ArticlePDF Available

Abstract and Figures

The cat flea (Ctenocephalides felis) is the most common parasite of domestic cats and dogs worldwide. Due to the morphological ambiguity of C. felis and a lack of — particularly largescale — phylogenetic data, we do not know whether global C. felis populations are morphologically and genetically conserved, or whether human-mediated migration of domestic cats and dogs has resulted in homogenous global populations. To determine the ancestral origin of the species and to understand the level of global pervasion of the cat flea and related taxa, our study aimed to document the distribution and phylogenetic relationships of Ctenocephalides fleas found on cats and dogs worldwide. We investigated the potential drivers behind the establishment of regional cat flea populations using a global collection of fleas from cats and dogs across six continents. We morphologically and molecularly evaluated six out of the 14 known taxa comprising genus Ctenocephalides, including the four original C. felis subspecies (Ctenocephalides felis felis, Ctenocephalides felis strongylus, Ctenocephalides felis orientis and Ctenocephalides felis damarensis), the cosmopolitan species Ctenocephalides canis and the African species Ctenocephalides connatus. We confirm the ubiquity of the cat flea, representing 85% of all fleas collected (4357/5123). Using a multigene approach combining two mitochondrial (cox1 and cox2) and two nuclear (Histone H3 and EF-1α) gene markers, as well as a cox1 survey of 516 fleas across 56 countries, we demonstrate out-of-Africa origins for the genus Ctenocephalides and high levels of genetic diversity within C. felis. We define four bioclimatically limited C. felis clusters (Temperate, Tropical I, Tropical II and African) using maximum entropy modelling. This study defines the global distribution, African origin and phylogenetic relationships of global Ctenocephalides fleas, whilst resolving the taxonomy of the C. felis subspecies and related taxa. We show that humans have inadvertently precipitated the expansion of C. felis throughout the world, promoting diverse population structure and bioclimatic plasticity. By demonstrating the link between the global cat flea communities and their affinity for specific bioclimatic niches, we reveal the drivers behind the establishment and success of the cat flea as a global parasite.
Ctenocephalides spp. reference material. (A, B) Photographs of Ctenocephalides type specimens available in the Rothschild collection of fleas held at the Natural History Museum, London, UK. Stars represent taxa with new vouchers collected in this study (A), and those for which not material was not available for molecular confirmation (B). Scale bars represent 200 mm. (C) The maximum intra-and minimum inter-specific mtDNA cox1 distances for each morphotype were calculated in three different taxonomic scenarios (a-c) based on previous literature. The first scenario (a) considers only Ctenocephalides felis, and does not separate Ctenocephalides felis felis, Ctenocephalides felis strongylus, Ctenocephalides felis ''transitional" and Ctenocephalides felis damarensis. The second scenario (b) delineates C. f. damarensis as suggested by Ménier and Beaucournu (1998) based on the morphology of the male aedeagus, whilst retaining as a single category C. f. felis, C. f. strongylus and C. felis ''transitional". The final scenario (c) treats each morphotype separately as presented in the original descriptions catalogued in Hopkins and Rothschild (1953). The evolutionary distances were calculated using the Kimura 2 (K2) parameter as recommended for closely related taxa and using cox1 mtDNA sequences. In all graphs, a 3% threshold is enforced, dividing the charts into four separate quadrants, each representative of different species statuses (Hebert et al., 2003): top left: species concordant with current taxonomy; bottom left: species that are synonymous or have recently undergone divergence or hybridization; bottom right: probable species misidentification; top right: probable composite species, candidates for taxonomic split.
… 
Phylogenetic analysis of the fleas in the genus Ctenocephalides inferred from mitochondrial and nuclear DNA markers. The evolutionary history was inferred using the Minimum Evolution (ME), Maximum Likelihood (ML) and Bayesian framework (BF) with either linked or unlinked parameters (A). A multigene alignment was created by concatenation of cox1, cox2, histone H3 and EF-1a corresponding to 24 selected Ctenocephalides specimens and Echidnophaga ambulans ambulans as an outgroup (2428 nucleotides). Substitution models with the lowest Bayesian Information Criterion (BIC) scores were selected using model selection (MEGA 7.0.14). The GTR + G + I model was used for the ML tree. The ME tree was reconstructed using K2 distance matrix. The BF tree was created using either linked GTR + G + I parameters (BF-linked) or unlinked GTR + G + I parameters (BF-linked) across the gene partitions. Branch support is shown, including bootstrap support (%) using 2000 replicates (ME) and 1000 replicates (ML), followed by posterior probability (PP) based on 75,000 trees (BF). Specimens are identified by their voucher identifier followed by species or subspecies/morphotype. At least one representative of each morphotype was selected. Sequence alignment of the amino acid variable sites for each clade and species is shown on the right. Residues identical to top sequence are displayed as dots (.). Question marks (?) denote codons containing variable nucleotides. Trees for individual partitions were reconstructed using ML using substitution models with the lowest BIC (cox1, cox2, Histone H3: T92 + G, EF-1a: K2 + G) with bootstraps from 500 replicates (B). Branch values lower than 50% were discarded. Trees were rooted using Echidnophaga ambulans ambulans (Siphonaptera: Pulicidae) -not shown.
… 
Content may be subject to copyright.
Out-of-Africa, human-mediated dispersal of the common cat flea,
Ctenocephalides felis: The hitchhiker’s guide to world domination
q
Andrea L. Lawrence
a,b,c
, Cameron E. Webb
b,c
, Nicholas J. Clark
d,e
, Ali Halajian
f
, Andrei D. Mihalca
g
,
Jorge Miret
h
, Gianluca D’Amico
g
, Graeme Brown
a
, Bersissa Kumsa
i
, David Modry
´
j,k,l
, Jan Šlapeta
a,
a
Sydney School of Veterinary Science, Faculty of Science, University of Sydney, Sydney, New South Wales 2006, Australia
b
Medical Entomology, NSW Health Pathology, ICPMR, Westmead Hospital, Westmead, New South Wales 2145, Australia
c
Marie Bashir Institute of Infectious Diseases and Biosecurity, University of Sydney, Sydney, New South Wales 2006, Australia
d
School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
e
Environmental Futures Research Institute, Griffith University, Gold Coast, Queensland, Australia
f
Department of Biodiversity (Zoology), University of Limpopo, Sovenga 0727, South Africa
g
Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania
h
Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Campus UNA, San Lorenzo, Paraguay
i
Department of Parasitology, Faculty of Veterinary Medicine, Addis Ababa University Debre Zeit, Ethiopia
j
Department of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
k
Biology Centre, Institute of Parasitology, Czech Academy of Sciences, C
ˇeské Bude
ˇjovice, Czech Republic
l
CEITEC-VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
article info
Article history:
Received 15 October 2018
Received in revised form 28 January 2019
Accepted 31 January 2019
Available online 9 March 2019
Keywords:
Siphonaptera
Phylogeography
Dog
Cat
Niche modelling
Ctenocephalides felis
DNA barcoding
abstract
The cat flea (Ctenocephalides felis) is the most common parasite of domestic cats and dogs worldwide. Due
to the morphological ambiguity of C. felis and a lack of — particularly largescale — phylogenetic data, we
do not know whether global C. felis populations are morphologically and genetically conserved, or
whether human-mediated migration of domestic cats and dogs has resulted in homogenous global pop-
ulations. To determine the ancestral origin of the species and to understand the level of global pervasion
of the cat flea and related taxa, our study aimed to document the distribution and phylogenetic relation-
ships of Ctenocephalides fleas found on cats and dogs worldwide. We investigated the potential drivers
behind the establishment of regional cat flea populations using a global collection of fleas from cats
and dogs across six continents. We morphologically and molecularly evaluated six out of the 14 known
taxa comprising genus Ctenocephalides, including the four original C. felis subspecies (Ctenocephalides felis
felis,Ctenocephalides felis strongylus, Ctenocephalides felis orientis and Ctenocephalides felis damarensis), the
cosmopolitan species Ctenocephalides canis and the African species Ctenocephalides connatus. We confirm
the ubiquity of the cat flea, representing 85% of all fleas collected (4357/5123). Using a multigene
approach combining two mitochondrial (cox1 and cox2) and two nuclear (Histone H3 and EF-1
a
) gene
markers, as well as a cox1 survey of 516 fleas across 56 countries, we demonstrate out-of-Africa origins
for the genus Ctenocephalides and high levels of genetic diversity within C. felis. We define four bioclimat-
ically limited C. felis clusters (Temperate, Tropical I, Tropical II and African) using maximum entropy
modelling. This study defines the global distribution, African origin and phylogenetic relationships of glo-
bal Ctenocephalides fleas, whilst resolving the taxonomy of the C. felis subspecies and related taxa. We
show that humans have inadvertently precipitated the expansion of C. felis throughout the world, pro-
moting diverse population structure and bioclimatic plasticity. By demonstrating the link between the
global cat flea communities and their affinity for specific bioclimatic niches, we reveal the drivers behind
the establishment and success of the cat flea as a global parasite.
Ó2019 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an
open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Parasites are an evolutionary success story that represent
around half of all species on Earth (Windsor, 1998; Weinstein
and Kuris, 2016). Domesticated cats and dogs provide a means of
dispersal for the parasites that infest them, including the cat flea
https://doi.org/10.1016/j.ijpara.2019.01.001
0020-7519/Ó2019 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
q
Note: Nucleotide sequence data reported in this paper are available in the
GenBank under accession numbers MG586243-MG586782 and the Barcode of Life
Data (BOLD) Systems under accession numbers: CTENO001-18 to CTENO551-18
and ALFLE001-14 to ALFLE024-14.
Corresponding author.
E-mail address: jan.slapeta@sydney.edu.au (J. Šlapeta).
International Journal for Parasitology 49 (2019) 321–336
Contents lists available at ScienceDirect
International Journal for Parasitology
journal homepage: www.elsevier.com/locate/ijpara
Ctenocephalides felis (Bouché, 1835) and its close relative, the dog
flea, Ctenocephalides canis (Curtis, 1826). The cat flea is assumed
to be the most globally pervasive flea species on Earth (Clark
et al., 2018). Its affinity for domestic cat and dog hosts and its con-
sequently synanthropic life history has permitted widespread geo-
graphical dispersal of the species (Hopkins and Rothschild, 1953;
Rust, 2017).
An African emergence of the cat flea is assumed a priori, but the
origin of the species has not been empirically investigated (Hopkins
and Rothschild, 1953; Rust and Dryden, 1997). The theory assumes
co-evolution of the cat flea and its dominant host, the domestic cat,
Felis silvestris catus Linnaeus, 1758, which likely originated in Africa
and the Near East fromancestral African wildcats,Felis silvestris lybica
Forster, 1780 (Driscoll et al., 2007; Hu et al., 2014).
The cat flea species historically includes four geographically
defined subspecies: the cosmopolitan Ctenocephalides felis felis
(Bouché, 1835), an Asian subspeciesCtenocephalides felis orientis (Jor-
dan, 1925) and two subspecies restricted to the African continent:
Ctenocephalides felis strongylus (Jordan, 1925) and Ctenocephalides
felis damarensis Jordan, 1936. Since their originaldescription, C. f. ori-
entis and C. f. damarensis have been morphologically reclassified as
full species (C. orientis and C. damarensis, respectively), but the
genetic identity of the C. felis subspecies remains elusive (De
Meillon et al., 1961; Louw and Horak, 1995; Beaucournu and
Menier, 1998; Ménier and Beaucournu, 1998). Due to morphological
ambiguity and the paucity of available genetic data for taxa in the
genus Ctenocephalides Stiles and Collins, 1930, it remains unknown
whether worldwide C. felis populations are genetically homogenous
(Beaucournu and Menier, 1998; Lawrence et al., 2014, 2015a).
Isolation of C. felis populations around the world was probably
confounded by historical human-mediated migration of cats and
dogs (Koch et al., 2016). The rapid mutation rates of mtDNA com-
pared with nDNA enables analysis of recent divergences within
and between species and can give insights into flea dispersal pat-
terns that may reveal signatures of human migration (Avise
et al., 1987; Avise, 2009). This data can also be used to reveal spe-
cies origins as ancestral populations exhibit higher genetic diver-
sity values compared with populations that have recently
expanded into novel territory (Savolainen et al., 2002; Ma et al.,
2012). The implementation of a combined morphological and
molecular taxonomic approach to define global cat flea populations
and elucidate their origins is imperative given the claims suggest-
ing temperature-dependent biological differences between sub-
species (Yao et al., 2006).
We aimed to discover the ancestral origin of the ubiquitous cat
flea, as a consequence of human-mediated dispersal of domesti-
cated cats and dogs. To achieve this, we collected and analysed
the most common Ctenocephalides flea species and subspecies
infesting cats and dogs across six continents and 56 countries. Glo-
bal cat flea communities were used to clarify the taxonomic status
of the four original continental C. felis subspecies. We assessed the
efficiency of the cytochrome c oxidase I (cox1) mtDNA barcoding
marker together with cytochrome c oxidase II (cox2) and two
nuclear markers, histone H3 and elongation factor 1 alpha (EF-
1
a
) for delineating Ctenocephalides taxa and for inferring the global
population structure of C. felis. We show how thermal zones have
shaped the distribution of the cat flea, in order to explain the wide-
spread and climatically diverse distribution of the species.
2. Materials and methods
2.1. Specimen collection, morphological taxonomy and isolation of
total DNA
Fleas were obtained from 57 countries, primarily from cat and
dog hosts (Supplementary Table S1). All fleas were collected oppor-
tunistically and donated for this study by colleagues listed in Sup-
plementary Table S2. All fleas were stored in 70% (v/v) ethanol,
transported to the University of Sydney, Australia and identified
to species level using a dissection microscope following morpho-
logical keys and descriptions (Hopkins and Rothschild, 1953;
Dunnet and Mardon, 1974; Segerman, 1995; Beaucournu and
Menier, 1998). The only flea from Canada was Pulex irritans Lin-
naeus, 1758; the remaining 56 countries included specimens of
the genus Ctenocephalides.
From the initial set of Ctenocephalides spp. specimens (n= 4771
from 56 countries), a subset of fleas (n= 572) underwent DNA iso-
lation (Supplementary Table S1). Some Ctenocephalides spp. speci-
mens (n= 154) were previously characterised by us and were
included in this study (Lawrence et al., 2014, 2015a, 2015b; Hii
et al., 2015; Chandra et al., 2017; Šlapeta and Šlapeta, 2016;
Šlapeta et al., 2018). In total, 430 new Ctenocephalides spp. speci-
mens were analysed molecularly. DNA was extracted while pre-
serving the exoskeleton as previously described (Lawrence et al.,
2014). The voucher exoskeletons were clarified in KOH and dehy-
drated in an ethanol series and slide-mounted in Euparal (Aus-
tralian Entomological Supplies, Australia) as previously described
(Lawrence et al., 2014). Species confirmation and subspecies iden-
tification were performed on the mounted fleas using keys and
descriptions (Hopkins and Rothschild, 1953; Segerman, 1995;
Beaucournu and Menier, 1998; Ménier and Beaucournu, 1998).
2.2. Amplification of mitochondrial cytochrome c oxidase subunit I,
cytochrome c oxidase subunit II and nuclear histone H3 and elongation
factor 1 alpha
A subset (n= 24/572) including representative specimens from
all collected taxa (n= 6 taxa; C. f. felis, C. f. strongylus, C. orientis,
C. damarensis, C. canis, and C. connatus) were selected for PCR
amplification using mtDNA cox1 and cox2, and two nDNA markers
histone H3 and EF-1
a
. In addition, Echidnophaga ambulans ambu-
lans Olliff, 1886 (Siphonaptera: Pulicidae) was used as an outgroup.
A 601 bp fragment of cox1 was amplified using LCO1490 (5
0
- GGT
CAA CAA ATC ATA AAG ATA TTG G-3
0
) and HCO2198 (5
0
-TAA ACT
TCA GGG TGA CCA AAA AAT CA-3
0
)(Folmer et al., 1994) and/or
Cff-F [S0367] (5
0
-AGA ATT AGG TCA ACC AGG A-3
0
) and Cff-R
[S0368] (5
0
-GAA GGG TCA AAG AAT GAT GT-3
0
)(Lawrence et al.,
2014). A 727 bp fragment of cox2 was amplified using F-Leu (5
0
-
TCT AAT ATG GCA GAT TAG TGC-3
0
) and R-Lys (5
0
-GAG ACC AGT
ACT TGC TTT CAG TCA TC-3
0
)(Whiting, 2002). A 349 bp fragment
of histone H3 was amplified using Hex-AF (5
0
- ATG GCT CGT ACC
AAG CAG ACG GC -3
0
) and Hex-AR (5
0
- ATA TCC TTG GGC ATG
ATG GTG AC 3
0
)(Zhu et al., 2015). A 933 bp of EF-1
a
was ampli-
fied with newly designed primers (Primer3 4.0.0): EF-1
a
_flea_F
(5
0
-AAT TGA AGG CCG AAC GTG AG-3
0
) and EF-1
a
_flea_R (5
0
-GAT
TTG CCA GTA CGA CGG TC-3
0
).
For all PCRs, reaction volumes of either 25
l
Lor30
l
L
contained MyTaq Red Mix (Bioline, Australia) and approximately
1–10 ng of genomic DNA template (2
l
L). All PCRs were run on a
Veriti thermocycler (Life Sciences, Australia) or on a Mastercycler
Personal (Eppendorf, Australia) with a negative control of PCR-
grade water and a positive control of flea DNA known to amplify
at conditions from previous studies (Šlapeta et al., 2011;
Lawrence et al., 2014). The cycling conditions for cox1, EF-1
a
and
histone H3 amplification started with 95 °C for 60 s followed by
35 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 10 s, and
extended for five min at 72 °C; and for cox2 started with 95 °C
for three min followed by 37 cycles of 94 °C for 30 s, 42 °C for
30 s, 72 °C for 15 s, and extended for five min at 72 °C. Products
of expected sizes, verified using agarose gel electrophoresis, were
bidirectionally sequenced using amplification primers (Macrogen
Ltd, Seoul, Korea). All raw sequences were assembled and
322 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
chromatographs visually inspected for ambiguities using CLC Main
Workbench 6.9.1 (CLC bio, Denmark).
2.3. Sequence and haplotype analysis, phylogenetic diversity and
ancestral state analyses
A multigene alignment was created by concatenation of cox1,
cox2, histone H3 and EF-1
a
corresponding to 24 selected Cteno-
cephalides specimens and E. a. ambulans as an outgroup (2428
nucleotides). Using a partitioned alignment, the phylogeny was
reconstructed in a Bayesian framework (BF) with either linked or
unlinked parameters for General Time Reversible +
c
substitution
+ invariant sites model (GTR + G + I) across gene partitions using
MrBayes v3.2.6 (Huelsenbeck and Ronquist, 2001). Two runs with
four chains of 20 million generations were performed, sampled
every 200 generations and with a burn-in period set for the first
25% of trees, resulting in 75,000 total trees in the posterior distri-
bution used to calculate posterior probability (PP). Chain mixing
and convergence were visualised using the program TRACER v1.6
(Drummond and Rambaut, 2007). Nucleotide substitution models
were tested using Maximum Likelihood (ML), and the GTR + G + I
was selected because it had the lowest Bayesian Information Crite-
rion (BIC) scores for the tree reconstructed using the concatenated
alignment. For topological comparisons, ML and Kimura-2 (K2) dis-
tance Minimum Evolution (ME) trees with bootstrap support (1000
replicates) were also reconstructed, using MEGA 7.0.14 (Kumar
et al., 2016). As a further comparison, trees for individual partitions
were reconstructed using ML in MEGA7, with the best model
selected using BIC scores (cox1, cox2, Histone H3: T92 + G, EF-1
a
:
K2 + G) with bootstraps from 500 replicates.
A total of 584 cox1 sequences from Ctenocephalides spp. were
included in an alignment with E. a. ambulans as an outgroup. Spec-
imens where a DNA sequence was available, but a voucher
exoskeleton was not, were excluded from further analysis. Phy-
logeny was reconstructed in BF with GTR + G + I in MrBayes from
two runs with four chains of 20 million generations, sampled every
500 generations and with a burn-in period set for the first 25% of
trees, resulting in 40,000 total trees in the posterior distribution.
As above, an ME tree was reconstructed from a K2 distance matrix
with bootstrap (2,000 replicates) in MEGA7 for topological com-
parisons. The Cff-F/Cff-R amplified cox1 region (513 bp) was used
for calculating haplotype and diversity metrics on a dataset
reduced to 516 sequences due to overrepresentation of Australia
and New Zealand (Supplementary Table S1). Within and between
taxon (or lineage) distances were calculated using K2 distance
matrix. The cox1 alignment was processed using DNAcollapser in
FaBox (Villesen, 2007). The number of haplotypes (Nh), haplotype
diversity (h) and nucleotide diversity (
p
) were calculated using
DnaSP v5.10.01 (Librado and Rozas, 2009). To test whether genetic
variation between groups conferred equal fitness, selective neu-
trality tests (Tajima’s Dand Fu’s Fs) were also conducted in DnaSP.
We used ancestral state reconstruction of cox1 sequences to
estimate the probability that the most recent common ancestor
of all sampled fleas originated in each of the sampled continents.
Using an alignment of unique cox1 sequences, the phylogeny was
reconstructed in a BF with a Yule speciation prior with a
Tamura-Nei +
c
substitution model in BEAST v1.8.1 (Drummond
and Rambaut, 2007). Two chains of 50 million generations were
run, sampled every 25,000 generations and with a burn-in period
of 15 million (resulting in 2,800 total trees in the posterior distri-
bution). Chain mixing and convergence were visualised using TRA-
CER v1.5 (Drummond and Rambaut, 2007). From the posterior
distribution of 2800 trees, we calculated the core ancestor cost
(CAC) to estimate the node position of each biogeographical com-
munity’s most recent common ancestor (Tsirogiannis, C., Sandel,
B., 2015. PhyloMeasures: fast and exact algorithms for computing
phylogenetic biodiversity measures. R package version 1.1.). Larger
CAC values indicate a community contains comparatively ‘older’
lineages of fleas. Next, for each biogeographical community we cal-
culated the net relatedness index (NRI), a standardised phyloge-
netic diversity index that will be lower (i.e. more negative) in
communities that are more phylogenetically diverse. To account
for influences of phylogenetic uncertainty on diversity measures,
the CAC and NRI indices were calculated across the full distribution
of Bayesian posterior trees.
2.4. Ecological niche modelling for global Ctenocephalides populations
To identify whether ecological factors play a role in the distribu-
tion of globally ubiquitous flea species (C. felis and C. canis) and C.
felis clusters, we employed the maximum entropy method in Max-
ent v3.4.1 (Phillips, S.J., Dudík, M., Schapire, R.E., 2017. Maxent
software for modeling species niches and distributions (Version
3.4.1). Available at: http://biodiversityinformatics.amnh.org/
open_source/maxent/. Accessed on: 18/07/2017). Maxent is a
machine learning modelling approach effective in predicting spe-
cies’ distributions from incomplete ‘presence-only’ data (Phillips
et al., 2006; Phillips et al., 2017). We used the geographical coordi-
nates of each collection site for all Ctenocephalides specimens col-
lected. We used two datasets, the first dataset comprised of C.
felis and C. canis (n= 483 sequences) and a second dataset com-
prised of C. felis only (n= 387 sequences). Models were run using
default parameters appropriate to large-scale presence-only data-
sets for qualitative exploratory analyses (Phillips et al., 2006;
Merow et al., 2013). Predictions of habitat suitability were
obtained using a global bioclimatic envelope containing 19 gridded
climatic variables (Supplementary Table S3) downloaded from the
WorldClim Version2 (http://worldclim.org/version2) at a grid cell
resolution of 2.5 min or approximately 5 km
2
(Supplementary
Table S3)(Fick and Hijmans, 2017). The bioclimatic data were con-
verted into ESRI ASCII Raster format using QGIS (QGIS Develop-
ment Team, 2017, QGIS Geographic Information System. Open
Source Geospatial Foundation Project. https://www.qgis.org/).
Variables relating to humidity and ambient temperature are likely
to be limiting due to their importance for Ctenocephalides flea sur-
vival and reproduction (Silverman et al., 1981). To evaluate
whether the models performed significantly better than random,
25% of each sample set was randomly selected as test data to create
Pvalues (significance at 0.05) relevant in ascertaining the reliabil-
ity of the model. The area under curve (AUC) value for the test data
on the ROC plots was used to assess the performance of each model
(Fielding and Bell, 1997). The relative importance of each biocli-
matic variable to the model prediction was assessed using contri-
bution percentages and a jackknife test on training, test and AUC
data.
2.5. Data accessibility
Images of representative specimens for each species, morpho-
type, sex and clade (https://doi.org/10.17632/tjfr3ddc52.1) and a
voucher specimen list (https://doi.org/10.17632/2f3hchym9v.1)
were deposited in Mendeley Data and associated analytical data
are available at LabArchives (http://dx.doi.org/10.25833/2dmy-
0655). Voucher specimens were deposited in the CSIRO Australian
National Insect Collection (ANIC) in Canberra, Australia. All
sequences were deposited in GenBank: cox1: MG586243-
MG586672 (n= 430), cox2: MG586673-MG586732 (n= 60), his-
tone H3: MG586758-MG586782 (n= 25), EF-1
a
: MG586733-
MG586757 (n= 25); Supplementary Table S1. Sequences were also
deposited in Barcode of Life Data (BOLD) Systems: CTENO001-18 to
CTENO551-18, ALFLE001-14 to ALFLE024-14.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 323
3. Results
3.1. Identification of Ctenocephalides fleas from six continents
A total of 4771 Ctenocephalides fleas were collected and identi-
fied across 56 countries (Table 1,Supplementary Table S1): five
countries in Oceania (n= 1446), 16 countries in Europe
(n= 1215), 17 countries in Asia (n= 805), 10 countries in Africa
(n= 571), four countries in South America (n= 412) and four coun-
tries in North America (n= 322). An average of 85 Ctenocephalides
fleas were collected from each country, ranging between 1 (Costa
Rica) and 996 (Australia) (median = 24). Six out of the 14 known
taxa for Ctenocephalides were identified with the aid of vouchers
for 11 out of the 14 Ctenocephalides taxa at the Natural History
Museum, London, including five holotypes and five lectotypes
(Table 1,Fig. 1). Morphological identification utilised the curvature
of the cephalic profile (Fig. 2) in combination with chaetotaxic fea-
tures (Fig. 3), followed by further morphological discernment of
sex-specific features (Fig. 4). A flowchart diagram was created
detailing the identification process of Ctenocephalides fleas (Sup-
plementary Fig. S1).
Fleas in the genus Ctenocephalides accounted for 93.1% of all
fleas collected (4771/5123; Table 1,Supplementary Table S1). Only
C. felis and C. canis were found across all six continents.The most
common flea was C. felis (85.0% of total fleas collected,
4353/5123; 91.2% of total Ctenocephalides collected, 4353/4771)
inclusive of two subspecies: C. f. felis (80.8%, 3856/4771) and C. f.
strongylus (0.7%, 32/4771). This group included 29 fleas (29/4771)
from Africa that could not be classified as either C. f. felis or C. f.
strongylus due to intermediate cephalic morphology visible upon
slide-mounting and were therefore designated as C. f. ‘‘transi-
tional”. The second most common was C. canis (4.4%, 208/4771)
followed by C. orientis (4.1%, 194/4771), C. connatus (0.3%,
12/4771) and C. damarensis (0.1%, 4/4771). A total of 436 C. felis
were not processed for slide-mounting and thus were not classified
to subspecies level.
3.2. Multigene analysis confirms taxonomic classification of
Ctenocephalides spp.
A concatenated cox1,cox2, histone H3 and EF-1
a
nucleotide (nt)
alignment (2428 nt, 808 amino acide (aa)) was used to resolve
Ctenocephalides phylogeny when rooted with E. a. ambulans
(Fig. 5). In all analyses (BF, ML, ME), C. orientis,C. canis and C. con-
natus were monophyletic, together forming a sister group to C. felis
(Fig. 5A). Two specimens, Ctenocephalides sp. ‘‘AL909” (morphology
corresponding to C. f. felis – AL909-1 and C. felis ‘‘transitional”
AL909-2) formed a unique highly supported group (C), sister to C.
felis (dashed line, Fig. 5A). Within C. felis (excluding Ctenocephalides
sp. ‘‘AL909”) we identified two lineages (A, B) supported by high
ME and ML bootstrap (>90%), and high PP (>90%) for A and low
PP (>55%) for B (bolded lines, Fig. 5). Within C. felis group A, four
strongly supported (>97%) clades were resolved. No clades were
consistently resolved in group B using either tree reconstruction
methods. When model parameters were unlinked in BF across
the concatenated partitions it provided strong PP (>90%) support
Table 1
Summary of species within the genus Ctenocephalides including taxonomic information, numbers of fleas analysed herein and current taxonomic assignments.
Taxa Authority Year Primary host Type
locality
NHM
type
identifier
No#
collected in
this study
No# cox1
sequenced
No#
multigene
sequenced
Current
taxonomic
assignment
C. felis felis Bouché 1835 Cats and dogs Germany 916140 3854 + 2
e
415 + 2
e
9+2
e
C. f. felis
C. felis strongylus Jordan 1925 Cats and dogs Kenya 916159 32 + 29
f
32 + 29
f
2+3
f
C. f. strongylus
C. felis damarensis Jordan 1936 Scrub hares (Lepus saxatilis) and
their predators: dogs and wild
canids (jackals, foxes), cats
Namibia 916156 4
g
4
g
1
g
C. damarensis
C. felis orientis Jordan 1925 Dogs Sri Lanka 916161 194 28 2 C. orientis
C. canis Curtis 1826 Dogs and wild canids (jackals,
foxes, wolves)
British Isles 916154 208 58 2 C. canis
C. connatus Jordan 1925 Cape ground squirrels
(Xerus inauris) and mongooses
(Herpestidae)
South
Africa
916144 12 9 3 C. connatus
C. rosmarus Rothschild 1907 Dassie/Hyrax (Procaviidae) Ethiopia 916151 0 0 0 C. rosmarus
C. craterus Jordan and
Rothschild
1913 Dassie/Hyrax (Procaviidae) Kenya 916146 0 0 0 C. craterus
C. arabicus Jordan 1925 Dassie/Hyrax (Procaviidae) Yemen 916148 0 0 0 C. arabicus
C. crataepus Jordan 1925 Ground squirrels (Xerus spp.)
and hedgehogs (Atelerix spp.)
Kenya 916142 0 0 0 C. crataepus
C. paradoxuri Wagner 1936 Civets (Paradoxurus spp.) and
mongooses (Herpestes vitticollis)
Sri Lanka 916152 0 0 0 C. paradoxuri
C. brygooi
a
Beaucournu 1975 Malagasy civet (Fossa fossana) Madagascar NA 0 0 0 C. brygooi
C. chabaudi
b
Beaucournu
and Bain
1982 Unknown
b
Gabon NA 0 0 0 C. chabaudi
C. grenieri
c
Beaucournu
and Rodhaln
1995 Dassie/rock hyrax (Procavia
capensis)
Cameroon NA 0 0 0 C. grenieri
Total 4335 + 436
d
577 + 7
h
24
NHM, Natural History Museum, London, UK.
a
Known only from type material – 7 females, 15 males.
b
Known only by a small number of specimens collected on the bay duiker (Cephalophus dorsalis) (type host) and the Gambian pouched rat (Cricetomys gambianus).
c
Known only from 4 specimens including type material.
d
An additional 436 C. felis were not processed for slide-mounting and were not classified to subspecies level.
e
Two specimens from Jordan (AL909-1, AL909-2) were identified as C. f. felis and C. f. ‘‘transitional” but later designated Ctenocephalides sp. ‘‘AL909” based on high genetic
divergence.
f
Total of 29 specimens from Africa had intermediate morphology between C. f. felis and C. f. strongylus and were designated C. f. ‘‘transitional”.
g
All specimens are male – female specimens are identical to C. f. felis and both species live sympatrically in South Africa, so any C. f. damarensis females are identified as C. f.
felis.
h
C. felis cox1 sequences for whose morphological voucher was not available (see Supplementary Table S1).
324 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
for all Ctenocephalides spp. as well as Ctenocephalides sp. ‘‘AL909”
and C. felis group A, but it did not resolve within-group relation-
ships (Fig. 5A). Individually, cox1 was the most diverse marker
resolving phylogeny closely resembling the concatenated tree,
similarly the cox2 tree had high bootstrap support (ML, 95%) for
the C. felis group A (Fig. 5B).The nuclear genes resolved monophyly
(ML, >90%) of C. orientis,C. canis,C. connatus and C. felis (including
Ctenocephalides sp. ‘‘AL909”) (Fig. 5B).
At the amino acid level, cox2 contained the highest amino acid
diversity with 11/228 unique variable sites, followed by the
nuclear gene EF-1
a
with 7/264 unique sites and cox1 with 2/200
unique sites (Fig. 5A). There were no variable amino acid residues
at histone H3 marker (116 aa). The sequences of C. canis, C. orientis,
C. connatus and Ctenocephalides sp. ‘‘AL909” resolved unique amino
acid substitutions, but C. damarensis was not resolved monophylet-
ically by any gene marker and did not exhibit a unique amino acid
profile (Fig. 5A). Therefore, we revert the taxon to subspecies level,
C. f. damarensis, and use this nomenclature henceforth. There was
no discernible amino acid signal between morphologically identi-
fied C. f. felis,C. f. strongylus, C. f. damarensis and C. f. ‘‘transitional”.
The two identified C. felis sister groups (A, B), as well as all clades in
group A were resolved using amino acid residues at cox2 (Fig. 5A).
Fig. 1. Ctenocephalides spp. reference material. (A, B) Photographs of Ctenocephalides type specimens available in the Rothschild collection of fleas held at the Natural History
Museum, London, UK. Stars represent taxa with new vouchers collected in this study (A), and those for which not material was not available for molecular confirmation (B).
Scale bars represent 200 mm. (C) The maximum intra- and minimum inter-specific mtDNA cox1 distances for each morphotype were calculated in three different taxonomic
scenarios (a-c) based on previous literature. The first scenario (a) considers only Ctenocephalides felis, and does not separate Ctenocephalides felis felis, Ctenocephalides felis
strongylus, Ctenocephalides felis ‘‘transitional” and Ctenocephalides felis damarensis. The second scenario (b) delineates C. f. damarensis as suggested by Ménier and Beaucournu
(1998) based on the morphology of the male aedeagus, whilst retaining as a single category C. f. felis,C. f. strongylus and C. felis ‘‘transitional”. The final scenario (c) treats each
morphotype separately as presented in the original descriptions catalogued in Hopkins and Rothschild (1953). The evolutionary distances were calculated using the Kimura 2
(K2) parameter as recommended for closely related taxa and using cox1 mtDNA sequences. In all graphs, a 3% threshold is enforced, dividing the charts into four separate
quadrants, each representative of different species statuses (Hebert et al., 2003): top left: species concordant with current taxonomy; bottom left: species that are
synonymous or have recently undergone divergence or hybridization; bottom right: probable species misidentification; top right: probable composite species, candidates for
taxonomic split.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 325
Fig. 2. Cephalic profile of six Ctenocephalides taxa and two morphotypes. (A) Fleas are labelled with species, subspecies or morphotype, specimen ID and sex. Female
specimens of Ctenocephalides damarensis are indistinguishable from Ctenocephalides felis felis females and are therefore not represented in this figure. The Ctenocephalides sp.
‘‘AL909” fleas found in Jordan were morphologically identical to C. f. felis and no males were collected. Identification was based on genetic divergence alone. Degree of frons
curvature is denoted by a curved stoppered line. Variation in frons curvature is demonstrated by the two species with the most disparate profiles: C. f. felis, almost straight and
Ctenocephalides canis, heavily rounded. (B) Magnified images of the post-ocular area including the occiput (a) and post-antennal fossa (b) of four species. Black arrows denote
the number of setae on the occiput of C. f. felis (two setae), C. orientis (two setae), C. canis (three setae) and C. connatus (one seta). White arrows denote the presence of micro-
setae behind the antennal fossa of C. orientis females only. All scales are 100 mm.
326 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
3.3. Incongruence between morphology and genetic signature for C.
felis subspecies
The cox1 gene region provided the highest nucleotide diversity
(Fig. 5B) and thus cox1 sequences were PCR amplified from 430
morphologically identified Ctenocephalides spp. vouchers (Supple-
mentary Table S1). Phylogenetic analysis of the cox1 DNA align-
ment demonstrated monophyly for C. felis as a species group
with both high PP (100%) and bootstrap (98%) support (Fig. 6).
However, the morphologically identified C. felis subspecies (i.e. C.
f. felis,C. f. strongylus,C. f. damarensis) and the C. f. ‘‘transitional”
morphotype were not conserved monophyletically by any gene
marker (Fig. 5). Ctenocephalides sp. ‘‘AL909” formed a strongly sup-
ported monophyletic sister lineage (C) to C. felis (Fig. 6) (PP: 100%,
bootstrap: 96%). In addition, cox1 sequences of C. canis,C. orientis
and C. connatus were unambiguously resolved into monophyletic
groups (PP: 100%, bootstrap: >99%). Within C. felis,cox1 sequences
were not clustered according to their morphological traits, i.e. sub-
species identity. Both sister lineages A and B within C. felis included
specimens morphologically classified as C. f. felis,C. f. strongylus,C.
f. damarensis and the C. f. ‘‘transitional” morphotype. The phyloge-
netic trees demonstrated the existence of eight clades within C.
felis (Clades 1–8), however the PP or bootstrap support was low
for most clades (Fig. 5B and Fig. 6).
To investigate whether cox1 correctly identifies Ctenocephalides
spp. and subspecies, the maximum intra- and minimum interspeci-
fic cox1 distances (%) were calculated with morphologically con-
firmed Ctenocephalides spp. and subspecies (Fig. 1) The calculated
distances were plotted on a grid split into four quadrants using
an arbitrary 3% cox1 barcode distance threshold (Hebert et al.,
2003)(Fig. 1C). The top left quadrant was occupied by C. canis, C.
orientis and C. connatus, implying support of taxonomic species sta-
tus by cox1 (Fig. 1Ca-c). The top right quadrant denoting probable
composite species was occupied by C. felis; subspecies were
lumped together (Fig. 1Ca). To assess support for C. f. damarensis,
the specimens were treated individually whilst the remaining C.
felis subspecies were grouped (Fig. 1Cb). Separation of C. f.
damarensis resulted in <3% inter- and intraspecific distance denot-
ing recent species divergence, hybridization or synonymy. Regard-
less of whether C. f. felis, C. f. strongylus and C. f. ‘‘transitional” were
grouped together or treated individually, the intraspecific distance
was high whilst the interspecific distance was effectively zero,
demonstrating that the cox1 signal does not correspond to mor-
phological traits (Fig. 1Cb-c).
3.4. High genetic diversity of C. felis fleas indicates out-of-Africa
origins
Out of a total of 90 unique cox1 haplotypes, 55 belonged to C.
felis specimens (Fig. 7A). Across the Ctenocephalides spp. sampled,
C. felis exhibited the highest genetic diversity (Table 2). The largest
number of cox1 haplotypes for C. felis was found in Africa (n= 24),
followed by Asia and Oceania (Table 2). Three C. felis cox1 haplo-
types (h1, h2 and h17) represented 46% of the total sequences with
h1 (C. felis) being the most common and widespread haplotype
(n= 145, 28% of total sequences) (Fig. 7B). Haplotypes h1, h2 and
h17 were distributed globally, but with continental predominance
to Europe, Asia and the Americas, respectively (Fig. 7B). A total of
54 cox1 haplotypes (10% of total sequences) were singletons. Phy-
logeny of cox1 haplotypes in general agreed with Fig. 6, except for
Clade 5 that was paraphyletic (Fig. 7).
A number of morphologically defined C. felis subspecies shared
a single cox1 haplotype in the African region (h6, h17, h21, h22,
h27). Morphological inconsistencies across the C. felis haplotype
primarily consisted of variations in the cephalic profile for both
sexes and in the aedeagal morphology (Ménier and Beaucournu,
1998) and tarsal chaetotaxy in male fleas (Beaucournu and
Menier, 1998)(Table 3 and Table 4). Moreover, a single haplotype
h6 was found in the Central African Republic, Seychelles and
Georgia USA representing C. f. strongylus,C. f. ‘‘transitional” and
C. f. felis, respectively (Fig. 7). The subspecies C. f. felis and C. f.
strongylus exhibit a tubus interior that may be toothed in four
out of five specimens (Table 3). Our data show that the rate is
much higher with the tubus interior toothed in half of the spec-
imens (Table 4).
Africa displayed the highest genetic diversity and the highest
probability for being the geographical origin of the ancestral root
of Ctenocephalides spp. at 20.1% compared with 15.016.8% for
all other continents (Table 2,Supplementary Fig. S2). The African
C. felis community had the highest CAC values (95% Confidence
Interval (CI): 61.44, 70.35; indices for all other continents were
<14.78) and most negative NRI values (95% CI: 9.36, 7.56;
indices for all other continents were >6.96), denoting a more
Fig. 3. Morphology of hind tibia (A) and lateral metanotal area (LMA) (B) for
Ctenocephalides felis, Ctenocephalides canis, Ctenocephalides orientis and Cteno-
cephalides sp. ‘‘AL909”. Fleas are labelled with species or subspecies and specimen
ID. (A) Arrows denote the number of setae bearing notches present on the
dorsoposterior margin of the hind tibia of Ctenocephalides felis felis (six), C. canis
(eight), C. orientis (seven), and Ctenocephalides sp. ‘‘AL909” (six). (B) Arrows denote
the number of setae on the LMA with C. f. felis, C. orientis and Ctenocephalides sp.
‘‘AL909” all bearing two setae and C. canis bearing three. All specimens are female.
All scales are 100 mm.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 327
ancient and phylogenetically diverse flea community compared
with other continents. Within C. felis, a large portion of fleas show
a more recent European ancestral line. Similarly, C. canis and C. ori-
entis show recent European and Asian ancestry, respectively (Sup-
plementary Fig. S2). There was no significant difference between
‘‘ancient” (Africa, Europe, Oceania and Asia) and ‘‘recently
colonised” (North and South America) regions (P> 0.1) using an
analysis of molecular variance (AMOVA) in an attempt to deter-
mine if patterns of genetic diversity reflected patterns of phyloge-
netic diversity.
Using mtDNA cox1 sequences we calculated neutrality values
to evaluate population structure and determine whether
Ctenocephalides populations are evolving non-randomly (Table 2).
Only C. canis had statistically significant negative neutrality values,
Fig. 4. Sex-specific morphology of Ctenocephalides fleas. (A) Plantar surface of fore tarsi V of male fleas. Number of spiniform bristles on the plantar surface is denoted by black
arrows: two for Ctenocephalides felis felis, six for Ctenocephalides damarensis and Ctenocephalides connatus. (B) Ventral sternites of female fleas. Number of sub-ventral setae on
sternites (3 – 6) are indicated with black arrows for C. f. felis (two), Ctenocephalides sp. ‘‘AL909” (two) and C. connatus (three). (C) Manubrium (m) of male fleas. Ctenocephalides
f. felis and C. damarensis possess a manubrium with a constricted apex, whilst for C. canis and C. orientis the apex of the manubrium is dilated, denoted by the stoppered line.
Fleas are labelled with species or subspecies and specimen ID. Scale for (A) is 50 mm and scale for (B) and (C) is 100mm.
328 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
suggesting recent population expansion (Fig. 7A). Within C. felis,
fleas from North and South America had statistically significant
population structure with positive neutrality values due to the
presence of a small number of distinct haplotypes and a low num-
ber of singletons, suggesting multiple introductions and sustained
isolation (Fig. 7A).
Fig. 5. Phylogenetic analysis of the fleas in the genus Ctenocephalides inferred from mitochondrial and nuclear DNA markers. The evolutionary history was inferred using the
Minimum Evolution (ME), Maximum Likelihood (ML) and Bayesian framework (BF) with either linked or unlinked parameters (A). A multigene alignment was created by
concatenation of cox1, cox2, histone H3 and EF-1
a
corresponding to 24 selected Ctenocephalides specimens and Echidnophaga ambulans ambulans as an outgroup (2428
nucleotides). Substitution models with the lowest Bayesian Information Criterion (BIC) scores were selected using model selection (MEGA 7.0.14). The GTR + G + I model was
used for the ML tree. The ME tree was reconstructed using K2 distance matrix. The BF tree was created using either linked GTR + G + I parameters (BF-linked) or unlinked GTR
+ G +I parameters (BF-linked) across the gene partitions. Branch support is shown, including bootstrap support (%) using 2000 replicates (ME) and 1000 replicates (ML),
followed by posterior probability (PP) based on 75,000 trees (BF). Specimens are identified by their voucher identifier followed by species or subspecies/morphotype. At least
one representative of each morphotype was selected. Sequence alignment of the amino acid variable sites for each clade and species is shown on the right. Residues identical
to top sequence are displayed as dots (.). Question marks (?) denote codons containing variable nucleotides. Trees for individual partitions were reconstructed using ML using
substitution models with the lowest BIC (cox1, cox2, Histone H3: T92 + G, EF-1
a
: K2 + G) with bootstraps from 500 replicates (B). Branch values lower than 50% were
discarded. Trees were rooted using Echidnophaga ambulans ambulans (Siphonaptera: Pulicidae) – not shown.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 329
3.5. Niche modelling on a global scale reveals ecologically discrete C.
felis clusters and temperate distribution of C. canis
Discrete geographical clusters were observed when the coordi-
nates of C. felis collection sites were plotted on a world map and
associated with their cox1 Clades 1–8 (Fig. 6); arbitrarily labelled:
‘Temperate’ (Clade 1 and Clade 2), ‘Tropical I’ (Clade 3 and Clade 4),
‘Tropical II’ (Clade 5 and Clade 6) and ‘African’ (Clade 7 and Clade 8)
clusters (Fig. 6 and Fig. 8). The distribution pattern of C. canis was
climatically restricted to the temperate zone in both the northern
Fig. 6. Phylogenetic reconstruction of Ctenocephalides spp. at cox1. Sequences from fleas (n= 520) from 56 countries across six continents were rooted with Echidnophaga
ambulans ambulans sequences (n= 4, not shown). The evolutionary history was inferred using Minimum Evolution (ME) with distances computed using the Kimura 2 (K2)
method and Bayesian framework (BF) with GTR + G + I parameters. Branch support is shown, including bootstrap support (%) using 2000 replicates (ME) followed by posterior
probability (PP) based on 40,000 trees (BF). Branch values lower than 50% were discarded. Clade identity is denoted by colour with collection sites for each sequenced flea
plotted on a world map. Maps are divided by lineage for C. felis or per individual species.
330 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
(not found lower than 27°N) and southern hemispheres (no fur-
ther north than 31°S) (Fig. 6). The species C. connatus was restricted
to South Africa. The oriental dog flea (C. orientis) was restricted to
Asia and located throughout the tropical and northern subtropical
zones (Fig. 6).
We tested the probability of the geographical clusters being
constrained by bioclimatic factors (Fig. 8). Models predicting local-
ities for C. felis and C. canis as well as C. felis (Temperate, Tropical I
and Tropical II) displayed high predictive power (AUC > 0.9). All
distribution models predicted test localities significantly better
than random (P< 0.001). The C. felis model displayed a highly cos-
mopolitan distribution whilst the model for C. canis demonstrated
a temperately constricted distribution with virtually no suitable
habitats highlighted along the equatorial belt, except at high alti-
tudes such as the Himalayas (Fig. 8B). A Jackknife test showed that
the C. felis Temperate cluster and C. canis shared the same biocli-
matic predictors (Supplementary Fig. 3A, E). Variables that related
to cold temperatures such as minimum temperature of the coldest
Fig. 7. Phylogenetic relationships and geographic distribution of haplotypes across Ctenocephalides morphologies. (A) Phylogenetic reconstruction of all unique cox1
haplotypes recovered in the study, inferred using Minimum Evolution (ME) and the K2 distance matrix. Bootstrap support values from 1000 replicates are shown at the
branches, values lower than 50% were discarded. The terminal node is labelled with the haplotype ID, followed by number of sequences found for each haplotype. The
radiating circles show the continent (colour-coded) and the number of sequences found per continent. The three most abundant haplotypes are shaded grey. For
Ctenocephalides felis, subspecies or morphotypes that deviate from the nominal subspecies Ctenocephalides felis felis are annotated. Species lineages are denoted with colour-
coded branches. (B) Phylogenetic relationships and amino acid translations for the most common haplotypes of each C. felis cox1 clade except Clade 8 (only two fleas, different
haplotypes). Two methods were used to infer evolutionary history: ME with K2 distance model and Maximum Likelihood (ML) with the T92 +
c
model. Bootstrap values
(1000 replicates) for both runs are shown and those lower than 50% were discarded. Amino acid translations for the mtDNA sequences show unique sites differentiating C.
felis from all other species. (C) Morphology was not conserved across identical haplotypes. For example, haplotype h6 contains fleas identified as C. f. felis and Ctenocephalides
felis strongylus, respectively. These subspecies are differentiated by the degree of curvature of the frons, as indicated by the stoppered line. The scales represent 100 mm. (D)
The three most globally abundant haplotypes (h1, h2 and h17) are shown with the proportion of members found in each continent.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 331
month (BIO06) and mean temperature of the coldest quarter
(BIO11) contributed the most information to the model and were
the best predictors of distribution when used in isolation from all
other variables (Supplementary Fig. S3A, E). Conversely, variables
relating to precipitation (annual precipitation – BIO12 and precip-
itation of the warmest quarter – BIO18) were the best predictors of
habitat distribution for the two Tropical clusters of C. felis (Supple-
mentary Fig. S3B-C). The most significant contributors to the C. felis
species distribution model were a mixture of factors relating to
cold temperature such as mean temperature of the coldest quarter
(BIO11) and precipitation, such as precipitation in the coldest quar-
ter (BIO19) (Supplementary Fig. S3D).
4. Discussion
We demonstrate ‘out-of-Africa’ origins of C. felis and elucidate
its taxonomy using a global collection of Ctenocephalides spp. from
56 countries. Our data provide empirical evidence of the ubiquity
of the cat flea on a global scale, where cat and dog domestication
and subsequent human-mediated migration has led to a world-
wide distribution of C. felis. We challenge the dogma that C. felis
specimens collected in regional studies from different countries
around the world are morphologically, phylogenetically and bio-
logically identical.
We demonstrate that C. felis originated in Africa because (i) the
continent holds the highest C. felis genetic diversity and (ii) C. felis
from Africa possess the highest CAC values, denoting a more
ancient population compared with other continents. Our analysis
demonstrates an African common ancestor for all Ctenocephalides
taxa collected in our study, therefore aligning with the proposed
African origin of the Pulicidae (Traub, 1985; Medvedev, 1998).
The evolution of pulicid fleas, including C. felis, likely occurred
simultaneously with their carnivorous hosts, thereby providing
the means for early dispersal (Zhu et al., 2015). The African wildcat
(F. s. lybica) had a distribution range that extended throughout
Africa and the Near East, supporting our hypothesis of an African
origin of C. felis (Driscoll et al., 2007). After Africa, the most ancient
flea communities were found in Asia, Europe, then Oceania while
the Americas held the most recently evolved cat flea populations.
These flea populations correspond to the origins of domestication
and the translocation of cats and dogs into new geographical loca-
tions by humans (Milham and Thompson, 1976; Savolainen et al.,
2004; Oskarsson et al., 2011).
The earliest evidence of cat domestication was discovered in
China and the group of now extinct ancestral wolves that gave rise
to the gray wolf (Canis lupus) and the domestic dog (Canis lupus
familiaris) originated in Europe (Thalmann et al., 2013; Hu et al.,
2014). Dog domestication is thought to have occurred over two
independent events, one in western Eurasia and another in eastern
Eurasia approximately 15,000 years ago; they were the first
domesticated animal to undergo human-mediated dispersal to all
continents (Oskarsson et al., 2011; Larson et al., 2012; Frantz
et al., 2016). These early domestication events are reflected in
the older, genetically diverse flea populations of Asia and Europe.
Table 2
Summary of genetic diversity and demographic statistics for Ctenocephalides spp., Ctenocephalides felis clades and continental populations of C. felis.
Genetic diversity statistics Tests of selective neutrality
nHnSh(SD)
p
(SD) Tajima’s DStat. sig. Fu and Li’s D
a
Stat. sig. Fu and Li’s F
a
Stat. sig.
By species
C. felis 424 55 49 0.846 (0.014) 0.01562 (3.9 10
-5
) 0.23 No 0.51 No 0.20 No
C. canis 54 20 25 0.630 (0.078) 0.00222 (4.4 10
4
)2.56 Yes
c
4.59 Yes
b
4.60 Yes
b
C. orientis 28 9 12 0.783 (0.065) 0.00351 (7.1 10
4
)1.38 No 1.75 No 1.91 No
C. connatus 9 5 7 0.722 (0.159) 0.00509 (1.4 10
3
) 0.06 No 0.05 No 0.03 No
Ctenocephalides sp.‘‘AL909” 2 1 0 NA NA NA NA NA NA NA NA
By clade
Clade 1 179 17 15 0.342 (0.046) 0.00087 (1.4 10
4
)2.13 Yes
a
2.63 Yes
a
2.93 Yes
a
Clade 2 19 3 3 0.292 (0.127) 0.00098 (4.6 10
4
)1.13 No 0.11 No 0.44 No
Clade 3 64 6 5 0.207 (0.067) 0.00042 (1.4 10
4
)1.84 Yes
a
2.93 Yes
a
3.03 Yes
a
Clade 4 35 4 3 0.166 (0.084) 0.00033 (1.7 10
4
)1.73 No 2.79 Yes
a
2.88 Yes
a
Clade 5 8 2 3 0.250 (0.180) 0.00146 (1.1 10
3
)1.45 No 1.57 No 1.69 No
Clade 6 82 10 12 0.780 (0.036) 0.00371 (2.8 10
4
)0.58 No 1.54 No 1.43 No
Clade 7 35 11 13 0.825 (0.05) 0.00629 (3.8 10
4
) 0.07 No 0.81 No 0.62 No
Clade 8 2 2 0 NA NA NA NA NA NA NA NA
By continent
Africa 91 24 36 0.918 (0.012) 0.01800 (5.3 10
4
) 0.95 No 0.05 No 0.48 No
Asia 85 14 25 0.793 (0.033) 0.01224 (8.6 10
4
) 0.79 No 0.38 No 0.64 No
Europe 104 9 13 0.51 (0.055) 0.00379 (5.7 10
4
)0.59 No 0.31 No 0.02 No
North America 48 4 21 0.689 (0.036) 0.01703 (8.9 10
4
) 2.73 Yes
b
1.70 Yes
b
2.44 Yes
b
Oceania 66 13 24 0.789 (0.026) 0.01331 (8.7 10
4
) 1.11 No 0.66 No 0.02 No
South America 30 4 16 0.524 (0.074) 0.01281 (1.7 10
3
) 2.13 Yes
a
0.81 No 1.44 No
n, number of sequences = number of fleas.
Hn, number of haplotypes.
S, number of variable sites.
h(SD), haplotype diversity plus/minus S.D.
p
(SD), nucleotide diversity plus/minus S.D.
a
P< 0.05.
b
P< 0.02.
c
P< 0.001.
Table 3
Distinguishing features of male Ctenocephalides felis subspecies and Ctenocephalides
felis damarensis as per previous literature.
Species Number of SPB
a
on fore tarsi
Phallosome structures
TI
b
toothed Hamulus length:
width
C. f. felis/strongylus 2 4 out of 5 are
toothed
2.5longer than
wide
C. f. damarensis 4–6 Small tooth approximately equal
a
Spiniform plantar bristles.
b
Tubus interior.
332 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
Given the close phylogenetic relationship of C. canis and C. orientis,
and their dual affinity for canine hosts, the European and Asian dis-
tribution and ancestry of C. canis and C. orientis, respectively, could
be a reflection of these domestication events. Evidence suggests
that humans began migrating out of Africa as early as
120,000 years ago but did not reach the Americas until
15,000 years ago, possibly explaining the more recently evolved
community of cat fleas in the Americas, characterised by low
genetic diversity (Goebel et al., 2008; Stringer, 2011, 2016;
Groucutt et al., 2015).
Maritime exploration and trade from the 15th to the 19th cen-
tury allowed the dispersal of cats and probably dogs, providing an
Table 4
Table of conflicting diagnostic features of all male Ctenocephalides felis and Ctenocephalides felis damarensis fleas collected from Africa.
Specimen information Number of SPB
a
on fore tarsi Phallosome structures
Morphotype Clade ID Haplotype ID Specimen ID Country TI
b
toothed? Hamulus ratio length:width
C. f. felis Clade 1 h1 AL796-4 Algeria 2 No 1.6
C. f. felis Clade 1 h1 AL796-5 Algeria 2 No 1.5
C. f. felis Clade 1 h1 AL798-1 Algeria 2 No 1.6
C. f. felis Clade 1 h1 AL913-1 Algeria 2 No 2.3
C. f. felis Clade 1 h1 AL917-1 Algeria 2 Yes 1.8
C. f. felis Clade 7 h27 AL300-1 South Africa 2 Yes 2.2
C. f. ‘‘transitional” Clade 3 h2 AL340-1 Kenya 2 Yes 1.3
C. f. ‘‘transitional” Clade 3 h2 AL341-1 Kenya 2 No 1.8
C. f. ‘‘transitional” Clade 7 h21 AL336-4 Kenya 2 Yes 2.3
C. f. ‘‘transitional” Clade 7 h21 AL820-3 Zimbabwe 2 Yes 2.3
C. f. strongylus Clade 5 h22 AL222-1 Guinea Bissau 2 No 1.8
C. f. strongylus Clade 5 h22 AL229-1 Guinea Bissau 2 No 2.7
C. f. strongylus Clade 4 h6 AL725-1 CAR
c
2 Yes 2.0
C. f. strongylus Clade 4 h6 AL728-1 CAR
c
2 Yes 2.0
C. f. strongylus Clade 6 h19 AL1113-2 Liberia 2 Yes 1.4
C. f. strongylus Clade 6 h19 AL1113-3 Liberia 2 Yes 1.8
C. f. strongylus Clade 6 h19 AL1113-5 Liberia NA
d
Yes 1.8
C. f. strongylus Clade 6 h17 AL1113-10 Liberia 2 Yes 2.0
C. f. strongylus Clade 7 h73 AL937-1 South Africa 2 No 1.8
C. f. strongylus Clade 7 h80 AL965-1 Kenya 2 Yes 1.3
C. f. damarensis Clade 7 h27 AL946-2 South Africa 5 Yes 1.1
C. f. damarensis Clade 7 h77 AL957-4 South Africa 6 Yes 1.3
C. f. damarensis Clade 7 h27 AL957-5 South Africa 6 Yes 1.7
C. f. damarensis Clade 7 h27 AL957-6 South Africa 5 Yes 1.6
a
Spiniform plantar bristles.
b
Tubus interior.
c
Central African Republic.
d
Unable to determine due to specimen damage.
Fig. 8. Predicted global geographic distribution of suitable habitat zones for Ctenocephalides felis and Ctenocephalides canis. Three subgroups of C. felis (see Fig. 6) distributions
were modelled – Temperate, Tropical I and Tropical II. The prediction is based on Ctenocephalides flea collection sites and 19 variables forming a bioclimatic envelope. A colour
scale is used to show the predicted probability of habitat suitability with red indicating high probability that the bioclimatic conditions in the area are suitable for the species
or lineage, green indicating conditions that are similar to those where the specimens are found and light blue indicating low probability of suitable conditions. White markers
represent the presence of a single flea.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 333
opportunity for rapid flea dispersal and admixture between conti-
nental populations (Koch et al., 2015, 2016). The near-boundless
global distributions of the genetically homogeneous C. felis Clade
1 and of C. canis could be a reflection of European exploration
and colonisation. Our data show that C. canis was not evolving neu-
trally and negative selective neutrality values suggest rapid recent
global expansion. This mirrored the population structure of Clade 1
of C. felis, a similarly temperately distributed population. Fleas (C.
felis) belonging to Clade 1 are the most globally ubiquitous and
these fleas are now dominant in Australia and New Zealand
(Šlapeta et al., 2011; Chandra et al., 2017). This genetic homogene-
ity within globally distributed populations likely denotes rapid
expansion and consequent founder effects in each area as a result
of modern human movement and globalisation (Avise, 2009).
Africa is the only continent that holds all eight clades of C. felis
and recent human-mediated dispersal of C. felis could, in part,
explain the genetic homogeneity found among continental popula-
tions that are geographically separated. This includes the C. felis in
Clade 4 (haplotype h6, 32/35 fleas) from the Central African Repub-
lic, the Seychelles and Georgia, USA. Both the Seychelles and south-
ern USA were involved in the transatlantic slave trade from the
16th to the 19th century with human cargo being sourced from
West Africa, including the region now called the Central African
Republic; although the Seychelles was more heavily influenced
by traders coming from eastern Africa (Avery, 2017; Lovejoy,
2011). These historic trade routes affected the dispersal of domes-
tic animals and their parasites throughout the world and likely
resulted in the introduction of flea populations into novel environ-
ments from Africa.
Similar to all ectoparasites with ‘off-host’ life stages, Cteno-
cephalides flea growth and reproduction is highly dependent upon
environmental conditions and climatic factors such as air temper-
ature and precipitation or humidity (Silverman et al., 1981; Dryden
and Rust, 1994; Silverman and Rust, 1983; Dryden and Rust, 1994).
The results of our analysis showed that, as a species, C. felis demon-
strates high levels of ecological plasticity with a broad spectrum of
suitable bioclimatic regions highlighted across multiple climatic
zones. Conversely, no C. canis were found between the two Tropics
and the species displayed strict temperate habitat distribution.
Analysis of the relative importance of environmental variables
used in a species distribution model allows insight into which
environmental factors represent the most significant drivers for
species distribution (Booth et al., 2014). Our results showed that
variables relating to cooler temperatures were best for predicting
the distribution of C. canis fleas. This corroborates laboratory bioas-
says that reported narrower temperature ranges for optimal
growth in C. canis (Baker and Elharam, 1992). Cat fleas (C. f.
strongylus) from equatorial Africa including the Ivory Coast, Guinea
Bissau and Liberia, fell within the Tropical I cluster and showed
ecological predilection for warmer, more humid habitats. This
aligns with experimental data demonstrating that the subspecies
C. f. strongylus completes its lifecycle slower at temperatures lower
than 27 °C when compared with C. f. felis; the optimal temperature
for development being 29 °C compared with 27 °C for C. f. felis (Yao
et al., 2006). Our work supports in vitro biological observations,
despite the dataset possibly being subject to spatial bias due to
our opportunistic sampling methods (Lobo Jorge et al., 2007;
Fourcade et al., 2014). The relative contribution of bioclimatic val-
ues, however, is known to be relatively heuristic due to the nature
of the algorithm and should be interpreted with care when envi-
ronmental variables are highly correlated (Phillips et al, 2006;
Phillips et al, 2017).
Our results confirm that the cat flea, C. felis, is the most common
flea infesting domestic cats and dogs around the world. The taxo-
nomic classification of the global subspecies has not been system-
atically addressed using molecular data. Using a multigene
approach coupled with comprehensive morphological analysis,
we resolve the taxonomic ambiguity of C. felis as a species and
demonstrate the presence of three subspecies: the cosmopolitan
C. f. felis, recovered from every continent, and two subspecies
restricted to the African region: C. f. strongylus and C. f. damarensis.
The subspecies C. f. damarensis was raised to full species level
during the 1990 s based on morphological investigations and his-
torical observations of host preferences (De Meillon et al., 1961;
Louw and Horak, 1995; Ménier and Beaucournu, 1998). We show
little to no genetic distance between C. f. damarensis morphotypes
and other C. felis morphotypes from Africa.Similarly, despite the
morphological differences between C. f. felis and C. f. strongylus
morphotypes, many of these specimens shared the same or very
similar genetic identity. Previous studies have attempted to syn-
onymise C. f. strongylus with C. f. felis due to difficulties in differen-
tiating the two in Africa, where the subspecies reside sympatrically
(Ménier and Beaucournu, 1998; Vobis et al., 2004). Our work
demonstrates that despite the presence of discernible morpholog-
ical differences between C. f. felis, C. f. strongylus and C. f. damaren-
sis, the subspecies lack genetic monophyly.
Most taxomomists agree that the definition of a subspecies dic-
tates that the group of taxa must be recognisable by at least one
feature or set of features, and that the taxa are geographically
defined in some way (Patten, 2015; Wallin et al., 2017). Unlike in
full species, reproductive isolation and reciprocal monophyly is
not a prerequisite for the definition of a subspecies (Mayr, 1942;
Patten, 2015). As such, when geographical barriers separating the
subspecies are overcome, hybridisation can occur, leading to
genetic homogeneity (Mayr, 1942; Patten, 2015). Due to the cos-
mopolitan distribution of the nominated subspecies C. f. felis,
including distribution throughout Africa, hybridisation between
this subspecies and both African subspecies is highly probable, par-
ticularly since they can share the same hosts. The presence of ‘in-
termediate’ specimens in Africa classified here as C. f. ‘‘transitional”
are possibly evidence of existing hybridisation between C. f. felis
and C. f. strongylus.
The mitochondrial gene markers provided the highest phyloge-
netic resolution compared with the nuclear genes for Cteno-
cephalides fleas. Since the nuclear genome evolves at a slower
rate compared with the mitogenome, the diversity seen in cox1
and cox2 may represent more recent divergences and thus utilisa-
tion of all four genes reveals the most comprehensive phylogenetic
picture (Avise, 2009). Using the multigene approach, C. connatus, C.
canis and C. orientis retained monophyly regardless of the gene
marker, supporting full species status for these taxa. The species
C. orientis was observed to have a strong canine host preference
and a close phylogenetic relationship with C. canis. The species is
also known to infest small ruminants such as goats and sheep
throughout Asia, supporting the retention of C. orientis as a full spe-
cies (Ashwini et al., 2017). The fact that there are very few histor-
ical cases of C. orientis collected from cats further corroborates this
theory (Hopkins and Rothschild, 1953; Beaucournu and Menier,
1998). Collection and genetic profiling of the remaining species
in genus Ctenocephalides, particularly those in Africa, is pertinent
and pressing in order to resolve the taxonomy and phylogeny of
the genus as a whole. This remains a monumental task, given that
many of these species are very rare with uncertain host affinities
(Beaucournu and Menier, 1998).
In summary, we confirm the full species status of C. orientis —
originally described as an Asian subspecies of C. felis — which
together with robust morphological separation and discerning host
preferences, formed a discrete monophyletic unit. Despite the tax-
onomic challenge presented by the intermediate specimens in
Africa, the C. felis subspecies nonetheless exhibit conserved identi-
fiable features; therefore, we reinstate and confirm the subspecies
status of the following within C. felis: cosmopolitan C. f. felis,
334 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
C. f. strongylus in Africa and Arabian Peninsula, and C. f. damarensis
in South Africa.
With a global collection of Ctenocephalides spp., we demon-
strated African origins of the genus and subsequent human-
mediated dispersal of C. felis as a result of cat and dog domestica-
tion. Through synanthropic host hitchhiking, the cat flea has suc-
cessfully achieved global dominance. The success of C. felis can
be attributed to high phylogenetic diversity and discrete climatic
clusters, allowing ecological plasticity of the species as a whole.
Using an integrated morphological and molecular approach, we
taxonomically resolved C. felis, demonstrating the presence of
three subspecies: cosmopolitan C. f. felis and two African sub-
species C. f. strongylus and C. f. damarensis, whilst confirming the
full species status of C. canis,C. orientis and C. connatus. Our study
demonstrates the ancestral origin of the most common flea on
Earth and exposes the drivers behind its global dispersal, thereby
defining the factors associated with this parasite’s success.
Acknowledgements
We are grateful to the many colleagues, friends and enthusiastic
members of the public who collected and donated the flea samples
for this study. Supplementary Table S2 contains the names and
contributions of every person, without whom this study would
not exist. We thank Theresa Howard (National History Museum,
London, UK) for access and guidance to the museum specimens
in the Rothschild Collection of Fleas. ALL was supported by the
Australian Postgraduate Award and the University of Sydney, Aus-
tralia, alumni scholarship. ALL was a recipient of the travel grants
from the Australian Society for Parasitology, the University of Syd-
ney and the Australian Biological Resources Study (ABRS) National
Taxonomy Research Grant Programme (NTRGP) to visit the Natural
History Museum, London. The study was supported by the Faculty
of Veterinary Science Intramural Collaboration Fellowship with
The Marie Bashir Institute for Infectious Diseases and Biosecurity
(MBI), University of Sydney. Aside from this, this research did not
receive any specific grant from funding agencies in the public,
commercial or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ijpara.2019.01.001. These data include
Google maps of the most important areas described in this article.
References
Ashwini, M.S., Puttalakshmamma, G.C., Mamatha, G.S., Chandranaik, B.M.,
Thimmareddy, P.M., Placid, E., Jalali, S.K., Venkatshan, T., 2017. Studies on
morphology and molecular characterization of oriental cat flea infesting small
ruminants by barcoding. J. Entomol. Zool. Stud. 5, 301–305.
Avery, S., 2017. Seychelles Political History and Governance. Lulu Press Inc,
Morrisville, NC.
Avise, J.C., 2009. Phylogeography: retrospect and prospect. J. Biogeogr. 36, 3–15.
Avise, J.C., Arnold, J., Ball, R.M., Bermingham, E., Lamb, T., Neigel, J.E., Reeb, C.A.,
Saunders, N.C., 1987. Intraspecific phylogeography: the mitochondrial DNA
bridge between population genetics and systematics. Annu. Rev. Ecol. Syst. 18,
489–522.
Baker, K.P., Elharam, S., 1992. The biology of Ctenocephalides canis in Ireland. Vet.
Parasitol. 45, 141–146.
Beaucournu, J.C., Menier, K., 1998. Le genre Ctenocephalides Stiles et Collins, 1930
(Siphonaptera, Pulicidae). Parasite 5, 3–16.
Booth, T.H., Nix, H.A., Busby, J.R., Hutchinson, M.F., 2014. bioclim: the first species
distribution modelling package, its early applications and relevance to most
current MaxEnt studies. Divers. Distrib. 20, 1–9.
Chandra, S., Forsyth, M., Lawrence, A.L., Emery, D., Šlapeta, J., 2017. Cat fleas
(Ctenocephalides felis) from cats and dogs in New Zealand: molecular
characterisation, presence of Rickettsia felis and Bartonella clarridgeiae and
comparison with Australia. Vet. Parasitol. 234, 25–30.
Clark, N.J., Seddon, J.M., Šlapeta, J., Wells, K., 2018. Parasite spread at the domestic
animal - wildlife interface: anthropogenic habitat use, phylogeny and body
mass drive risk of cat and dog flea (Ctenocephalides spp.) infestation in wild
mammals. Parasit. Vectors 11, 8.
De Meillon, B., Davis, D.H.S., Hardy, F., 1961. The Siphonaptera (excluding
Ischnopsyllidae). Plague in Southern Africa. Govt. Printer. of South Africa,
Pretoria, Republic.
Driscoll, C.A., Menotti-Raymond, M., Roca, A.L., Hupe, K., Johnson, W.E., Geffen, E.,
Harley, E.H., Delibes, M., Pontier, D., Kitchener, A.C., Yamaguchi, N., Brien, S.J.,
Macdonald, D.W., 2007. The Near Eastern origin of cat domestication. Science
317, 519.
Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evol. Biol. 7, 214.
Dryden, M.W., Rust, M.K., 1994. The cat flea: biology, ecology and control. Vet.
Parasitol. 52, 1–19.
Dunnet, G., Mardon, D., 1974. A monograph of Australian fleas (Siphonaptera). Aust.
J. Zool. Suppl. Ser. 22, 1–273.
Fick, S.E., Hijmans, R.J., 2017. WorldClim 2: new 1-km spatial resolution climate
surfaces for global land areas. Int. J. Climatol. 37, 4302–4315.
Fielding, A.H., Bell, J.F., 1997. A review of methods for the assessment of prediction
errors in conservation presence/absence models. Environ. Conserv. 24, 38–49.
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.
Fourcade, Y., Engler, J.O., Rodder, D., Secondi, J., 2014. Mapping species distributions
with MAXENT using a geographically biased sample of presence data: a
performance assessment of methods for correcting sampling bias. PLoS One 9,
e97122.
Frantz, L.A.F., Mullin, V.E., Pionnier-Capitan, M., Lebrasseur, O., Ollivier, M., Perri, A.,
Linderholm, A., Mattiangeli, V., Teasdale, M.D., Dimopoulos, E.A., Tresset, A.,
Duffraisse, M., McCormick, F., Bartosiewicz, L., Gál, E., Nyerges, É.A., Sablin, M.V.,
Bréhard, S., Mashkour, M., Ba
˘la
˘sßescu, A., Gillet, B., Hughes, S., Chassaing, O.,
Hitte, C., Vigne, J.-D., Dobney, K., Hänni, C., Bradley, D.G., Larson, G., 2016.
Genomic and archaeological evidence suggest a dual origin of domestic dogs.
Science 352, 1228.
Goebel, T., Waters, M.R., Rourke, D.H., 2008. The late Pleistocene dispersal of
modern humans in the Americas. Science 319, 1497.
Groucutt, H.S., Petraglia, M.D., Bailey, G., Scerri, E.M.L., Parton, A., Clark-Balzan, L.,
Jennings, R.P., Lewis, L., Blinkhorn, J., Drake, N.A., 2015. Rethinking the dispersal
of Homo sapiens out of Africa. Evol. Anthropol. 24, 149–164.
Hebert, P.D., Cywinska, A., Ball, S.L., deWaard, J.R., 2003. Biological identifications
through DNA barcodes. Proc. Biol. Sci. 270, 313–321.
Hii, S.-F., Lawrence, A.L., Cuttell, L., Tynas, R., Abd Rani, P., Šlapeta, J., Traub, R., 2015.
Evidence for a specific host-endosymbiont relationship between ’Rickettsia sp.
genotype RF2125’ and Ctenocephalides felis orientis infesting dogs in India.
Parasit. Vectors 8, 169.
Hopkins, G.H.E., Rothschild, M., 1953. Volume 1: Tungidae and Pulicidae. An
illustrated catalogue of the Rothschild collection of fleas (Siphonaptera) in the
British Museum (Natural History): with keys and short descriptions for the
identification of families, genera, species and subspecies. The Trustees of the
British Museum, London.
Hu, Y., Hu, S., Wang, W., Wu, X., Marshall, F.B., Chen, X., Hou, L., Wang, C., 2014.
Earliest evidence for commensal processes of cat domestication. Proc. Natl.
Acad. Sci. 111, 116–120.
Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic
trees. Bioinformatics 17, 754–755.
Koch, K., Algar, D., Schwenk, K., 2016. Feral cat globetrotters: genetic traces of
historical human-mediated dispersal. Ecol. Evol. 6, 5321–5332.
Koch, K., Algar, D., Searle, J.B., Pfenninger, M., Schwenk, K., 2015. A voyage to Terra
Australis: human-mediated dispersal of cats. BMC Evol. Biol. 15, 262.
Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.
Larson, G., Karlsson, E.K., Perri, A., Webster, M.T., Ho, S.Y.W., Peters, J., Stahl, P.W.,
Piper, P.J., Lingaas, F., Fredholm, M., 2012. Rethinking dog domestication by
integrating genetics, archeology, and biogeography. Proc. Natl. Acad. Sci. 109,
8878–8883.
Lawrence, A.L., Brown, G.K., Peters, B., Spielman, D.S., Morin-Adeline, V., Šlapeta, J.,
2014. High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two
mitochondrial DNA markers. Med. Vet. Entomol. 28, 330–336.
Lawrence, A.L., Hii, S.-F., Jirsová, D., Panáková, L., Ionica
˘, A.M., Gilchrist, K., Modry
´, D.,
Mihalca, A.D., Webb, C.E., Traub, R.J., Šlapeta, J., 2015a. Integrated morphological
and molecular identification of cat fleas (Ctenocephalides felis) and dog fleas
(Ctenocephalides canis) vectoring Rickettsia felis in central Europe. Vet. Parasitol.
210, 215–223.
Lawrence, A.L., Hii, S.F., Chong, R., Webb, C.E., Traub, R., Brown, G., Šlapeta, J., 2015b.
Evaluation of the bacterial microbiome of two flea species using different DNA-
isolation techniques provides insights into flea host ecology. FEMS Microbiol.
Ecol., 91
Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA
polymorphism data. Bioinformatics 25, 1451–1452.
Lobo Jorge, M., Jiménez-Valverde, A., Real, R., 2007. AUC: a misleading measure of
the performance of predictive distribution models. Global Ecol. Biogeogr. 17,
145–151.
Louw, J.P., Horak, M.L., 1995. Fleas, lice and mites on scrub hares (Lepus saxatilis)in
northern and eastern Transvaal and in KwaZulu-Natal, South Africa.
Onderstepoort. J. Vet. Res. 62, 133–137.
Lovejoy, P.E., 2011. Transformations in Slavery: A History of Slavery in Africa.
Cambridge University Press, Cambridge, UK.
A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336 335
Ma, C., Yang, P., Jiang, F., Chapuis, M.P., Shali, Y., Sword, G.A., Kang, L.E., 2012.
Mitochondrial genomes reveal the global phylogeography and dispersal routes
of the migratory locust. Mol. Ecol. 21, 4344–4358.
Mayr, E., 1942. Systematics and the Origin of Species, from the Viewpoint of a
Zoologist. Harvard University Press, Cambridge, MA.
Medvedev, S.G., 1998. Classification of fleas (order Siphonaptera) and its theoretical
foundations. Entomol. Rev. 78, 1080–1093.
Ménier, K., Beaucournu, J.-C., 1998. Taxonomic study of the genus Ctenocephalides
Stiles & Collins, 1930 (Insecta: Siphonaptera: Pulicidae) by using aedeagus
characters. J. Med. Entomol. 35, 883–890.
Merow, C., Smith, M.J., Silander, J.A., 2013. A practical guide to MaxEnt for modeling
species’ distributions: what it does, and why inputs and settings matter.
Ecography 36, 1058–1069.
Milham, P., Thompson, P., 1976. Relative antiquity of human occupation and extinct
fauna at Madura Cave, southeastern Western Australia. Aust. J. Anthropol. 10,
175–180.
Oskarsson, M.C.R., Klütsch, C.F.C., Boonyaprakob, U., Wilton, A., Tanabe, Y.,
Savolainen, P., 2011. Mitochondrial DNA data indicate an introduction
through mainland Southeast Asia for Australian dingoes and Polynesian
domestic dogs. Proc. R. Soc. Lond. B. Biol. Sci. rspb20111395.
Patten, M.A., 2015. Subspecies and the philosophy of science. The Auk 132, 481–
485.
Phillips, S.J., Anderson, R.P., Dudík, M., Schapire, R.E., Blair, M.E., 2017. Opening the
black box: an open-source release of Maxent. Ecography 40, 887–893.
Phillips, S.J., Anderson, R.P., Schapire, R.E., 2006. Maximum entropy modeling of
species geographic distributions. Ecol. Model. 190, 231–259.
Rust, K.M., 2017. The biology and ecology of cat fleas and advancements in their
pest management: A review. Insects 8.
Rust, M.K., Dryden, M.W., 1997. The biology, ecology, and management of the cat
flea. Annu. Rev. Entomol. 42, 451–473.
Savolainen, P., Leitner, T., Wilton, A.N., Matisoo-Smith, E., Lundeberg, J., 2004. A
detailed picture of the origin of the Australian dingo, obtained from the study of
mitochondrial DNA. Proc. Natl. Acad. Sci. USA 101, 12387–12390.
Savolainen, P., Zhang, Y.-P., Luo, J., Lundeberg, J., Leitner, T., 2002. Genetic evidence
for an East Asian origin of domestic dogs. Science 298, 1610–1613.
Segerman, J., 1995. Siphonaptera of Southern Africa: Handbook for the
Identification of Fleas. South African Institute for Medical Research,
Johannesburg, South Africa.
Silverman, J., Rust, M.K., 1983. Some abiotic factors affecting the survival of the cat
flea, Ctenocephalides felis (Siphonaptera: Pulicidae). Environ. Entomol. 12, 490–
495.
Silverman, J., Rust, M.K., Reierson, D.A., 1981. Influence of temperature and
humidity on survival and development of the cat flea, Ctenocephalides felis
(Siphonaptera: Pulicidae). J. Med. Entomol. 18, 78–83.
Šlapeta, J., King, J., McDonell, D., Malik, R., Homer, D., Hannan, P., Emery, D., 2011.
The cat flea (Ctenocephalides f. felis) is the dominant flea on domestic dogs and
cats in Australian veterinary practices. Vet. Parasitol. 180, 383–388.
Šlapeta, J., Lawrence, A., Reichel, M.P., 2018. Cat fleas (Ctenocephalides felis) carrying
Rickettsia felis and Bartonella species in Hong Kong. Parasitol. Int. 67, 209–212.
Šlapeta, Š., Šlapeta, J., 2016. Molecular identity of cat fleas (Ctenocephalides felis)
from cats in Georgia, USA carrying Bartonella clarridgeiae,Bartonella henselae
and Rickettsia sp. RF2125. Vet. Parasitol. Reg. Stud. Rep. 3, 36–40.
Stringer, C., 2011. The Origin of Our Species. Penguin, UK.
Stringer, C., 2016. Human migration: Climate and the peopling of the world. Nature
538, 49–50.
Thalmann, O., Shapiro, B., Cui, P., Schuenemann, V.J., Sawyer, S.K., Greenfield, D.L.,
Germonpré, M.B., Sablin, M.V., López-Giráldez, F., Domingo-Roura, X., Napierala,
H., Uerpmann, H.P., Loponte, D.M., Acosta, A.A., Giemsch, L., Schmitz, R.W.,
Worthington, B., Buikstra, J.E., Druzhkova, A., Graphodatsky, A.S., Ovodov, N.D.,
Wahlberg, N., Freedman, A.H., Schweizer, R.M., Koepfli, K.P., Leonard, J.A.,
Meyer, M., Krause, J., Pääbo, S., Green, R.E., Wayne, R.K., 2013. Complete
mitochondrial genomes of ancient canids suggest a European origin of domestic
dogs. Science 342, 871.
Traub, R., 1985. Coevolution of Fleas and Mammals. In: Lim, K.C. (Ed.), Coevolution
of Parasitic Arthropods and Mammals. Wiley, New York, pp. 295–437.
Villesen, P., 2007. FaBox: an online toolbox for fasta sequences. Mol. Ecol. Notes 7,
965–968.
Vobis, M., D’Haese, J., Mehlhorn, H., Mencke, N., Blagburn, B.L., Bond, R., Denholm, I.,
Dryden, M.W., Payne, P., Rust, M.K., Schroeder, I., Vaughn, M.B., Bledsoe, D.,
2004. Molecular phylogeny of isolates of Ctenocephalides felis and related
species based on analysis of ITS1, ITS2 and mitochondrial 16S rDNA sequences
and random binding primers. Parasitol. Res. 94, 219–226.
Wallin, H., Kvamme, T., Bergsten, J., 2017. To be or not to be a subspecies:
description of Saperda populnea lapponica ssp. n. (Coleoptera, Cerambycidae)
developing in downy willow (Salix lapponum L.). ZooKeys 691, 103–148.
Weinstein, S.B., Kuris, A.M., 2016. Independent Origins of Parasitism in Animalia.
Biol, Lett, p. 12.
Whiting, M.F., 2002. Mecoptera is paraphyletic: multiple genes and phylogeny of
Mecoptera and Siphonaptera. Zool. Scr. 31, 93–104.
Windsor, D.A., 1998. Controversies in parasitology, most of the species on earth are
parasites. Int. J. Parasitol. 28, 1939–1941.
Yao, K.P., Ngoran, K.E., Franc, M., 2006. Étude de quelques paramètres écologiques
de Ctenocephalides felis strongylus (Jordan, 1925) (Siphonaptera: Pulicidae).
Parasite 13, 159–164.
Zhu, Q., Hastriter, M.W., Whiting, M.F., Dittmar, K., 2015. Fleas (Siphonaptera) are
Cretaceous, and evolved with Theria. Mol. Phylogen. Evol. 90, 129–139.
336 A.L. Lawrence et al. / International Journal for Parasitology 49 (2019) 321–336
... In addition to the relationship between bacterial species carried by vectors, studies examining mosquitos and plant pathogen vectors document the importance of vector genotype on pathogen transmission dynamics [35,36]. Ctenocephalides felis is a highly diverse species with four bioclimatically limited clusters originating from Africa [37]. Most published studies either have not determined the infection status of fleas surveyed solely for phylogenetic diversity or investigated only a small number of fleas from which both genotype and pathogen carriage was established [37][38][39]. ...
... Ctenocephalides felis is a highly diverse species with four bioclimatically limited clusters originating from Africa [37]. Most published studies either have not determined the infection status of fleas surveyed solely for phylogenetic diversity or investigated only a small number of fleas from which both genotype and pathogen carriage was established [37][38][39]. ...
... Analysis of flea phylogenetic sequences required the creation of a neighbor-joining tree. Detected haplotypes were compared to those reported by Lawrence et al. [37]. α-Diversity was analyzed via calculation of net relatedness index (NRI) [51]. ...
Article
Full-text available
Background Ctenocephalides felis, the cat flea, is the most common ectoparasite of cats and dogs worldwide. As a cause of flea allergy dermatitis and a vector for two genera of zoonotic pathogens (Bartonella and Rickettsia spp.), the effect of the C. felis microbiome on pathogen transmission and vector survival is of substantial medical importance to both human and veterinary medicine. The aim of this study was to assay the pathogenic and commensal eubacterial microbial communities of individual C. felis from multiple geographic locations and analyze these findings by location, qPCR pathogen prevalence, and flea genetic diversity. Methods 16S Next Generation Sequencing (NGS) was utilized to sequence the microbiome of fleas collected from free-roaming cats, and the cox1 gene was used for flea phylogenetic analysis. NGS data were analyzed for 168 individual fleas from seven locations within the US and UK. Given inconsistency in the genera historically reported to constitute the C. felis microbiome, we utilized the decontam prevalence method followed by literature review to separate contaminants from true microbiome members. Results NGS identified a single dominant and cosmopolitan amplicon sequence variant (ASV) from Rickettsia and Wolbachia while identifying one dominant Bartonella clarridgeiae and one dominant Bartonella henselae/Bartonella koehlerae ASV. Multiple less common ASVs from these genera were detected within restricted geographical ranges. Co-detection of two or more genera (Bartonella, Rickettsia, and/or Wolbachia) or multiple ASVs from a single genus in a single flea was common. Achromobacter, Peptoniphilus, and Rhodococcus were identified as additional candidate members of the C. felis microbiome on the basis of decontam analysis and literature review. Ctenocephalides felis phylogenetic diversity as assessed by the cox1 gene fell within currently characterized clades while identifying seven novel haplotypes. NGS sensitivity and specificity for Bartonella and Rickettsia spp. DNA detection were compared to targeted qPCR. Conclusions Our findings confirm the widespread coinfection of fleas with multiple bacterial genera and strains, proposing three additional microbiome members. The presence of minor Bartonella, Rickettsia, and Wolbachia ASVs was found to vary by location and flea haplotype. These findings have important implications for flea-borne pathogen transmission and control. Graphical Abstract
... Three species of fleas are known to be the most common ones identified in domestic dogs and cats worldwide, apart from Antarctica, namely Ctenocephalides canis (Curtis, 1826), Ctenocephalides felis (Bouché, 1835) and Ctenocephalides orientis (Jordan, 1925) [8]. In Australia, Central and Southwestern Europe, South and Southeast Asia, the dominant species in dogs is C. felis [8][9][10][11][12][13]. ...
... Three species of fleas are known to be the most common ones identified in domestic dogs and cats worldwide, apart from Antarctica, namely Ctenocephalides canis (Curtis, 1826), Ctenocephalides felis (Bouché, 1835) and Ctenocephalides orientis (Jordan, 1925) [8]. In Australia, Central and Southwestern Europe, South and Southeast Asia, the dominant species in dogs is C. felis [8][9][10][11][12][13]. Interestingly, in dogs of several Eastern Europe countries, a more common infestation by C. canis and even Pulex irritans Linnaeus, 1758, occurs [14]. ...
... Interestingly, in dogs of several Eastern Europe countries, a more common infestation by C. canis and even Pulex irritans Linnaeus, 1758, occurs [14]. The dog flea, C. canis, was dominant also in dogs from South Korea [15], while in India the most common species detected in dogs was C. orientis, a species previously associated with other animal species, such as ruminants [8,16,17]. The differentiation of C. canis from C. orientis requires fine morphological observation or molecular confirmation, and it is likely that past studies from Asia and Southeast Asia may have misidentified C. orientis as C. canis [8,16]. ...
Article
Full-text available
Background The Silk Road connected the East and West for over 1500 years. Countries in Central Asia are valuable in addressing the hypothesis that parasites on domestic animals were introduced along the Silk Road. Adult fleas are obligate parasites, having worldwide distribution. In dogs, Ctenocephalides canis, C. felis and C. orientis are the most common species identified. The distribution of the Oriental cat flea, C. orientis, is restricted to southeast Asia. The purpose of this study was to determine the diversity of dog fleas from Uzbekistan, a country in Central Asia, with particular reference to C. orientis. Methods Fleas were collected from 77 dogs from 5 locations in Uzbekistan. The cox1 gene sequences from Ctenocephalides spp. were compared to global collection of Ctenocephalides cox1 haplotypes. Landmark-based geometric morphometrics have been applied to the head and curvature to compare C. canis and C. canis using canonical variate analysis and discriminant function analysis. Results Overall, 199 fleas were collected and identified as C. canis (n = 115, 58%), C. orientis (n = 53, 27%) and Pulex irritans (n = 22, 11%). None of the fleas were C. felis. All Ctenocephalides spp. fleas were subject to cox1 amplification and 95% (166/175) yielded DNA sequence. There were 25 cox1 haplotypes; 14 (22/25, 88%) were C. canis cox1 haplotypes and 3 (3/25, 12%) were C. orientis cox1 haplotypes. Molecular analysis confirmed the absence of C. felis. Four (4/22) and one (1/3) cox1 haplotypes were identical to cox1 haplotypes belonging to C. canis and C. orientis cox1 haplotypes identified elsewhere, respectively. Overall morphometric analysis confirmed significant differences between the head shape of C. canis and C. orientis and improved four–fivefold the species identification compared to traditional morphological key. Conclusion We report for the first time the presence of C. orientis in Uzbekistan. Differentiation of C. orientis from C. canis and C. felis remains difficult in regions where these species coexist. Studies in Central and Southeast Asia should confirm species identity using cox1 locus to enable retracing of the distribution of the Ctenocephalides in Asia. The presence of C. orientis suggests that this species may have been introduced from the east along the ancient Silk Road. Graphical Abstract
... With the development of genetic technologies, which complement, to some extent, the limitations of traditional morphology, molecular biology methods have been widely used in taxonomy, systematics, and population genetics, including those of fleas [13,14]. The genetic diversity of fleas has also been studied using nuclear markers, such as histone H3, EF-1α, ITS1, and ITS2 [13,[15][16][17]. ...
... With the development of genetic technologies, which complement, to some extent, the limitations of traditional morphology, molecular biology methods have been widely used in taxonomy, systematics, and population genetics, including those of fleas [13,14]. The genetic diversity of fleas has also been studied using nuclear markers, such as histone H3, EF-1α, ITS1, and ITS2 [13,[15][16][17]. Mitochondria caught the attention of evolutionary biologists in the 1960s because of their small size, high abundance in cells, and simple mode of inheritance [18,19]. ...
... The genetic diversity of C. felis has so far been investigated in Africa [13], the USA [26,27], Europe [25], Asia [28,29], and Australia-New Zealand [10,30,31]. In 2020, Azrizal-Wahid et al. used cytochrome c oxidase subunit I (cox1) and II (cox2) to study the genetic lineages of the C. felis population in Malaysia and revealed two main lineages, with Malaysian haplotypes closely related to tropical haplotypes [12]. ...
Article
Full-text available
Background Fleas are the most economically significant blood-feeding ectoparasites worldwide. Ctenocephalides felis and Pulex irritans can parasitize various animals closely related to humans and are of high veterinary significance. Methods In this study, 82 samples were collected from 7 provinces of China. Through studying the nuclear genes ITS1 and EF-1α and two different mitochondrial genes cox1 and cox2, the population genetics and genetic variation of C. felis and P. irritans in China were further investigated. Results The intraspecies differences between C. felis and P. irritans ranged from 0 to 3.9%. The interspecific variance in the EF-1α, cox1, and cox2 sequences was 8.2–18.3%, while the ITS1 sequence was 50.1–52.2%. High genetic diversity was observed in both C. felis and P. irritans, and the nucleotide diversity of cox1 was higher than that of cox2. Moderate gene flow was detected in the C. felis and P. irritans populations. Both species possessed many haplotypes, but the haplotype distribution was uneven. Fu's Fs and Tajima's D tests showed that C. felis and P. irritans experienced a bottleneck effect in Guangxi Zhuang Autonomous Region and Henan province. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found. Conclusions Using sequence comparison and the construction of phylogenetic trees, we found a moderate amount of gene flow in the C. felis and P. irritans populations. Both species possessed many haplotypes, but the distribution of haplotypes varied among the provinces. Fu’s Fs and Tajima’s D tests indicated that both species had experienced a bottleneck effect in Guangxi and Henan provinces. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found. This study will help better understand fleas' population genetics and evolutionary biology. Graphical Abstract
... [3,12], C. felis and C. canis are well-studied fleas through morphological and molecular techniques, which usually allow for the differentiation of both species [13][14][15]. However, variations in morphological characteristics were observed among these fleas, hindering their correct identification, and giving rise to several uncertainties about its taxonomic diversity [16]. The cat flea species historically includes four geographically defined subspecies, but their remarkable morphological ambiguity and the assumption of interbreeding between subspecies make differentiation even more complex if not impossible [5,9,17,18]. ...
... In addition, Lawrence et al. [16] reported that C. felis was most phylogenetically diverse in Africa, with genetic assemblages that do not belong strictly to any subspecies designations. This fact is in accordance with the intermediate size presented by C. felis from South Africa in females (Figure 3), whose factor map overlaps with both C. felis from Spain and C. canis from Iran. ...
Article
Full-text available
Fleas (Siphonaptera) are one of the most important ectoparasites that represent a potential danger for the transmission of pathogens in our environment. The cat flea, Ctenocephalides felis (Bouché,1835), and the dog flea, Ctenocephalides canis (Curtis, 1826) are among the most prevalent and most frequently studied species throughout the world. However, the variations observed in their morphological characteristics complicate their correct identification, especially when there is a lack of access to the equipment and funds required to carry out molecular biology techniques. With the objective to provide an additional tool to help in the differentiation of Ctenocephalides species, a principal component analysis was carried out for the first time in the present work on populations of C. felis and C. canis from countries in three continents, namely Spain (Europe), South Africa (Africa) and Iran (Asia). The factor maps assisted in the differentiation of both species and the detection of differences in overall size, although morphological ambiguity prevented the delimitation in populations of the same species. Thus, morphometrics represents a complementary tool to other traditional and modern techniques, with great potential to assist in the differentiation of fleas, particularly species that have historically been difficult to identify.
... In the second scenario, pulicids colonized jerboas, switching from gerbils (Gerbillidae) that originated in Africa and dispersed to Asia no later than in the Miocene (Wessels 1998). Another example is represented by the cat flea Ctenocephalides felis, which originated in Africa and dispersed worldwide with cat and dog domestication (Lawrence et al. 2019). ...
Article
Full-text available
We applied a step-down factor analysis (SDFA) and multi-site generalised dissimilarity modelling (MS-GDM) to local flea communities harboured by small mammals (i.e., collected at small sampling sites over a short time period) in two South American regions (Patagonia and the Northwestern Argentina) with the aim of understanding whether these communities were assembled via niche-based or dispersal-based processes. The SDFA allows us to determine whether clusters of flea assemblages across different types of climates, vegetation and soils can be distinguished (suggesting niche-based assembly). MS-GDM allows us to determine whether a substantial proportion of the variation in flea species turnover is explained by specific climate-associated, vegetation-associated and soil-associated variables (indicating niche-based assembly) or host turnover (indicating dispersal-based assembly). Mapping of assemblages on climate, vegetation and soil maps, according to their loadings on axis 1 or axis 2 of the SDFA, did not provide clear-cut results. Clusters of similar loadings could be recognized within some, but not other, climate, vegetation and soil types. However, MS-GDM demonstrated that the effect of environmental variables (especially air temperature) on flea compositional turnover was much stronger than that of host turnover, indicating the predominance of niche-based processes in local community assembly. A comparison of our results with those on the mechanisms that drive species assembly in regional communities allows us to conclude that local and regional communities result from the joint action of niche-based and dispersal-based processes, with the former more important at a smaller spatial scale and the latter at a larger spatial scale.
... For that reason, if we want to infer a robust and feasible phylogeny, we should include multiple independent loci combining nuclear and mitochondrial data (Edwards & Bensch, 2009). This fact has been largely proved in flea's field as for genus and species level (Lawrence et al., 2019; as family and higher stratum (Whiting et al., 2008;Zhu et al., 2015). ...
Article
The taxonomic status of Leptopsyllidae family has remained controversial over the years. Thus, some entomologists placed this group of fleas within Ceratophyllidae family, considering it at level of Leptopsyllinae subfamily or even appearing as a paraphyletic group within Siphonaptera phylogeny. This fact is emphasized by the lack of molecular and phylogenetic data of Leptopsyllidae taxa available in public databases. The aim of this study was to carry out a comparative morphological, phylogenetic and molecular study of two species of Leptopsylla genus with zoonotic relevance (Leptopsylla segnis and Leptopsylla taschenbergi) isolated from rodents collected from different geographical areas of Europe in order to molecularly characterize both taxa and to establish their taxonomic and phylogenetic status within Leptopsyllidae family. For this purpose, we have analysed and compared several morphological traits between L. segnis and L. taschenbergi and compared five different molecular markers (ITS2, EF1‐α, cox1, cox2 and cytb) among these both species and others belonging to Leptopsyllidae family. Based on the morphological results, we found a phenotypic plasticity phenomenon in one female specimen showing morphological characters of L. segnis but molecular sequences distinctive for L. taschenbergi. Furthermore, the molecular and phylogenetic analysis could easily discriminate among both species providing, by the first time, a monophyletic origin of Leptopsyllidae family. Lastly, with this work, we demonstrate one more time, the usefulness of the combination of mitochondrial and nuclear markers to solve taxonomic and phylogenetic issues within Siphonaptera field by the use of concatenated dataset.
Preprint
Full-text available
While fleas are often perceived simply as a biting nuisance and cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat ( Ctenocephalides felis ) and dog ( C. canis ) fleas, as well as C. orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp. of the so-called "transitional group". The latter includes R. felis (agent of flea-borne spotted fever) and R. asembonensis , an emerging pathogen. The relatively depauperate flea microbiome can also contain arthropod-specific endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis -associated pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain ( w Cori), and the C. orientis mitochondrion; all obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which ( w CfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades ("supergroups"), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors, and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the US. Analysis of loci under positive selection in the transitional group revealed genes involved in host-pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited largescale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides -associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.
Chapter
Als Arthropoda (= Gliederfüßer) werden Tiere mit heteronomer Segmentierung zusammengefasst. Ihnen ist gemeinsam, dass sie ein mehr oder minder starres Exoskelett aus Chitin (und anderen Elementen) besitzen, das bei Wachstumsprozessen regelmäßig gehäutet werden muss. Die heteronomen Körper- und Beinsegmente sind durch häutige Elemente verbunden und dadurch beweglich.
Article
Seasonality of fleas (Siphonaptera) may be due to species competition, prompting the idea that flea species partition temperature, along with correlated variables such as moisture (thermal-niche partitioning hypothesis). I compared the fleas of five rodent-flea communities described from the literature for thermal-niche optima by fitting non-linear LRF (Lobry-Rosso-Flandrois) curves to examine whether flea species in a community show distinct, partitioned thermal niches. LRF curves estimate physiological parameters of temperature minimum, optimum, maximum, and maximum abundance, and facilitate comparison between species by summarizing seasonal data. Flea-communities were on Nearctic Southern flying squirrel (Glaucomys volans volans), Richardson's ground-squirrel (Urocitellus richardsonii), North American deer-mouse (Peromyscus maniculatus), and Palearctic Midday jird (Meriones meridianus), and Wagner's gerbil (Dipodillus dasyurus). Flea communities appeared to show seasonality consistent with thermal-niche partitioning. Several flea families and genera had characteristic thermal niches: Ceratophyllidae had broad tolerance to extreme temperature, Leptopsyllidae (one species in this study) to cold, and Pulicidae to hot. In contrast, at the local, species level, climatic speciation could be significant in flea diversification. Non-competition hypotheses (environmental filtering, neutrality) require testing, too. Thermal-niche partitioning may increase flea species richness on hosts and could occur in other insect and plant communities. Implications for biodiversity conservation and disease ecology under global warming are wide-ranging.
Article
The cat flea “Ctenocephalides felis” has veterinary and medical importance since it is a vector for numerous important pathogens. In this study, a total of 249 flea samples were collected from goats bred in eight different farms (located in İzmir and Şanlıurfa provinces of Turkey) and morphologically identified under microscopy. Later, the genetic diversity was investigated in 117 of C. felis samples that were morphologically identified by sequencing the mitochondrial cox1 gene, followed by phylogenetic tree, haplotype, genetic differentiation and gene flow analyses. In addition, Rickettsia spp. and Bartonella spp. which are zoonoses were screened in 27 pools comprising 249 flea samples by PCR. The phylogenetic tree showed that 117 flea samples were clustered in Clade 1 together with isolates from Australia, New Zealand, the Czech Republic, and India. Four haplotypes (haplotypes I, II, III and IV) were detected within the C. felis species. The most prevalent haplotype was haplotype I (57/117; 48.7%). Among the population of flea samples in İzmir and Şanlıurfa, the Fst and Nm values were 0.16261 and 2.57, respectively, indicating a moderate genetic differentiation and high gene flow. Rickettsia spp. was detected in four of C. felis pool samples whereas Bartonella spp. was detected in 25 of them. BLAST analysis identified R. raoultii as well as B. henselae and B. elizabethae. In conclusion, the findings showed that C. felis samples collected from goats in Turkey were classified within Clade 1 representing four different haplotypes with a moderate genetic diversity for the first time. Also, R. raoultii, B. henselae and B. elizabethae were demonstrated for the first time in cat flea samples collected in Turkey.
Article
Full-text available
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
Article
Full-text available
[Background] Spillover of parasites at the domestic animal - wildlife interface is a pervasive threat to animal health. Cat and dog fleas (Ctenocephalides felis and C. canis) are among the world’s most invasive and economically important ectoparasites. Although both species are presumed to infest a diversity of host species across the globe, knowledge on their distributions in wildlife is poor. We built a global dataset of wild mammal host associations for cat and dog fleas, and used Bayesian hierarchical models to identify traits that predict wildlife infestation probability. We complemented this by calculating functional-phylogenetic host specificity to assess whether fleas are restricted to hosts with similar evolutionary histories, diet or habitat niches. [Results] Over 130 wildlife species have been found to harbour cat fleas, representing nearly 20% of all mammal species sampled for fleas. Phylogenetic models indicate cat fleas are capable of infesting a broad diversity of wild mammal species through ecological fitting. Those that use anthropogenic habitats are at highest risk. Dog fleas, by contrast, have been recorded in 31 mammal species that are primarily restricted to certain phylogenetic clades, including canids, felids and murids. Both flea species are commonly reported infesting mammals that are feral (free-roaming cats and dogs) or introduced (red foxes, black rats and brown rats), suggesting the breakdown of barriers between wildlife and invasive reservoir species will increase spillover at the domestic animal - wildlife interface. [Conclusions] Our empirical evidence shows that cat fleas are incredibly host-generalist, likely exhibiting a host range that is among the broadest of all ectoparasites. Reducing wild species’ contact rates with domestic animals across natural and anthropogenic habitats, together with mitigating impacts of invasive reservoir hosts, will be crucial for reducing invasive flea infestations in wild mammals.
Article
Full-text available
The cat flea Ctenocephalides felis felis (Bouché) is the most important ectoparasite of domestic cats and dogs worldwide. It has been two decades since the last comprehensive review concerning the biology and ecology of C. f. felis and its management. Since then there have been major advances in our understanding of the diseases associated with C. f. felis and their implications for humans and their pets. Two rickettsial diseases, flea-borne spotted fever and murine typhus, have been identified in domestic animal populations and cat fleas. Cat fleas are the primary vector of Bartonella henselae (cat scratch fever) with the spread of the bacteria when flea feces are scratched in to bites or wounds. Flea allergic dermatitis (FAD) common in dogs and cats has been successfully treated and tapeworm infestations prevented with a number of new products being used to control fleas. There has been a continuous development of new products with novel chemistries that have focused on increased convenience and the control of fleas and other arthropod ectoparasites. The possibility of feral animals serving as potential reservoirs for flea infestations has taken on additional importance because of the lack of effective environmental controls in recent years. Physiological insecticide resistance in C. f. felis continues to be of concern, especially because pyrethroid resistance now appears to be more widespread. In spite of their broad use since 1994, there is little evidence that resistance has developed to many of the on-animal or oral treatments such as fipronil, imidacloprid or lufenuron. Reports of the perceived lack of performance of some of the new on-animal therapies have been attributed to compliance issues and their misuse. Consequentially, there is a continuing need for consumer awareness of products registered for cats and dogs and their safety.
Article
Full-text available
A new subspecies of the European cerambycid Saperdapopulnea (Linnaeus, 1758) is described: Saperdapopulnealapponicassp. n. based on specimens from Scandinavia. The male genitalia characters were examined and found to provide support for this separation, as well as differences in morphology, geographical distribution and bionomy. The preferred host tree for the nominate subspecies S.populneapopulnea is Populustremula L., whereas S.populnealapponicassp. n. is considered to be monophagous on Salixlapponum L. DNA sequence data of mitochondrial cytochrome oxidase subunit I (COI) was generated from Scandinavian specimens of S.populneapopulnea and specimens representing S.populnealapponicassp. n. The two subspecies were not reciprocally monophyletic and genetic distances in COI were small. All synonyms of S.populneapopulnea have been considered, and species similar to S.populneapopulnea have been examined, and not found to be related to S.populnealapponicassp. n. A male lectotype has been designated for each of the two following synonyms: Cerambyxdecempunctatus De Geer, 1775, and Saperdasalicis Zetterstedt, 1818. The synonymised species from Asia, S.balsamifera (Motshulsky, 1860), is elevated to subspecies: S.populneabalsamiferastat. n. We end with a discussion on the definition of subspecies under the unified species concept.
Article
Full-text available
The study was carried out to identify the flea species infesting small ruminants in Karnataka state, India. The morphological differentiation of species of genus Ctenocephalides was often confused due to loss of specific characters. Therefore, DNA barcoding of COX1 mitochondrial gene was carried and PCR product yielded a specific amplicon at 700 bp. The COX1 sequences of Ctenocephalides orientis from sheep were showing 99-100% homology to C. orientis from goats whereas, C. felis felis from dogs showed 99% homology to C. felis felis from cat. The accession number of KX467332 and KX467333 for C. orientis from Sheep and goats, KX467334 and KX467335 for C. felis felis from dog and cats respectively was allotted by NCBI. In the phylogenetic analysis, C. orientis formed a separate cluster to the subspecies of C. felis and clustered as sister species with C. canis. Thus, supporting C. orientis as separate species as previously suggested by morphological and molecular approaches.
Article
Full-text available
p>Gaps in the fossil record have limited our understanding of how Homo sapiens evolved. The discovery in Morocco of the earliest known H. sapiens fossils might revise our ideas about human evolution in Africa. See Letters p.289 & p.293</p