of the 163 bat carcasses tested, 18 carcasses had not been previously identified with morphological criteria. The sequence analysis by BLAST and/or phylogeny showed for the 18 undetermined bats the following species: P. pipistrellus (n = 6), P. kuhlii (n = 7), M. daubentonii (n = 1), R. hipposideros (n = 1), Hypsugo savii (n = 2) and P. pipistrellus or P.pygmaeus (n = 1).

Bat faeces

The analyses of cyt b sequences led to a specific identification of the 31 samples of bat species from one faecal pellet for the seven bat species tested (Table 6).

Table 6. Results of PCR on the partial cytochrome b gene and species misidentification of bat guano samples compared with morphological identification.

Family	Bat species^*	Nb. bat faecal specimens	Morphological misidentification of bat species	Clarifications	Species
Family	Bat species^*	2019	Morphological misidentification of bat species	Clarifications	Species
Vespertilionidae	Eptesicus serotinus	1	1/1		Rh (n = 1)
	Myotis emarginatus	3	0/2
	Myotis myotis	20	0/22
	Pipistrellus pipistrellus	1	0/1
	Plecotus auritus and/or Plecotus austriacus	2	1/2		Rh (n = 1)
Rhinolophidae	Rhinolophus hipposideros	3	0/3
	Total of samples tested	32	0/31

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*: identification based on morphological criteria.

Abbreviations: Rhinolophus hipposideros (Rh).

The 31 genetically identified samples represented 2 out of the 3 families currently distributed throughout France with bat species belonging to the families Rhinolophidae (n = 1) and Vespertilionidae (n = 3), respectively (Table 6).

BLAST analysis allowed the identification of the bat species, R. hipposideros with ~96% of nucleotide similarity with the GenBank KU531352 and KC978344 sequences. ~ 94% of similarity were shown between bats morphologically identified as M. emarginatus and the AF376849 GenBank sequence representative of M. emarginatus. Within the two species P. pipistrellus and M. myotis, a mean of 99% were shown between the faecal samples and the Genbank sequences KJ765388.1 (M. myotis) and AH006588.2 (P. pipistrellus).

Interestingly, and as for bat carcasses, the samples that had previously been morphologically determined as Plecotus sp (n = 11) could not be identified by BLAST sequence analysis of the cyt b amplicons but was identified by phylogeny with a bootstrap of 99 (Fig 1).

Comparison between morphological identification and cyt b PCR analysis

Bat carcasses

The comparison between morphological and genetic identification carried out on the 163 bat samples showed the same results for 142 samples tested and identified a total of 21 misidentifications. These 21 morphological misidentifications represented ~12.5% of total bat carcasses tested. The misidentifications were confirmed by bat specialists who performed a second morphological identification on these bat samples using another species determination key. Results are detailed in Table 4. Morphological identification errors were reported for 11 species: M. brandtii (n = 1), M. daubentonii (n = 1), M. myotis (n = 2), M. mystacinus (n = 2), N. noctula (n = 1), P. kuhlii (n = 2), P. nathusii (n = 1), P. pipistrellus (n = 3), P. pygmaeus (n = 5), P. auritus (n = 2) and P. austriacus (n = 1) (S1 Table).

Genetic identification allowed clarifications for 26 bats tested (18 bats morphologically identified as not determined and 8 bats morphologically identified as Pipistrellus sp.) (Table 5).

BLAST analysis allowed the distinction of the 8 Pipistrelle bats tested in the study with the genetic identification of 4 P. pipistrellus (~96% similarity with the Genbank AH006588.2 and AJ504443.1 sequences) and 4 P. Kuhlii (95% similarity with the Genbank MN045571.1 sequence representative of P. Kuhlii). Of the 18 bat carcasses morphologically not determined, we reported the identification of 6 species belonging in the Vespertilionidae family with P. pipistrellus (n = 6), P. kuhlii (n = 7), M. daubentonii (n = 1), R. hipposideros (n = 1), Hypsugo savii (n = 2) and P. pipistrellus or P. pygmaeus (n = 1).

Bat faeces

The genetic identification of bat species from the guano samples showed 2 morphological misidentifications out of the 31 guano samples tested. Misidentifications were reported in two sites: the site 22 among Plecotus sp. and R. hipposideros and the site 31 among E. serotinus and R. hipposideros (S1 Table).

Discussion and conclusion

To our knowledge, this study is the first to evaluate published universal primers targeting the cyt b gene [27] from two different bat matrices, wing punch and guano, to genetically identify autochthonous bat species. Of the 35 species reported in France, 14 species are uncommon in France. Our study showed 12.5% of misidentification for 11 out of the 22 bat species tested. Our results corroborate the Nadin-Davis (2012) study, which also showed non-negligible percentages of morphological bat species misidentification of between 10 and 15%.

It is rare and very complicated to collect samples for research or rabies diagnosis from autochthonous bats. The fact that all bat carcasses included in this study came from a sample collection compiled for rabies diagnosis at ANSES Laboratory led to an over representation of P. pipistrellus in our sampling. In France, P. pipistrellus is a very common bat species compared with other bat species. On average, there is one P. pipistrellus colony in each town in France (Laurent Arthur, personal communication). P. pipistrellus represents on average between 45 and 50% of the total number of carcasses in the rabies diagnosis sample collection. In our study, P. pipistrellus represented 16% of the total number of samples.

The species could not be identified for 11 of the 163 samples tested. These samples were morphologically identified as E. serotinus (n = 6), E. nilssonii (n = 1), V. murinus (n = 2) and Plecotus sp (n = 2). One hypothesis of species non-identification is that the cyt b PCR was not able to identify these 8 samples due to DNA degradation. Two published studies investigated the genetic structure of E. serotinus bats by amplifying the partial D-loop region [25, 26]. Thus, the amplification of the partial D-loop region on the five E. serotinus was successful and our results on Sanger sequencing confirmed the morphological species determination as E. serotinus.

Regarding bat faecal specimens, results and analyses of the 31 amplicons showed that the cyt b PCR allowed specific identification of bat species from just one faecal pellet of bat guano. Bat species have previously been genetically identified from guano samples by amplification of a segment of the cox1 mitochondrial gene using real-time PCR [21]. Some studies have demonstrated the advantages of using real-time PCR compared with conventional PCR: real-time PCR is more sensitive, specific and rapid as a diagnostic method for detecting Vibrio vulnificus and Samonella spp. compared with conventional PCR [55, 56]. Both PCR techniques are equally effective for detection of the genome of visceral leishmaniasis [57]. The discrepancy between the results obtained in our study and those of the Walker et al. study likely arises from using a traditional PCR with the cyt b gene universal primers [21, 27]. In our study, the genetic determination of bats was based on universal primers of the cytb gene, described by Lopez-Oceja et al., as highly specific, especially for highly degraded DNA samples (Lopez-Oceja et al., 2016). Species identification from bat faecal samples can also be undertaken by DNA mini-barcode assay based on the amplification of a segment of the mitochondrial gene cytochrome c oxidase I (COI) [21]. New primers targeting a 580 bp fragment of the COI gene were described for the identification of bat species [21]. Interestingly, the comparison between the cytb and COI genes was studied by Tobe et al. for reconstructing mammalian phylogenies [58]. Their results tend to support the use of Cytb over that of COI. Conventional PCR allowed us to obtain nucleotide sequences from amplicons and to genetically determine bat species using BLAST and/or phylogeny. In addition, the cost of real-time PCR is higher than conventional PCR. In our study, we demonstrated the efficacy of using universal cyt b primers to genetically identify autochthonous bats from faecal samples, a non-invasive method.

The cyt b PCR made it possible to determine 18 bat samples that could not initially be identified based on morphological criteria. Non-determination of bats can be attributed to the state of decomposition of bat carcasses, the age of the bat, especially for juveniles or pups, or inexperienced bat naturalists. Morphological identification of bat species is usually carried out on living bats. Some morphological features disappear if the carcasses are not fresh, and identification becomes more complicated, creating a source of errors [59, 60].

It is important to identify bat species to preserve bats, which play a key role in the environment. Bats play an important biological and ecological role and many studies have suggested that they are reservoirs in the transmission of many zoonoses and infectious diseases from animals to humans [3, 9, 61]. To better understand bats and their role in the circulation of pathogens, specific and precise identification of bat species is required. Our results here showed that genetic identification is an efficient way to identify bat species in France and is a rapid and reliable tool to use compared with morphological identification.

Supporting information

S1 Table. Raw data analysis: Bat identification from bat wing punches and faecal samples.

(XLSX)

Click here for additional data file.^{(31.9KB, xlsx)}

S1 Raw images

(TIF)

Click here for additional data file.^{(867KB, tif)}

Acknowledgments

We thank our collaborators and bat naturalists for providing the bat specimens used in this study. We also thank Alexis Petitdemange, Mélanie Badré Biarnais and Jean Luc Schereffer for their technical assistance in this study.

Abbreviations

Cox: Cytochrome c oxidase subunit 1
Cyt b: Cytochrome b
DNA: Deoxyribonucleic acid
LPO: League for the Protection of Birds
LRFSN: Nancy Laboratory for Rabies and Wildlife
mt: Mitochondrial
n.d.: Not determined
PCR: Polymerase Chain Reaction

Data Availability

All files are available from the Genbank database (accession number(s) MZ066766 to MZ066790). All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by the French National Agency for Food, Environmental and Occupational Health & Safety and VetAgro Sup laboratory for Leptospira.

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PLoS One. doi: 10.1371/journal.pone.0261344.r001

Decision Letter 0

Camille Lebarbenchon

1 Jul 2021

PONE-D-21-14577

Genetic identification of bat species for pathogens surveillance across France

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This study describes the test of previously published primers for the barcoding of french bat species, using both wing punch from bat carcasses and faecal pellets. Results showed that the method is successful in identifying at least 22 from the 35 bat species described in France. This technique allows to correct 12.5% of morphological misidentifications among 167 samples, and identify 24 samples that could not be morphologically identify in the field. This study provides new and important tools for the correct identification of bat species, which is of primary importance for ecological and pathogen-associated studies of bats.

I enjoyed reading this manuscript which is globally well written. However, I recommend the authors to add the line numbers on the manuscript to help the reviewing process. My two major concern are the lack of information for the phylogenetic analysis and D-loop PCR. The authors should better explain why they perform the phylogenetic analysis in addition to the BLAST, and better present the results of the grouping in the tree. Also throughout the manuscript, it is not clear why the authors also performed PCR on the D-loop, but only for 1 bat species and on a very limited number of samples. Please see below for more detailed comments.

1) Page 1, title: Please correct “pathogens surveillance” to “pathogen surveillance”.

2) Page 5: please correct ‘bats species’ to ‘bat species’.

3) Methods, page 6: were the faecal pellets collected directly from bat handling or from the environment?

4) Methods, sequencing and phylogenetic analysis: please provide more details on which criteria were used in the BLAST output to identify bat species? And for the phylogenetic tree analysis? A better explanation of phylogenetic results would also be valuable, for example, whether or not the tested samples clustered with previous reference sequences (=are the species-clusters are well supported or not). Did you assume correct identification if the sample clustered with reference sequences?

5) Page 11: genetic distances were calculated but I did not see the results of this analysis.

6) Results, page 12: please remove ‘by’ in ‘by followed…’

7) Results, page 12: what do the authors mean by ‘optimization’? I don’t see any description of the(PCR?) optimization in the methods. I don’t understand if the protocol was different for the 37 samples used for optimization. Please provide more details in the methods and results.

8) Why only serotine bats were tested for D-loop and only 6 individuals? I understood later from the discussion that the D-loop PCR was performed because there were no amplification with the cytb, and this allowed confirming the ID for E. serotinus. But why the D-loop PCR was not done for the other non-cytb amplified samples (one E. nilsonni and one V. murinus)?

9) Page 12: Pl. auritus should be P. auritus.

10) Results, page 12: Genetic identification of bat carcasses: ‘the panel of genetically…M. schreibersii (n=1)’. It looks like that the authors give here the results of the cytb genetic identification. Is it not just the listing of the samples used in this analysis (same as in table 1A). If so, this is a bit redundant with information presented in the methods. I suggest to remove this sentence and present directly the comparison of morphological/genetic identification.

11) Figure 3: It is not clear what are the sequences produced in this study. Please used bold or color to highlight them. Please also add posterior values at the nodes. Is the tree well supported? It may also be clearer if the different bat species were better delineated (using colored boxes for example).

12) For the 25 sequences included in the tree, was the BLAST results the same as the tree classification? Why only 25 sequences were included in the phylogenetic tree, and not all the sequences produced (n=167)? Why all these sequences were not submitted to Genbank?

13) Page 14: ‘Sequences analyses using BLAST… following species…’. I don’t understand this result. Authors mentioned above that errors of morphological determination were observed for 11 species.

14) Page 13: Genetic identification of bat faeces: ‘1 was not determined’. Does this mean the BLAST and phylogeny analyses did not give any conclusive results?

15) Discussion, second paragraph: ’It is rare…’ I’m not sure to understand the relevance of this paragraph. Is this to justify the non-homogeneity of number of samples per bat species? If so, I would just state that : “the fact that all bat carcasses…ANSES Laboratory, leading to an over representation of P. pipistrellus in our sampling’.

16) Page 21: please give details on what is the discrepancy between the present study and that of Walker’s, to make it clearer for non-specialist readers.

17) Page 22: the authors suggest that the non-amplification of the cytb for 8 samples (including E. serotinus, E. nilssonii and V. murinus) could be due to the short length of the PCR fragment. Why exactly? But they were successful in amplifying these 3 species for other samples and for the same gene fragment. I would rather suggest that the non-amplification results from degraded DNA. It would have been valuable to test the integrity of DNA by gel electrophoresis.

Reviewer #2: This study identified bats in France from wing punches and fecal material. The results are straightforward and a useful contribution to the identification of bat remains in France. I have made a number of minor edits and comments on the pdf as notes/sticky notes/deletions.

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Attachment

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PLoS One. 2022 Jan 4;17(1):e0261344. doi: 10.1371/journal.pone.0261344.r002

Author response to Decision Letter 0

5 Aug 2021

See below the responses to the remarks given by the two reviewers:

Corrections for the paper entitled “Genetic identification of bat species for pathogens surveillance across France” by Y. Arnaoult et al.

General comments:

The manuscript was completely revised according to the general and the specific comments of the two reviewers. Corrections and clarifications were given all along the manuscript, as suggested.

A supplementary Table (S Table 1) was added to follow the remarks of the reviewer N°1. The two figures 1 and 2 were deleted to follow the reviewing remarks. The figure 3, renumbered 1, was slightly modified by the adding of a blue colored dot (differentiation between the Genbank sequences and the sequences of this study).

Responses to the remarks given by the two reviewers are written in blue following each question/remark.

Following the e-mail dated of 19 July 2021, the three tables 3-5 were renumbered, as follows:

- Table 3 was renumbered Table 4 (See Page 20)

- Table 4 was renumbered Table 5 (See Page 21)

- Table 5 was renumbered Table 3 (See Page 13-14).

The three tables are refered in the text.

Review Comments to the Author

Reviewer 1:

1) Page 1, title: Please correct “pathogens surveillance” to “pathogen surveillance”.

The remark has been taken into consideration. We changed the title and correct “pathogens surveillance” to “pathogen surveillance”. See Page 1, line 1.

2) Page 5: please correct ‘bats species’ to ‘bat species’.

To follow the remark, we correct ‘bats species’ to ‘bat species’. See Page 5, line 106.

3) Methods, page 6: were the faecal pellets collected directly from bat handling or from the environment?

The faecal pellets were collected directly from the environment. The sampling was carried out by naturalists who are participating each year in the monitoring of bat population in the Grand Est region in France.

We clarified the manuscript (See page 6, line 131-133): “Faecal pellets were collected directly from the environment in three different sites in the Grand Est region in France.”

We agree with the remark of the reviewer and we clarified the manuscript by adding the criteria used to identify bat species in the BLASTN analysis. See page 12, lines 214-217.

The following sentence was added: “The nucleotide sequences were identified using BLASTN with the following parameters: standard nucleotide database and standard algorithm parameters by default (threshold of 0.05 and mismatch scores of 1,-2). In each case, the top BLAST hit was retained if the BLAST alignment covered more than 95% of the query length and the BLAST high-scoring segment pair identity was greater than �90%.”

We have also clarified the phylogenetic analysis by detailing in the methods the calcul of bootstrap probabilities of each node to assess the robustness of the phylogeny tree. See page 6 lines 2010-213.

The following sentence was added: The bootstrap probabilities of each node were calculated using 500 replicates to assess the robustness of the maximum likelihood method. Bootstrap values over 70% were regarded as significant for phylogenetic analysis.

We also detailed in the results a deeper analysis of the phylogeny for each species. See the results section.

5) Page 11: genetic distances were calculated but I did not see the results of this analysis.

We deleted the mention “calculate the distance” as these results are not presented in this paper that are based on the BLAST analysis and the phylogenetic tree for the genetic identification of bat species.

6) Results, page 12: please remove ‘by’ in ‘by followed…’

The sentence was deleted to follow the remark of the reviewer 2.

This sentence was deleted to follow the remark of the reviewer 2. To answer to the query, the amplification of the partial cytB described by Lopez-Oceja et al., was slightly modified in the laboratory and validated against these 37 samples for determining the diagnosis sensitivity of the PCR. The method developed in the laboratory has been fully described in the Methods, in Page 10.

The remark of the reviewer is relevant. Indeed, it should have been interesting to check the specificity of the primers used for amplifying the D-loop region with V. murinus and/or E. nilssoni. However, the PCR was not done, because the PCR described by Moussy et al. was been tested on Eptesicus serotinus, only, and described as specific of this species.

9) Page 12: Pl. auritus should be P. auritus.

The section (lines 254 - 259) was deleted to follow the remark 10 (See Page 16).

To follow the remark, we deleted the sentence.

We agree with the remark of the reviewer. The figure 3 was slightly modified by differentiating the Genbank sequences from the sequences of this study. Before each bat sequence of the study, was added a blue colored dot. See Figure 1. (To follow the remark of the reviewer 2, the two figures 1 and 2 were deleted. Fig 3 is so renumbered as Fig 1 in the new version of the manuscript).

We clarified the manuscript following the remarks 4, 12 and 13.

To answer to the different remarks and in particular the remark 12 we included in the phylogenetic analysis only 1 sequence representative of each bat family, in order to reduce the redundancy of sequences.

The manuscript was clarified accordingly to the remarks with the supplementary Table 1 detailing the BLAST results and phylogeny for all samples. Phylogeny results were added in the S. Table 1 for the 25 samples as well as for the samples representing Plecotus auritus and P. austriacus, analysed by phylogeny. The supplementary Table 1 describes the genetic determination for the two types of matrices tested: faecal and bat wing punches.

The section “Genetic identification of bat carcasses and bat faeces “ was modified to take account the remark. We added in this section, for both bat carcasses and bat faeces, the results of blast analysis for the 3 families, Rhinolophidae, Miniopteridae and Vespertilionidae.

14) Page 13: Genetic identification of bat faeces: ‘1 was not determined’. Does this mean the BLAST and phylogeny analyses did not give any conclusive results?

The mention “not determined” was deleted in the two tables 2 (page 9) and 5 (page 21). After checking the raws datas, there are” no undetermined” in the sampling.

The remark 15 has been taken into consideration. We changed the sentence by the following sentence: “It is rare and very complicated to collect samples for research or rabies diagnosis from autochthonous bats. The fact that all bat carcasses included in this study came from a sample collection compiled for rabies diagnosis at ANSES Laboratory led to an over representation of P. pipistrellus in our sampling.. In France, this bat species, P. pipistrellus is a very common bat species compared with other bat species…..”See page 27, lines 397 to 403.

16) Page 21: please give details on what is the discrepancy between the present study and that of Walker’s, to make it clearer for non-specialist readers.

The remark has been taken into consideration and we have added in Discussion the following sentence:

In our study, the genetic determination of bats was based on universal primers of the cytb gene, described by Lopez-Oceja et al., as highly specific, especially for highly degraded DNA samples (Lopez-Oceja et al., 2016). Species identification from bat faecal samples can also be undertaken by DNA mini-barcode assay based on the amplification of a segment of the mitochondrial gene cytochrome c oxidase I (COI) (Walker et al., 2016). New primers targeting a 580 bp fragment of the COI gene were described for the identification of bat species (Walker et al., 2016). Interestingly, the comparison between the cytb and COI genes was studied by Tobe et al. for reconstructing mammalian phylogenies (Tobe et al., 2010). Their results tend to support the use of Cytb over that of COI. See page 28, lines 424-431.

To follow the remark, we changed the sentence. See page 27, lines 409 to 412.

Reviewer 2:

Abstract:

The abstract has been modified. See Page 1, line 22, 25 and 30.

Introduction:

We have changed the introduction, as suggested by the reviewer as follows:

- “in” has been changed by “occur in”. See See Page 3, line 59.

- “1980s” has been changed by “the 1980s”. See See Page 4, line 75.

- “Since 1989” has been changed by “For example, since 1989” See See Page 4, line 75.

Results:

The two sentences have been deleted to follow the remark. See Page 12 Lines 220-228.

We added in Methods the sentence on the negative and positive controls that were well done for each run. See Page 11, line 186 and lines 196-197.

Genetic identification of bat faeces:

As suggested, we deleted the mention of the optimization of PCR in lines 266-268 and moved the sentence in the Materials section, in Page 6, lines 126-128.

Figure 3:

We agree with the remark of the reviewer. The figure 3 (renumbered Figure 1) was slightly modified by differentiating the Genbank sequences from the sequences of this study. Before each bat sequence of the study, was added a blue colored dot.

Figure 1 and Figure 2:

As suggested, the two figures were deleted. Fig 3 has been renumbered as Fig 1 in the new version of the manuscript.

Discussion and conclusion:

As suggested, we clarified the sentence by adding in Page 27, in line 392, “wing punch and guano”.

We deleted the sentence “This frequency can be explained ….vegetation” as well as the associated reference. See Page 27, lines 406 to 408.

In Page 29, line 442, we added as suggested by the reviewer, the two references (60,61), as follows:

- 59. Korstian JM, Hale AM, Bennett VJ, Williams DA. Using DNA barcoding to improve bat carcass identification at wind farms in the United States. Conserv Genet Resour. 1 mars 2016;8(1):27‑34.

- 60. Chipps AS, Hale AM, Weaver SP, Williams DA. Genetic diversity, population structure, and effective population size in two yellow bat species in south Texas. PeerJ. 2020;8:e10348.

In Page 28, line 414, we changed “six” by “five”.

Attachment

Submitted filename: 2021.08.05. Responses to Reviewers and Editor comments.docx

Click here for additional data file.^{(29.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0261344.r003

Decision Letter 1

Daniel Becker

1 Dec 2021

Genetic identification of bat species for pathogen surveillance across France

PONE-D-21-14577R1

Dear Dr. Picard-Meyer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have responded to almost all the comments, but there is still incomplete information in the results and some rephrasement to do. Please see my comments below:

1) Line 23 : I think “herpes virus” should be “herpesvirus”.

2) Line 82: “pygmaeus and P. nathusii, or to…”: “and” should not be in italics.

3) Line 131: it is still not clear how the faecal pellets were associated to a bat species, as no capture was done. I guess that the pellets were collected under a monospecific bat colony, and that the bat species was determined by inspected hanging individuals in the colony? Also, how fresh were the pellets ? From the day, or probably several days, weeks ? Were they collected directly on the ground, or some plastic sheets were used ? Might be good to add these details.

4) Table 2 “n.d” in footnotes but not seen in the table.

5) Line 195: please correct “…TCCT-3). With each run,…”

6) Line 196: please explain in the text why only serotine bats were amplified with the Dloop.

7) Please delete “P. pipistrellus”, because of a repetition with “this bat species”.

Reviewer #2: Only one edit: Replace reference - Chipps AS, Hale AM, Weaver SP, Williams DA. Genetic diversity, population

structure, and effective population size in two yellow bat species in south Texas. PeerJ. 2020;8:e10348.

WITH

Chipps AS, AM Hale, SP Weaver, and DA Williams. 2020. Genetic approaches are necessary to accurately understand bat‐wind turbine impacts. Diversity 12:236.

**********

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Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0261344.r004

Acceptance letter

Daniel Becker

15 Dec 2021

PONE-D-21-14577R1

Genetic identification of bat species for pathogen surveillance across France

Dear Dr. Picard-Meyer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Raw data analysis: Bat identification from bat wing punches and faecal samples.

(XLSX)

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S1 Raw images

(TIF)

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Click here for additional data file.^{(29.5KB, docx)}

Data Availability Statement

All files are available from the Genbank database (accession number(s) MZ066766 to MZ066790). All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Genetic identification of bat species for pathogen surveillance across France

Youssef Arnaout

Zouheira Djelouadji

Emmanuelle Robardet

Julien Cappelle

Florence Cliquet

Frédéric Touzalin

Giacomo Jimenez

Suzel Hurstel

Christophe Borel

Evelyne Picard-Meyer

Roles

Abstract

Introduction

Materials and methods

Bat specimens

Table 1. Characteristics of the 163 bat carcasses included for genetic identification.

Table 2. Characteristics of the 37 bat carcasses included for optimization.

Table 3. Characteristics of the 31 bat faecal specimens included in the study.

Ethics statement

DNA extraction

PCR: Amplification of the partial cytochrome b gene

PCR: Amplification of partial D-loop

Sequencing and phylogenetic analysis

Table 4. Characteristics of the partial cytochrome b gene reference sequences retrieved from GenBank and other sequences amplified from wing punches and bat guano from this study.

Results

Genetic identification of bat carcasses and bat faeces

Bat carcasses

Table 5. Results of PCR on the partial cytochrome b gene and species misidentification of bat wing punch samples compared with morphological species identification.

Fig 1. Phylogenetic tree of the partial cytochrome b (cyt b) gene of 52 referenced sequences and 25 bat sequences representing 15 autochthonous bat species.

Bat faeces

Table 6. Results of PCR on the partial cytochrome b gene and species misidentification of bat guano samples compared with morphological identification.

Comparison between morphological identification and cyt b PCR analysis

Bat carcasses

Bat faeces

Discussion and conclusion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Camille Lebarbenchon

Roles

Author response to Decision Letter 0

Decision Letter 1

Daniel Becker

Roles

Acceptance letter

Daniel Becker

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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