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Molecular and morphological analysis revealed a new Lipoptena species
(Diptera: Hippoboscidae) in southern Spain harbouring Coxiella burnetii and
bacterial endosymbionts
Mikel Alexander Gonz´
alez
a,b,*
, Ignacio Ruiz-Arrondo
c,d
, Sergio Magallanes
a,b
, Jozef Oboˇ
na
e
,
María Jos´
e Ruiz-L´
opez
a,b
, Jordi Figuerola
a,b
a
Estaci´
on Biol´
ogica de Do˜
nana (EBD, CSIC), Am´
erico Vespucio, s/n, Sevilla 41092, Spain
b
Ciber de Epidemiología y Salud Pública (CIBERESP), Av. Monforte de Lemos, 3-5, Madrid 28029, Spain
c
Department of Animal Pathology, Faculty of Veterinary Sciences, Instituto Universitario de Investigaci´
on Mixto Agroalimentario de Arag´
on (IA2), Universidad de
Zaragoza, C. Miguel Servet, 177, Zaragoza 50013, Spain
d
Centre of Rickettsiosis and Arthropod-Borne Diseases, Hospital Universitario San Pedro-CIBIR, C. Piqueras, 98, Logro˜
no, La Rioja 26006, Spain
e
University of Preˇ
sov, 17. novembra 3724/15, Preˇ
sov 080 01, Slovakia
ARTICLE INFO
Keywords:
Anatomical features
Blood-feeding ies
CO
2
traps
DNA barcoding
Endosymbiont organisms
Louse ies
Microbial pathogens
Novel species
ABSTRACT
Hippoboscid ies (Diptera: Hippoboscidae) are obligate bloodsucking ectoparasites of animals. In Europe,
limited research has been conducted on this family until the recent introduction of the deer ked Lipoptena for-
tisetosa Maa, 1965. A new species of the genus Lipoptena, Lipoptena andaluciensis sp. nov., was found in southern
Spain after extensive sampling with carbon-dioxide baited suction traps. A total of 52 females and 32 males were
collected at 29 out of 476 sites examined over eight months in 2023. Lipoptena andaluciensis sp. nov. was
characterized morphologically and molecularly. The new Lipoptena species can be differentiated from the closely
related L. fortisetosa by size, chaetotaxy of the dorsal and ventral thorax, abdominal plates, and genitalia. Based
on DNA-barcoding, our specimens showed the highest similarity with Melophagus ovinus (Linnaeus, 1758)
(88.4 %) and with L. fortisetosa (86–88 %). Individual screening of Lipoptena specimens (n =76) for seven
important zoonotic pathogens such as bacteria (Anaplasmataceae family: Bartonella spp., Borrelia spp., Coxiella
burnetii and Rickettsia spp.) and protozoans (Babesia spp. and Theileria spp.) by conventional PCR and RT-PCR was
performed. DNA of C. burnetii was detected in one specimen, while two other specimens harboured Ana-
plasmataceae (Wolbachia spp., 100 % homology and another endosymbiont probably related to Arsenophonus sp.,
95.3 % homology, respectively), all representing the rst records of these bacteria in the Lipoptena spp. from
Europe. Carbon dioxide traps probed its effectiveness as a reliable passive method for keds surveillance. Our
study highlights the existence of a new Lipoptena species, presumably widely distributed in southern Spain. The
role of this species in the transmission cycle of pathogens of medical-veterinary relevance needs to be considered
in the area.
1. Introduction
Hippoboscidae, commonly known as louse ies or keds, constitutes a
family of parasitic ies that primarily infest birds and mammals
(Hutson, 1984; Reeves and Lloyd, 2019). Around 213 hippoboscid
species are known worldwide, from which 32 species have been
described in Europe (Keve et al., 2024; Oboˇ
na et al., 2022, 2019).
Hippoboscidae ies are divided into the subfamilies Lipopteninae,
Ornithomyinae, and Hippoboscinae (Reeves and Lloyd, 2019). The
former subfamily includes the genus Lipoptena, also known as deer keds,
which are characterized by losing their wings after nding a suitable
host. There are about 30 species of Lipoptena worldwide (Dibo et al.,
2023) recorded mostly in America, Central and Northern Europe, South
Africa, and Occidental Asia. In Europe, ve species are present, Lip-
optena cervi (Linnaeus, 1758), Lipoptena couturieri S´
eguy, 1935, Lipoptena
capreoli Rondani, 1878, Lipoptena arianae Maa, 1969, and Lipoptena
* Corresponding author at: Estaci´
on Biol´
ogica de Do˜
nana (EBD, CSIC), Am´
erico Vespucio, s/n, Sevilla 41092, Spain.
E-mail addresses: malexander.gonzalez@ebd.csic.es (M.A. Gonz´
alez), iruizarr@unizar.es, irarrondo@riojasalud.es (I. Ruiz-Arrondo), sergio.magallanes@ebd.csic.
es (S. Magallanes), mjruiz@ebd.csic.es (M.J. Ruiz-L´
opez), jordi@ebd.csic.es, jozef.obona@unipo.sk (J. Figuerola).
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
https://doi.org/10.1016/j.vetpar.2024.110300
Received 8 July 2024; Received in revised form 24 August 2024; Accepted 29 August 2024
Veterinary Parasitology 332 (2024) 110300
Available online 3 September 2024
0304-4017/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/by-
nc/4.0/ ).
fortisetosa Maa, 1965. In recent years, the exotic species L. fortisetosa has
been reported in Europe, probably due to the introduction of sika deer
(Cervus nippon) from the Eastern Palaearctic. This ked has drawn
considerable attention due to its impact on various autochthonous
mammals, and its expanding distribution across Europe. The presence of
this species has been documented across more than 12 european coun-
tries, including Central, Eastern, and some regions in Southern Europe
(Dibo et al., 2023; Kurina et al., 2019). The southernmost records were
in the North of Italy (Salvetti et al., 2020) and the northwestern of
France (Guillou and Chapelin-Viscardi, 2020). Mihalca et al. (2019) also
emphasized that this species is probably overlooked and misidentied as
L. cervi in many cases. For these reasons, misidentication may likely
occur when identifying species of this genus, as many researchers are
assuming the discovery of L. fortisetosa as a new record in their countries
without having a detailed comparison with other related species.
Hippoboscid ies have been long overlooked by the scientic com-
munity, probably because the host range for most species is restricted to
wildlife (Bezerra-Santos and Otranto, 2020). Collection of these ies
from birds and mammal hosts can be difcult and frustrating, which
likely accounts for the absence of records. These challenges might
explain the lack of studies on this family, contributing to the limited
records. Studies on Hippoboscidae in Spain are scarce and mostly
focused on ornithophilic species from bird ringing programs (Gangoso
et al., 2019; Gonz´
alez et al., 2023), with a considerable gap on mam-
mophilic species. Around 19 hippoboscid species have been recorded
from the Iberian Peninsula and Islands (Oboˇ
na et al., 2019). There are 14
species that primarily feed on birds and ve species that mostly feed on
mammals. In Spain, the genus Lipoptena is composed by only two spe-
cies, L. cervi and L. couturieri (Carles-Tolr´
a and B´
aez, 2012). Over the last
50 years, L. cervi has only been formally recorded in Catalonia (Cordero
del Campillo et al., 1980), Le´
on (Martínez, 2015), and Galicia (V´
azquez,
2010). The older and unique record of L. couturieri was found in the
twentieth century in the Pyrenees (Huesca) (Beaucournu et al., 1985).
Certain species of the genus Lipoptena can profoundly impact animal
health, especially wildlife and livestock. Severe infestations of these
ectoparasites can cause skin irritation, lesions, dermatitis, allergic rhi-
noconjunctivitis, and in extreme cases, anaphylactic shock when they
bite humans (Ma´
slanko et al., 2020; Madslien et al., 2011). Besides
direct nuisance, deer keds are potential vectors of a variety of pathogens,
some of them important zoonotic agents such as Anaplasma phag-
ocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato (s.l.), Rick-
ettsia spp., and Theileria ovis, among others (Bezerra-Santos and Otranto,
2020).
In the absence of data on mammophilic Lipoptena species in Spain,
this study provides the most comprehensive systematic study conducted
in the country so far. Our work aimed to determine the ked species
composition captured with suction traps baited with carbon dioxide and
to evaluate the role of the new Lipoptena sp. as a potential vector of
pathogens through a molecular screening of seven pathogen groups with
sanitary interest. A comprehensive characterization of the new Lipoptena
species is presented here, including traditional morphometric and
morphological analysis, as well as DNA barcoding.
2. Materials and methods
2.1. Study area
A stratied random sampling was conducted in Andalusia (southern
Spain) covering the provinces of Sevilla, Huelva, C´
adiz, M´
alaga, and
C´
ordoba, which accounted for ca. 52,670 km
2
. Southern Spain is char-
acterized by a Mediterranean climate, with dry hot summers and mild
winters. In this region, daytime temperatures often soar well above 35ºC
and occasionally reach the mid-40 s during heatwaves. Winters are
generally mild, with daytime temperatures ranging from 15 to 20ºC
occasionally dropping lower at night. Precipitation levels vary, but
generally, experience less rainfall, especially during the summer
months, creating a predominantly dry and sunny climate throughout
much of the year. The area of the study features diverse habitats
including agricultural landscapes, riverine ecosystems, coastal plains,
and extensive forests, primarily composed of cork oaks, holm oaks, and
chestnut trees. Regarding fauna, around 75 species of mammals are
reported in Andalusia, including seven species of ungulates, with Cervus
elaphus, Capreolus capreolus, and Dama dama, being the most widely
distributed species in the study area (Miteco, 2024).
2.2. Design and trapping method
Specimens of the family Hippoboscidae described in this study were
collected as part of a broader project designed to collect ying Diptera,
particularly mosquitoes. The territory was stratied into 21 areas of
similar size. In each area, approx. 21 sampling points were selected
based on four main criteria: a) cover the territory homogeneously, b)
different land cover areas, c) available routes for easy access, and d) low
vandalism. The order of sampling of these predened tracks was
randomly selected to prevent any bias. Each point was sampled three
times (spring, summer, and autumn) from April to November, giving a
trapping effort of approx. 1,350-day (21 areas x 21 points x 3 seasons).
Twenty-six additional sites, 8 in Sevilla and 8 in C´
adiz sampled at
weekly intervals and 6 in C´
ordoba and 4 in M´
alaga sampled every two
weeks, in both cases from 15-June to 23-November (i.e. 436 trapping
days) were sampled. Hippoboscids were captured using BG-sentinel
traps (model 2 trap, BIOGENTS, Germany) supplemented with approx.
1 Kg of CO
2
(dry ice) operating over 24-h periods. Collection bags with
insects were immediately retrieved each morning and stored at −80◦C
for further analysis.
2.3. Taxonomic identication and morphological analysis
Hippoboscids were separated, counted, identied and sexed ac-
cording to the recent taxonomic keys designed for the identication of
European hippoboscid species (Oboˇ
na et al., 2022) and keys specically
elaborated for the identication of Lipoptena species (Andreani et al.,
2019; Salvetti et al., 2020). The original descriptions of L. fortisetosa
were also consulted (Maa, 1965). Our specimens were compared with
fresh-fed individuals from Slovakia and the four previous mentioned
sources.
Key external morphological features such as mesonotum chaetotaxy
(distribution and number of bristles), head (shape and antennae chae-
totaxy), body dimensions, abdominal terga, and genitalia were thor-
oughly checked. The body length and width were measured to be
compared with data from other European records. Body measurements
of males and females were performed under a binocular stereo micro-
scope coupled with a Leica camera (Zeiss, Stemi 2000-C, Ontario, NY).
The following morphometric measurements (Fig. 1S) were measured in
mm in 10 males and 10 females: i) body length: distance from top of the
head to end of abdomen, ii) head (length x width, in dorsal view), dorsal
thorax (length x width), ventral thorax (length x width) and wing (from
the base of the tegula to the outermost tip of the wing). The length of the
abdomen was calculated by dividing the total length minus the thorax
minus the head. The terminalia of females is considered a stable feature
among this genus, thus the number of setae in anal sclerite was counted
for each female (n =25). Some random specimens were subjected to
digestion in 15 % KOH for 24 h to enhance the visualization of certain
diagnostic structures, which were then photographed. There is no
consensus in the terminology to refer to chaetotaxy; setae, for ordinary
cuticular extensions arising from alveoli; bristles, for long, stout, erect or
suberect setae; spurs, robust setae at apex of tibia; spines, for short, very
stout and dark coloured.
2.4. Genomic DNA extraction
DNA was extracted for two purposes using the DNeasy® Blood and
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
2
Tissue kit (QIAGEN, Germany). First, for species identication, four
Lipoptena specimens were randomly selected. Genomic DNA was
extracted from two legs following the manufacturer’s instructions for
tissue extraction. Second, a total of 76 specimens (45 females and 31
males) were selected for pathogen screening. A selection of the most
common pathogens reported in the literature in Lipoptena genus in the
Palearctic region were analyzed (Bezerra-Santos and Otranto, 2020).
DNA was extracted from each specimen separately following the man-
ufacturer’s instructions for the purication of total DNA from ticks for
detection of Borrelia DNA (https://www.qiagen.com/de/resources/re
sourcedetail?id=20e82926-1a1b-4686–8cfd-3ee9974f624b&lang=en).
In brief, the body of the ked was moistened with buffer and then the
body was pulled with the tip of a pipette to the upper rim of the opened
microcentrifuge tube, after that, several cuts into the body were done
with a scalpel until the body was completely crushed.
2.5. Ked species identication
To identify the insect species at the molecular level, we amplied a
658 bp fragment of the mitochondrial cytochrome c oxidase subunit I
(COI) gene using the universal primers LCO1490 (5
′
-GGTCAA-
CAAATCATAAAGATATTGG-3
′
) and HCO2198 (5
′
-TAAACTTCAGGGT-
GACCAAAAAATCA-3
′
) (Folmer et al., 1994). Amplication reactions
were performed in a 25 µl volume containing approximately 1x Buffer,
3.9 mM MgCl2, 0.8 mM dNTPs (Bioline), 0.6 µM of each primer, and 2.5
Units of Taq polymerase (BIOTAQ™ DNA polymerase, Bioline). The
amplication reaction started with a polymerase activation phase at
94◦C for 4 minutes followed by 35 cycles at 94◦C for 1 minute, 40◦C for
1 minute, and 72◦C for 1 minute, followed by a nal extension for
10 minutes at 72◦C. The amplied products were sequenced on both
strands using Capillary Electrophoresis Sequencing by the genomic
service at Universidad Complutense de Madrid (Madrid, Spain). We
analyzed the sequences using Geneious software v2020.0.3 (Kearse
et al., 2012). To identify the species, consensus COI sequences were
compared, using the nucleotide Basic Local Alignment Search Tool
(Blastn), to sequences available on NCBI GenBank database (National
Center for Biotechnology Information; https://blast.ncbi.nlm.nih.gov/
Blast.cgi) and with those deposited in the Barcode of Life Data (BOLD)
Systems platform (http://boldsystems.org/views/login.php).
2.6. Pathogen screening
DNA from specimens was screened using specic PCR assays
(Table S1) for the presence of six different pathogens including (i)
bacteria: Anaplasmataceae family, Bartonella spp., Borrelia spp., Coxiella
burnetii, Rickettsia spp. and (ii) protozoans: Babesia spp. and Theileria
spp. In brief, Anaplasmataceae, Bartonella spp. and Babesia/Theileria
spp. pathogens were analyzed by conventional PCR, Borrelia spp., Cox-
iella burnetii, and Rickettsia spp. with RT-PCR and Rickettsia spp. by
nested PCR. Anaplasma phagocytophilum, Babesia microti, Bartonella
henselae, Borrelia miyamotoi, Coxiella burnetii and Rickettsia amblyom-
matis DNA were used as positive controls in the corresponding PCR as-
says. Negative controls containing distilled water were included in all
PCR assays. The sequencing and sequences comparison were conducted
as in ked species identication, but the sequencing was done at the
Center for Biomedical Research of La Rioja (CIBIR), Spain.
2.7. Phylogenetic analysis
The four high quality sequences (658 pb) from this study were
analyzed together with 16 partial COI sequences representative of
related species belonging to the families Hippoboscidae, Nycteribiidae,
and Glossinidae, along with four Lipopteninae species. Multiple align-
ment was carried out with Clustal W in CIPRES (Miller, 2010). Model
GTR+G was the best t substitution model according to jmoldeltest
2.1.10 (Darriba et al., 2012) in CIPRES (Miller, 2010). The phylogenetic
tree was generated using a maximum likelihood approach and a general
time reversible model (GTR+G) and 1000 bootstraps in MEGA11
(Tamura et al., 2021). We also estimated the genetic distances between
the samples in this study and all the other samples included in the an-
alyses using a Maximum Composite Likelihood model (Tamura et al.,
2004).
2.8. Mapping and host distribution
The distribution of L. andaluciensis nov. sp. along with three other
ked species was mapped using QGIS software (QGIS version 3.32). The
distribution of the three most common cervids (C. elaphus, C. capreolus,
and D. dama) was obtained from “Atlas and Red book of Mammals of
Spain” (10 ×10 Km UTM) (Palomo et al. 2007). Then, the distribution of
these cervids was overlapped with the records of keds from our study.
3. Results
3.1. Overall ndings
Lipoptena andaluciensis sp. nov. was discovered based on both
morphological and molecular evidence. Signicant differences in
morphological traits and high genetic diversity were found compared to
closely related species. A total of 84 winged Lipoptena andaluciensis sp.
nov. specimens (62 % females and 38 % males) were collected over a 8-
month period in the provinces of Sevilla, Huelva, and C´
adiz (southern
Spain) (Table 1; Fig. 1). Most of the collections (>75 %) occurred in
mountain areas with typical Mediterranean vegetation in Huelva. Lip-
optena andaluciensis sp. nov. keds were present in 29 sampling points
(6.1 % of the total sampled points) and were consistently collected over
the sampling period from 11-April to 16-November 2023, with a major
peak in spring season (Table 1). The spatial distribution of cervids
overlapped with L. andaluciensis sp. nov records in 16 out of the 29
positive sampling points (Fig. 1). Three accidental collections of other
hippoboscids were also recorded: Hippobosca equina Linnaeus, 1758,
Pseudolynchia canariensis (Macquart in Webb & Berthelot, 1839), and
Ornithoica turdi (Olivier in Latreille, 1811) (Fig. 1).
3.2. Description of Lipoptena andaluciensis sp. nov. Gonz´
alez, 2024
Following the International Code of Zoological Nomenclature
(ICZN), details of new species have been submitted to ZooBank with Life
Science Identier (LSID) zoobank.org.pub: https://zoobank.org/Refer
ences/52C43515-4A68-4484-BE57-1E868C3A2AF7.
3.2.1. Material examined
52 females and 32 males are examined under the binocular and
subsequently the DNA (n =76) was used for molecular purposes
(pathogen screening and/or COI barcoding). Holotype: 1 female and
allotype: 1 male (Huelva: coordinates 37.816527, −7.028300, date 30/
08/2023; Sevilla: coordinates 37.957468, –5.925663, date 6/10/2023,
respectively, Spain). Collected with suction traps baited with CO
2
. Par-
atypes: 2 females and 1 male, same data and location as holotype. Ho-
lotype and paratypes are individually preserved in dry (1.5 ml vials) at
−20 ºC at the Collection Services from Estaci´
on Biol´
ogica de Do˜
nana
(EBD-CSIC, Sevilla, Spain) (https://www.ebd.csic.es/). under the reg-
ister number: 2.024_073. The DNA of four specimens was also preserved
individually in 1.5 ml vials at - 80ºC.
3.2.2. Morphological description (Figs. 2-6)
All the specimens collected were unfed winged adults.
Body (Fig. 2). Similar in both sexes. Overall body amber, bright-
brown, or dark brownish. Legs slightly lighter than main body. Body
size length: Mean ±Standard Deviation (SD) – males: 2.65 ±0.08 mm
and females: 2.54 ±0.17 mm.
Head (Fig. 3). Head length – males: 0.58 ±0.02 x 0.89 ±0.03 mm,
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
3
females: 0.55 ±0.09 x 0.88 ±0.07 mm. Head rhomboidal shape,
anterior margin with marked curvature, almost semicircular; eyes
relatively large, distinctly narrowed behind (Fig. 3A). Eyes and post
vertex with ocelli in rounded dark brown plate, rest of head pale brown;
vertex hourglass-shape (Fig. 3A). Palpi short, dorsally barely visible.
Chaetotaxy of palpi (with very long apical setae, distinguishable from
the rest) (Fig. 3 B) and antennae (with nine typical aligned bristles)
(Fig. 3C). Each side of the eye bears four dorsal lateral bristles (two
fronto-orbital bristles, one ne and one long plus two long in the vertex)
(Fig. 3D). Ventrally with relatively abundant short setae particularly in
the inner rim of the eye and characteristic long pair bristles pointed
toward below eyes (Fig. 3E).
Thorax (Fig. 4). Dorsal thorax measures for males: 1.00 ±0.09 x
1.14 ±0.04 mm and females: 1.01 ±0.02 x 1.14 ±0.11 mm. Pronotum
ribbon-like. Median notal suture almost reaching scutellum. Mesonotal
chaetotaxy (Figs. 4A and 4B): 4 humeral bristles in each side; 2 – 3 pairs
of postalar bristles; two groups of laterocentral bristles, 2 pairs of ne
inner ones plus 3 – 4 strong setae; 3 pairs of scutellar bristles, rarely 2,
medium pair twice the size of the lateral ones. Acrostichal bristles on
both sides of the medionotal suture of the scutum are absent (rarely
bearing one pair) (Fig. 4A, yellow rectangle). Two or three laterocentral
bristles are present (rarely one or four) on both sides of the posterior
dorsocentral scutum (Fig. 4B, yellow rectangles).
Ventral thorax (Fig. 4C) measures – males: 1.01 ±0.07 x 0.748 ±
0.08 mm and females: 1.10 ±0.125 x 0.08 ±0.13 mm. Prosternum
anteriorly rounded (broadly convex); anterior laterals bear 4 – 5 (very
rarely 6) spines (at least one longer than others) on each side (Fig. 4C,
red rectangle). Mesosternum (approx. 2 times wider than the length)
with two groups of spines, evenly scattered (<30 on each side, 23–29)
and a row of setae bordering the lower rim of mesosternum (<20,
15–19), with a single and distinctive long bristle laterally (Fig. 4C,
yellow rectangle). Metasternum with 2 regular spines rows (both not
exceeding 25 spines in total), upper arrow bears 6 – 9 and the lower row,
slightly longer in size, with 10–15. The posterior plate of the meta-
sternum is bare, curved forward and with a median suture well-marked.
The length and robustness of the spines are similar in the three pro-
Table 1
Counts of the Lipoptena andaluciensis sp. nov. detected in 2023 in Andalusia (southern Spain) and numbers infected by bacteria.
Month Location nº sites Female Male Total Bacteria detected
C. burnetii Endosymbiont
April Sevilla and Huelva 7 29 12 41 1
1
1
2
May Sevilla, C´
adiz, Huelva 5 3 3 6 0 0
June C´
adiz and Huelva 4 3 4 7 0 0
July Huelva 3 2 1 3 0 0
August Huelva, C´
adiz 7 3 6 9 0 0
September Huelva, C´
adiz 1 4 2 6 0 1
3
October Huelva, C´
adiz, Sevilla 4 8 3 11 0 0
November Huelva 1 0 1 1 0 0
Total 32 52 32 84 1 2
1
Coxiella burnetii (female, coordinates: 37.782431, −6.291059),
2
Wolbachia sp. (male, 37.957468, −5.925663) and
3
Arsenophonus sp. (male, 37.3261695,
−7.2035444).
Fig. 1. Map of the 476 sampling sites in the ve western provinces of Andalusia (southern Spain). Empty circles: negative sampling sites for the presence of Lipoptena.
White circles: sampling sites with captures of Lipoptena andaluciensis sp. nov. Green, orange, and blue triangles: sampling sites with captures of Pseudolynchia
canariensis, Hippobosca equina , and Ornithoica turdi, respectively. Pink rectangles (10 ×10 km) represent the distribution of Cervidae (C. elaphus, C. capreolus, and
D. dama pooled).
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
4
meso-metathorax parts in both sexes.
Abdomen (Fig. 5). In both sexes, tegument of the ventral abdomen
highly setose in both sexes (with long setae uniform in length and
robustness except in lateral and apical margins, which is longer). Ter-
gites are similar in size, anterior tergites wider than posterior ones.
Male (Fig. 5A-D). Abdomen in dorsal (Fig. 5A) and ventral view
(Fig. 5B). Pleurite 1 large, kidney-shaped with 3 series of setae. Outer
series of setae (20 – 22 setae on each side) aligned in margin and at least
2 times longer in size compared to other two series. Disposition of setae
in series 2 and 3 not aligned. Tergite 3 is hardly visible (partly covered
by pleurite 1) with 10 – 12 setae. Tergite 4 with 10 – 12 setae, tergite 5
with 2 distinctive long-curved setae and a couple of ne setae (some-
times absent). Tergite 6 – 7 (terminalia of abdomen) with usually 6 – 8
long straight setae disposed on lateral margins (3 – 4 +3 – 4). 1st
abdominal sternite deeply emarginate. The outer margin distinctly
convex with one apical and one subapical bristle on each side; also, with
aligned spines and some scattered setae on the surface (Fig. 5D). Male
genitalia (fresh specimens): genitalia barely visible or retracted
(Fig. 5B). Male terminalia invaginated within a semi-disc-shaped
structure with anterior margins that are setose. Surstylus poorly devel-
oped, with a group of inconspicuous setae on each side. After pressing
the abdomen, the male terminalia become visible and has two orange
and bright lateral gonopods that guide the aedeagus (Fig. 5C). Gonopods
end in a rounded tip. Aedeagus is yellow-transparent in colour, with a
bifurcated tip. Ventral surface of the aedeagus covered in stretch marks
(under the microscope, 40x magnication, numerous misaligned shark-
Fig. 2. Habitus of winged Lipoptena andaluciensis sp. nov. in dorsal view (A) and ventral view (B).
Fig. 3. Head of Lipoptena andaluciensis sp. nov. Dorsal view (A). Palpi (B). Bristles of antenna (C). Dorsal chaetotaxy (D). Ventral chaetotaxy (E). Structures B, C, and
D photographed after KOH digestion.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
5
like teeth are observed). Dorsal tip of the aedeagus has a groove,
apparently bearing inconspicuous spines. Slide-mounted genitalia (after
KOH digestion) have a membranous appearance, except for the edges
and proximal region Gonopods and aedeagus slightly curved downward
and narrow in the apical region (Fig. 5C).
Female (Fig. 5E-H). Abdomen in dorsal (Fig. 5E) and ventral view
(Fig. 5F). Pleurite 1 like males in size and almost the same number and
disposition of setae. Tergite 3 with approx. 12 setae, tergite 4 with 12
–14 setae. Tergite 5 with 10 setae, tergite 6 with 2 distinctive long and
curved setae. Tergite 7 with 4 long straight setae, tergite 8 (terminalia of
the abdomen) is composed of usually 8 long setae (4 left +4 right)
(rarely 10) clearly exceeding the female abdomen. The median pre-
genital (central anal sclerite) plate is small (with variable development)
and rectangular-shaped, usually bearing 2 long setae. 1st abdominal
sternite similar to males. Female genitalia (Figs. 5G and 5H): The
number of setae in pregenital sclerite variable, 90 % of the specimens
possessed 2 long setae (n =45) (Fig. 5H), 8 % with 3 setae of different
sizes (n =4) and 2 % with 4 variable size setae (n =1) (Fig. 5G). Pre-
genital plate is inconspicuous. Hypoproct large, semi-circular, covered
by numerous spine-like setae (with different robustness) interspersed in
most of the surface area (except in the apical part). Tegument around
hypoproct bare. Setae on both sides of pregenital sclerite absent. The rim
of setae surrounding genitalia covered by abundant strong setae.
Legs (Fig. 6A-H). The legs are fairly setose. Coxa and trochanter of
fore, mid, and hind legs with a combination of ne and strong setae.
Each moderately enlarged femur (fore, mid, and hind) with 3, 3 and 3 – 5
major postero-dorsal bristles, respectively (Fig. 6A-C). Fore femur with a
distinctive curved bristle in base (Fig. 6A, yellow arrow). Anterior
margin of fore and mid femur densely covered by short-setae (Fig. 6 A,
red arrow) less obvious in hind femur. Fore femur with at least 2
distinctive bristles (in anterior and posterior proximal positions,
respectively) (Fig. 6D). Mid femur variable (usually 1–4 distinctive
bristles) and hind femur with a bristle at the base and another subapical
bristle (red arrows, Fig. 6E). Surface of ventral femur of fore and mid
legs slightly setose and in hind legs moderately setose. Apex of fore, mid,
and hind tibia with a strong hair-like setae (red arrows, Fig. 6F-H). Tibia
with variable distribution of ne setae. Hind tibia with 6 – 8 strong inner
setae with variable robustness and size (usually 2 – 3 medial strong setae
plus 3 – 4 apical strong setae along with bristle-like long setae) (Fig. 6I).
Tarsus (sum 1 – 4) similar length to tarsus 5. All tarsus with major and
minor apical spurs with variable numbers. The ventral surface of seg-
ments 3 – 5 bearing longer setae (at least 2 setae difference for each
tarsus 3 – 5) compared to tarsus 1 – 2. The dorsal surface of the tarsus
bears hardly visible minute setae.
Claws (Fig. 6J). Bid asymmetric claws sharply curved with a pale
basal lobe. One prominent pad-like pulvillus (the other is smaller and
shorter). Empodium is ne and elongated.
Wings (Fig. 6K). The wings are fully developed, clear, and hyaline,
with only one cross-vein (males: 2.95 ±0.03 mm, females: 2.75 ±
0.07 mm). Apical margin of the wing down pointed. Basal veins and
cells well-marked (end of R
4+5
rounded and sclerotized), distal cells
partially transparent (2 A, Cu+1 A, M
3+4
, M
1+2
), gradually curded and
reach the margin. Cell Sc ends at or before Cl (vein R
4+5
ends clearly
further than R
1
).
3.2.3. Etymology
The name andaluciensis derives from the region where the specimens
were collected: Andalucía, an autonomous community in Spain.
3.2.4. Distribution
Southern Spain (Sevilla, Huelva, and C´
adiz). Known only from the
type-locations (Fig. 1).
3.2.5. Hosts
Unknown.
3.2.6. Habitat of keds
Well-distributed in forested Mediterranean habitats with low
mountains (<1.500 m altitude).
3.2.7. Diagnosis and comparison
This new species shares many morphological features with
L. fortisetosa, but there are at least six key differential diagnostic traits
that distinguish both species. i) mesonotal chaetotaxy is almost identical
to L. fortisetosa (Fig. S2A) but in L. andaluciensis sp. nov. there are no
acrostichal bristles on both sides of the medionotal suture of the scutum
(rarely bearing one pair) and possess a group of extra 2 or 3 laterocentral
bristles (rarely 1 or 4) in both sides of the posterior dorsocentral scutum;
ii) the main differences are in chaetotaxy of the ventral thorax (pros-
ternum, mesosternum, and metasternum) (Fig. S2B). A higher number of
prosternal setae are observed in L. fortisetosa (5 – 8) compared to
L. andaluciensis sp. nov. (4 – 5) on each side. Excluding the lower rim,
L. andaluciensis sp. nov. shows lower (<30) number of spines in the
mesosternum compared to L. fortisetosa (>30). Three irregular rows of
metasternum spines in L. fortisetosa and two irregular rows in
L. andaluciensis sp. nov. Also, they differ in the size of the spines, almost
identical in L. andaluciensis sp. nov. and with variable size in
L. fortisetosa; iii) The outer margin of the 1st abdominal sternite in
L. andaluciensis sp. nov. has 1 apical and 1 subapical bristle (on each
side) where in L. fortisetosa there is only one apical bristle (Fig. S2C); iv)
chaetotaxy of pleurites and tergites also differ between both Lipoptena
species. Additionally, in ventral view, the abdomen of females appears
more setose than L. fortisetosa, v) in females of the new species, there is
an absence of a pair of isolated setae on both sides of the pregenital
Fig. 4. Thorax of Lipoptena andaluciensis sp. nov. Chaetotaxy of mesonotum
(yellow square in A denotes the absence of acrostichal bristles in the medionotal
suture and in B denotes the position of the 2 – 3 bristles) (A-B). Chaetotaxy of
pro-meso-metasternum (red square denotes the prosternal setae and yellow
square denotes the single and distinctive long bristle in the mesosternal lateral
side) (C). Structures B and C photographed after KOH digestion.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
6
sclerite, compared to L. fortisetosa, where both setae are located close to
pregenital sclerite (Fig. S2D) and vi) the body and wing length of
L. andaluciensis sp. nov is smaller compared to L. fortisetosa. Wing
venation similar to L. fortisetosa and L. cervi. We cannot discuss on male
terminalia features in comparison to sibling species, as previous works
used Scanning Electron Microscope (SEM) (Andreani et al. 2019; 2020).
Morphologically, the male genitalia appear to be more similar to
L. fortisetosa than to L. cervi. Therefore, it is necessary to update the key
for the European Lipoptena species (Oboˇ
na et al., 2022); p. 87 intro-
ducing the new species L. andaluciensis sp. nov. (Table S2).
3.3. Molecular identication and phylogenetic analysis
We identied two different COI sequences in the four individuals
sequenced. These sequences differed in a single nucleotide poly-
morphism (T-C) bp at position 637. Two carry the type (T) poly-
morphism (GenBank accession numbers: PQ176812–13) and two the
type (C) (PQ176810–11). The genetic distance between these two se-
quences was 0.001. The sequences from this study cluster within the
Lipopteninae and grouped with high condence with M. ovinus (Fig. 7).
Both sequences exhibited an 88.4 % similarity with M. ovinus
(ON129182) from Austria (Fig. 7). The percentage of identity with
L. fortisetosa ranged from the 88 % found for L. fortisetosa (MN807843)
from Estonia (Kurina et al., 2019), which was the second closest
sequence, to the 86 % found for L. fortisetosa (AB632572) from Japan
(Lee et al., 2016) (Fig. 7). The percentage of identity with Lipoptena
mazamae Rondani, 1878 and L. cervi was 87 % and 86 % respectively.
3.4. Screening of pathogens
Seventy-three Lipoptena specimens tested negative for the analysis of
pathogens. Two individuals were positive for Anaplasmataceae (Infec-
tion rate: 3.94%) (Table 1). The 16S rRNA gene sequence obtained from
one sample showed the highest identities (95.63–96.14 %) with the
bacterium endosymbiont of Leptocyclopodia brevicula (KC597729). The
following GenBank entries (95.32 %) corresponded to Arsenophonus sp.
(AB795344, MN594578). The other specimen yielded a sequence with
100 % identity with the Wolbachia endosymbiont of Phlebotomus perni-
ciosus (KY303724, KY303723), 100 % with Candidatus Wolbachia ino-
kumae (DQ402518) and 99.69 % with the Wolbachia endosymbiont of
Culex quinquefasciatus (KX611381) and Wolbachia pipientis (LC101758).
Detailed specimen records and sequence information of both endo-
symbiont isolated were submitted to the GenBank public database with
the accession numbers: PP922609-610. One of the Lipoptena was
Fig. 5. Abdomen of Lipoptena andaluciensis sp. nov. Male (A-D) and female (E-H). Dorsal abdominal segments (tergum) (A and E). Ventral abdominal segments
(sternum) (B and F). Gonopods and aedeagus of male genitalia (C). Below, genitalia in lateral (left) and ventral view (right). 1st abdominal sternite (D). Detail of
pregenital sclerites (G and H). Structures lower images in C and D photographed after KOH digestion.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
7
positive for Coxiella burnetii by qPCR, however, an amplicon could not be
obtained by conventional PCR, preventing us from obtaining the cor-
responding sequence. These records are novel for Lipoptena genus in
Europe.
4. Discussion
A new deer ked species, Lipoptena andaluciensis sp. nov. (Diptera:
Hippoboscidae), is described for the rst time in Andalusia (southern
Spain). The new species is morphologically similar to L. fortisetosa but
can be distinguished based on the chaetotaxy of mesonotum, pro-meso-
metasternum and abdominal plates as well as genitalia and size. Our
study also raises awareness about the taxonomic status of Lipoptena
species in Europe and suggests the need to conduct a detailed revision of
the different L. fortisetosa morphotypes discovered in Europe over the
last few years. Clearly, this genus has received little attention, and more
studies are necessary to determine the distribution of these species.In
addition, we also conducted a comprehensive screening of pathogens in
the new discovered species, marking the rst analysis for this genus in
Spain.
Regarding morphological identication, several morphometric and
morphological differences distinguish L. andaluciensis sp. nov. from
L. fortisetosa. First, the body and wing length of L. andaluciensis sp. nov. is
notably smaller compared to the closely related species L. fortisetosa
(Andreani et al., 2019; Ma´
slanko et al., 2020; Oboˇ
na et al., 2022; Salvetti
et al., 2020; Werszko et al., 2020). However, these measures might be
conditioned by the gonotrophic stage, because our specimens were all
unfed specimens. Given the results, L. andaluciensis sp. nov. is together
with L. arianae, the smallest species of the genus Lipoptena in Europe
(Oboˇ
na et al., 2019). The second key feature is chaetotaxy, a character
known as one of the most common and useful diagnosis tools to clarify
the taxonomy and phylogeny between taxa. Recently, various studies
have shown some kind of variation in the distribution of the bristles in
the mesonotum with respect to the original description of the Lipoptena
species (Andreani et al., 2019). Oboˇ
na et al. (2023) also noted
L. fortisetosa specimens collected from Slovakia with a great variation in
the chaetotaxy. Overall, all our specimens showed high consistency in
the distribution, number and size of the bristles and setae, with few
exceptions. Several morphological differences were found when
compared to L. fortisetosa, particularly in the ventral surface of the
thorax. Our specimens showed considerable similarity in the appearance
of the hypoproct, anal sclerite shape, and robustness of the two
distinctive setae. However, the specimens of L. andaluciensis sp. nov. lack
the two additional isolated setae in both sides of the anal sclerite,
Fig. 6. Legs, wing and claws of Lipoptena andaluciensis sp. nov. Fore, mid, and hind femur (red arrow in A denotes the rows of spines in anterior margin of fore femur,
yellow arrow in A denotes the distinctive curved bristle at the base of femur, and red arrows in D denote bristles at the posterior surface of proximal femur (A-D).
Hind femur (red arrows show two distinctive bristles) (E). Fore, mid, and hind tibia and tarsus (red arrow shows the apical strong hair-like setae of the tibia (F-H).
Detail of the apical and subapical spines of the hind tibia (I). Claws (J). Wing (K). Structure I photographed after KOH digestion.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
8
compared to the original description of Maa (Maa, 1965), which are also
visible in the specimens from Slovakia (our specimens) and Estonia
(Kurina et al., 2019). Previous studies have not considered the signi-
cance of the absence of these two isolated setae (Andreani et al., 2019).
Some variations in the abdomen might be also misinterpreted as it
typically would vary depending on the gonotrophic stage, with
blood-engorged specimens showing the abdomen inated and the setae
more dispersed.
Phylogenetic studies within the superfamily Hippoboscoidea are
scarce, largely due to the high genetic variance typical of these insects,
which complicates the construction of accurate phylogenetic trees. To
address these issues, it is essential to consider this genetic heterogeneity
by employing advanced analysis techniques, extensive sampling, and
multiple genetic loci, ensuring more robust phylogenetic inferences
(Petersen et al., 2007). To reconstruct the evolutionary history of the
family Hippoboscidae, multiple markers have been used (Petersen et al.,
2007). Here, we sequenced the COI gene because it was previously used
to identify Lipoptena species (Klepeckien ˙
e et al., 2020), and allows ac-
curate identication of L. fortisetosa and L. cervi (Tiawsirisup et al.,
2023). The phylogenetic tree obtained in our study is consistent with
results from previous works (Petersen et al., 2007; Tiawsirisup et al.,
2023) and conrms that L. andaluciensis sp. nov. clusters with other
Lipopteninae members such as L. mazamae. However, the new species
shares a higher percentage of identity with M. ovinus (88.4 %) than with
any other Lipoptena species and clusters with it in the phylogenetic tree
with high support, although this percentage is far from the values ex-
pected for species of the same genus (Gałęcki et al., 2021). This result is
not uncommon as other phylogenetic studies have also found that
M. ovinus is closely related to certain Lipoptena species (Petersen et al.,
2007; Tiawsirisup et al., 2023), despite M. ovinus being ecologically and
morphologically different. Based on its genetics, the second closest
related Lipoptena species is L. fortisetosa. Although previous studies have
found two clades of L. fortisetosa, the percentage of identity among these
clades is over 93 % (Tiawsirisup et al., 2023). These identity values are
much higher than the percentages of identity between L. andaluciensis
and L. fortisetosa which range between 86 % and 88 %. Considering this
together with the morphological variations described above, we can
conclude that our specimens are a different species.
While deer keds typically infest animals, their propensity to bite
humans introduces a health hazard that requires further investigation to
conrm their potential to transmit zoonotic diseases. Recently, the role
of keds as potential vectors of various pathogens has been reviewed in
Europe (Bezerra-Santos and Otranto, 2020). Our study represents the
rst screening for pathogens with medical and veterinary interest in
Lipoptena species in Spain, as previous efforts have mostly focused on
Central Europe (Bezerra-Santos and Otranto, 2020). Considering the life
cycle of keds (Reeves et al., 2019), the winged adults collected from our
study probably had not yet come into contact with their hosts. Thus, the
existence of zoonotic agents was not expected. However, vertical
transmission of Bartonella has also been reported in L. cervi (De Bruin
et al., 2015). Although DNA presence does not guarantee pathogen
transmission, it may highlight the potential risk for mechanical trans-
mission of pathogens to humans and healthy animals via the bite of
infected vectors (Wechtaisong et al., 2023). Coxiella burnetii is an obli-
gate intracellular bacterial pathogen and is responsible for causing Q
fever. The presence of C. burnetii represents the second record for keds
and the rst for Europe. Previous studies have only detected Coxiella
spp. in L. fortisetosa in Hydropotes inermis (Korean water deers) (Lee
et al., 2016). The endosymbiotic Wolbachia is a widely distributed
intracellular symbiotic bacteria presented globally and about 40 % of
arthropods and over 65 % of insect species naturally carry Wolbachia
(Zug and Hammerstein, 2012). Wolbachia bacterium have been detected
previously in the midgut of the sheep ked M. ovinus in Asia (Duan et al.,
2017; Liu et al., 2018), thus this nding represents the rst record for
Lipoptena genus. Our sample showed the closest similarity with the
bacterium endosymbiont L. brevicula, which belongs to the family Nyc-
teribiidae, within the superfamily Hippoboscoidae. The next closest
identity was with Arsenophonus, a group of symbiotic bacteria primarily
associated with insects, known for their wide host distribution range and
frequent presence in Hippoboscidae (Nov´
akov´
a et al., 2009). The low
identity (95–96 %) obtained in our sample might be due to the fact that
this endosymbiont genus has not yet been recorded in public databases
as most records of this bacteria belong to 16S rDNA sequences
(Nov´
akov´
a et al., 2009).
Despite the lack of standardized methods for collecting Hippo-
boscidae ying ectoparasites (Poh et al., 2020), our study utilizing
carbon-dioxide baited traps has proved to be an effective approach. So
far, the most common practice for collecting them relies on capturing
specimens from hunter-harvested hosts (require capturing or killing
cervids), which may not been feasible (Poh et al., 2020; Skvarla and
Machtinger, 2019), nor ethically desirable. The use of carbon dioxide to
attract hippoboscids has been tested in many studies but in general yield
low numbers and/or accidental collections (Yamauchi et al., 2011).
Particularly, there have been few studies specically targeting keds
using traps. Kortet et al. (2010) suggested that deer keds such as L. cervi
were not attracted to CO
2
. Recently, Andreani et al. (2021) showed
coloured sticky blue panels attracted the highest number of L. fortisetosa
compared to other colours. It would be interesting to assess if a syner-
gistic effect exists between CO
2
and the blue/black/white pattern of the
BG-traps used in our study. Our work demonstrates that substantial
numbers of L. andaluciensis sp. nov. can be trapped but we are unable to
assess the signicance of our collections in terms of productivity.
Therefore, the use of suction traps baited with carbon dioxide is pro-
posed as an easy and non-invasive solution for the surveillance of keds of
this Lipoptena species.
5. Conclusions
Given the limited number of published studies on Lipoptena genus, it
is not surprising that many species remain undiscovered. Here, we
describe a new species of Lipoptena based on both morphological char-
acters and DNA barcoding. This species appears widely distributed and
Fig. 7. Phylogenetic tree of COI sequences. The tree was constructed with the
Maximum likelihood method. Bootstrap values are given for 1000 replicates
and only those over 50 are shown. Sequences are identied by the GenBank
accession number and species. * Denotes the two types of sequences found in
L. andaluciensis sp. nov.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
9
presumably relatively abundant in the study region, which consists of
lower mountain ecosystems with pasture and meadow habitats featuring
typical Mediterranean ora. Data reported in this article contributes to
accurately identify this species and differentiate it from its sibling spe-
cies (L. cervi and L. fortisetosa). Our study also raises the potential of this
species to harbor bacteria with sanitary interest. It is worth suggesting
the need for future research to focus on the taxonomic status of this new
ked species in Europe. Additionally, identifying the mammal species
closely associated with the life cycle of these keds is essential. This
knowledge could be crucial for managing and controlling ked pop-
ulations in wildlife, and potentially mitigating their impact on other
species, including humans.
Funding
This study has been funded by Fundaci´
on “La Caixa” through the
project ARBOPREVENT (HR22-00123).
CRediT authorship contribution statement
Mikel Alexander Gonz´
alez: Writing – original draft, Methodology,
Investigation, Data curation, Conceptualization. Jordi Figuerola:
Writing – review & editing, Validation, Supervision, Resources, Project
administration, Funding acquisition, Conceptualization. Sergio Mag-
allanes: Writing – review & editing, Validation, Data curation. Ignacio
Ruiz-Arrondo: Writing – review & editing, Methodology, Formal
analysis, Data curation. María Jos´
e Ruiz-L´
opez: Validation, Supervi-
sion, Formal analysis. Jozef Oboˇ
na: Writing – review & editing, Visu-
alization, Validation, Investigation.
Declaration of Competing Interest
The authors declare the following nancial interests/personal re-
lationships which may be considered as potential competing interests:
Jordi Figuerola reports nancial support was provided by LaCaixa
Foundation. If there are other authors, they declare that they have no
known competing nancial interests or personal relationships that could
have appeared to inuence the work reported in this paper
Data availability
The datasets generated during and/or analyzed during the current
study are available from the corresponding author upon reasonable
request.
Acknowledgements
We thank Alvaro Solis, Cintia Vega, Cristina Diaz, Maria del Mar
´
Andujar, and Juan Jos´
e Talaver´
on for helping in the capture and sepa-
ration of the keds. Thanks to Joy Bromley for DNA extraction of ked
specimens. We thank Dr. Patrizia Sacchetti for sharing her knowledge on
L. fortisetosa. We thank Dr. Joaquim Ruiz (Universidad Cientíca del
Sur, Peru) for providing positive control of Bartonella henselae. Special
thanks to Dr. Shirin Taheri for the elaboration of the map.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.vetpar.2024.110300.
References
Andreani, A., Giangaspero, A., Marangi, M., Barlaam, A., Ponzetta, M.P., Roy, L.,
Belcari, A., Sacchetti, P., 2020. Asia and Europe: so distant so close? The Case of
Lipoptena fortisetosa in Italy. Korean J. Parasitol. 58 (6), 661–668. https://doi.org/
10.3347/kjp.2020.58.6.661.
Andreani, A., Rosi, M.C., Guidi, R., Jafrancesco, D., Farini, A., Belcari, A., Sacchetti, P.,
2021. Colour preference of the deer ked Lipoptena fortisetosa (Diptera:
Hippoboscidae). Insects 12 (9), 84512. https://doi.org/10.3390/INSECTS12090845.
Andreani, A., Sacchetti, P., Belcari, A., 2019. Comparative morphology of the deer ked
Lipoptena fortisetosa rst recorded from Italy. Med. Vet. Entomol. 33, 140–153.
https://doi.org/10.1111/mve.12342.
Beaucournu, J.-C., Beaucournu-Saguez, F., Guiguen, C., 1985. Nouvelles donn´
ees sur les
Dipt`
eres pupipares (Hippoboscidae et Streblidae) de la sous-r´
egion m´
editerran´
eenne
occidentale. Ann. Parasitol. Hum. Compar´
ee 60, 311–327. https://doi.org/10.1051/
parasite/1985603311.
Bezerra-Santos, M.A., Otranto, D., 2020. Keds, the enigmatic ies and their role as
vectors of pathogens. Acta Trop. 209, 105521 https://doi.org/10.1016/j.
actatropica.2020.105521.
Carles-Tolr´
a & B´
aez, M. 2012. Hippoboscidae, pp. 208. In: Carles-Tolr´
a Hjorth-Andersen,
M. (coord.), Cat´
alogo de los Diptera de Espana, Portugal y Andorra (Insecta),
Monograas S.E.A., 8, Zaragoza: Sociedad Entomol´
ogica Aragonesa.
Cordero del Campillo, M., Manga-Gonz´
alez, M.Y., Gonz´
alez Lanza, C., 1980. ´
Indice-
Cat´
alogo de Zoopar´
asitos Ib´
ericos. Ministerio de Sanidad y Seguridad Social
(Espa˜
na).
Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new
heuristics and parallel computing. Nat. Method 9 (8), 772.
De Bruin, A., Van Leeuwen, A.D., Jahfari, S., Takken, W., F¨
oldv´
ari, M., Dremmel, L.,
Sprong, H., F¨
oldv´
ari, G., 2015. Vertical transmission of Bartonella schoenbuchensis in
Lipoptena cervi. Parasit. Vectors 8, 176. https://doi.org/10.1186/S13071-015-0764-
Y.
Dibo, N., Yang, Y., Wu, X., Meng, F., 2023. A brief review on deer keds of the genus
Lipoptena (Diptera: Hippoboscidae). Vet. Parasitol. 313, 109850 https://doi.org/
10.1016/j.vetpar.2022.109850.
Duan, D.Y., Liu, G.H., Cheng, T.Y., Wang, Y.Q., 2017. Microbial population analysis of
the midgut of Melophagus ovinus via high-throughput sequencing. Parasites Vectors
10, 1–7. https://doi.org/10.1186/S13071-017-2323-1/TABLES/2.
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for
amplication of mitochondrial cytochrome c oxidase subunit I from diverse
metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.
Gałęcki, R., Xuan, X., Bakuła, T., Jaroszewski, J., 2021. Molecular Characterization of
Lipoptena fortisetosa from environmental samples collected in North-Eastern Poland.
Animals 11 (4), 1093. https://doi.org/10.3390/ANI11041093.
Gangoso, L., Guti´
errez-L´
opez, R., Martínez-de la Puente, J., Figuerola, J., 2019. Louse
ies of Eleonora’s falcons that also feed on their prey are evolutionary dead-end
hosts for blood parasites. Mol. Ecol. 28, 1812–1825. https://doi.org/10.1111/
MEC.15020.
Gonz´
alez, M.A., Hidalgo, J.C., Talabante, C., Bernal, I., 2023. New faunistic records and
host-parasite interactions of louse ies (Diptera: Hippoboscidae) from a community
of birds collected by mist-netting in the Spanish Central System. Graellsia 79, 8–11.
https://doi.org/10.3989/graellsia.2023.v79.383.
Hutson, A.M., 1984. Keds, at-ies and bat-ies: diptera, hippoboscidae and
nycteribiidae. handbooks for the identication of british insects. R. Entomol. Soc.
Lond. 10, 40.
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S.,
Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P.,
Drummond, A., 2012. Geneious basic: an integrated and extendable desktop
software platform for the organization and analysis of sequence data. Bioinformatics
28, 1647–1649. https://doi.org/10.1093/bioinformatics/bts199.
Keve, Gerg˝
o., Cs¨
org˝
o, T., Kov´
ats, D., Benke, A., Bende, A.T., ´
Agoston, H., M´
orocz, A.,
N´
emeth, ´
A., Tam´
as, E.A., Huber, A., Gyur´
acz, J., Keve, G.´
abor, Kontsch´
an, J.,
N´
emeth, A., Hornok, S., 2024. Contributions to our knowledge on avian louse ies
(Hippoboscidae: Ornithomyinae) with the rst European record of the African
species Ornithoctona laticornis. Parasit. Vectors 17, 237. https://doi.org/10.1186/
S13071-024-06303-8.
Klepeckien˙
e, K., Radzijevskaja, J., Raˇ
zansk˙
e, I., ˇ
Zukauskien˙
e, J., Paulauskas, A., 2020.
The prevalence, abundance, and molecular characterization of Lipoptena deer keds
from cervids. J. Vector Ecol. 45, 211–219. https://doi.org/10.1111/jvec.12392.
Kortet, R., H¨
ark¨
onen, L., Hokkanen, P., H¨
ark¨
onen, S., Kaitala, A., Kaunisto, S.,
Laaksonen, S., Kek¨
al¨
ainen, J., Ylnen, H., 2010. Experiments on the ectoparasitic deer
ked that often attacks humans; preferences for body parts, colour and temperature.
Bull. Entomol. Res. 100, 279–285. https://doi.org/10.1017/S0007485309990277.
Kurina, O., Kirik, H., ˜
Ounap, H., ˜
Ounap, E., 2019. The northernmost record of a blood-
sucking ectoparasite, Lipoptena fortisetosa Maa (Diptera: Hippoboscidae), in Estonia.
Biodivers. Data J. 7, e47857 https://doi.org/10.3897/BDJ.7.E47857.
Le Guillou, G., Chapelin-Viscardi, J.D., 2020. D´
ecouverte de Lipoptena fortisetosa Maa,
1965 en France (Diptera Hippoboscidae). L’Entomol. 76 (5), 277–280.
Lee, S.H., Kim, K.T., Kwon, O.D., Ock, Y., Kim, T., Choi, D., Kwak, D., 2016. Novel
detection of Coxiella spp., Theileria luwenshuni, and T. ovis endosymbionts in deer
keds (Lipoptena fortisetosa). PLoS One 11, 1–10. https://doi.org/10.1371/journal.
pone.0156727.
Liu, Y., He, B., Li, F., Li, K., Zhang, L., Li, X., Zhao, L., 2018. Molecular Identication of
Bartonella melophagi and Wolbachia Supergroup F from Sheep Keds in Xinjiang,
China. Korean J. Parasitol. 56, 365. https://doi.org/10.3347/KJP.2018.56.4.365.
Maa, T.C., 1965. A synopsis of the Lipopteninae (Diptera: Hippoboscidae). J. Med.
Entomol. 2, 233–248. https://doi.org/10.1093/jmedent/2.3.233.
Madslien, K., Ytrehus, B., Vikøren, T., Malmsten, J., Isaksen, K., Hygen, H.O., Solberg, E.
J., 2011. Hair-loss epizootic in moose (Alces alces) associated with massive deer ked
(Lipoptena cervi) infestation. J. Wildl. Dis. 47, 893–906. https://doi.org/10.7589/
0090-3558-47.4.893.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
10
Martínez, A., 2015. Contribuci´
on al conocimiento del estado sanitario del ciervo (Cervus
elaphus) en el norte de Espa˜
na: Procesos parasitarios. Doctoral Tesis. Universidad de
Le´
on. Spain.
Ma´
slanko, W., Bartosik, K., Raszewska-Famielec, M., Szwaj, E., Asman, M., 2020.
Exposure of humans to attacks by deer keds and consequences of their bites—a case
report with environmental background. Insects 11 (12), 1–9. https://doi.org/
10.3390/insects11120859.
Mihalca, A.D., P˘
astrav, I.R., S´
andor, A.D., Deak, G., Gherman, C.M., Sarmas
¸i, A.,
Votýpka, J., 2019. First report of the dog louse y Hippobosca longipennis in Romania.
Med. Vet. Entomol. 33 (4), 530–535. https://doi.org/10.1111/mve.12395.
Miller, M.A., 2010. CIPRES Science Gateway survey results. CIPRES Sci. Gatew. Surv.
results. Available at: http//www.phylo.org/tools/survey2.html http://www.phylo.
org/tools/survey2.html. Accesed on 15 September 2023.
Miteco, 2024. Ministerio para la Transici´
on Ecol´
ogica y el Reto Demogr´
aco. Inventario
Espa˜
nol de Especies Terrestres. Vertebrados. Mamíferos. Available at: https://www.
miteco.gob.es/es/biodiversidad/temas/inventarios-nacionales/inventario-especies-
terrestres/inventario-nacional-de-biodiversidad/bdn-ieet-atlas-vert-mamif.html.
Accesed on 15 August 2024.
Nov´
akov´
a, E., Hypˇ
sa, V., Moran, N.A., 2009. Arsenophonus, an emerging clade of
intracellular symbionts with a broad host distribution. BMC Microbiol 9, 143.
https://doi.org/10.1186/1471-2180-9-143.
Oboˇ
na, J., Csan´
ady, A., Hromada, M., Kuberka, P., Ox, K., Mlyn´
arov´
a, L., Manko, P.,
2023. The variability of chaetotaxy of Lipoptena fortisetosa Maa, 1965 (Diptera:
Hippoboscidae). Biodivers. Environ. 2023 15 (2), 17–21.
Oboˇ
na, J., Fogaˇ
sov´
a, K., Fulín, M., Greˇ
s, S., Manko, P., Repaský, J., Roh´
aˇ
cek, J.,
Sychra, O., Hromada, M., 2022. Updated taxonomic keys for European
Hippoboscidae (Diptera), and expansion in Central Europe of the bird louse y
Ornithomya comosa (Austen, 1930) with the rst record from Slovakia. Zookeys
1115, 81. https://doi.org/10.3897/ZOOKEYS.1115.80146.
Oboˇ
na, J., Sychra, O., Greˇ
s, S., Heˇ
rman, P., Manko, P., Roh´
aˇ
cek, J., ˇ
Sest´
akov´
a, A.,
ˇ
Slap´
ak, J., Hromada, M., 2019. A revised annotated checklist of louse ies (Diptera,
Hippoboscidae) from slovakia. Zookeys 2019 862, 129–152. https://doi.org/
10.3897/zookeys.862.25992.
Palomo, L.J., Gisbert, J. y, Blanco, J.C., 2007. Atlas y Libro Rojo de los Mamíferos
Terrestres de Espa˜
na. Direcci´
on. General para la Biodiversidad. SECEM-SECEMU,
Madrid, p. 588.
Petersen, F.T., Meier, R., Kutty, S.N., Wiegmann, B.M., 2007. The phylogeny and
evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four
molecular markers. Mol. Phylogenet. Evol. 45 (1), 111–122. https://doi.org/
10.1016/J.YMPEV.2007.04.023.
Poh, K.C., Skvarla, M., Evans, J.R., Machtinger, E.T., 2020. Collecting deer keds (Diptera:
Hippoboscidae: Lipoptena nitzsch, 1818 and Neolipoptena Bequaert, 1942) and ticks
(Acari: Ixodidae) from hunter-harvested deer and other cervids. J. Insect Sci. 20 (6),
19. https://doi.org/10.1093/jisesa/ieaa024.
Reeves, W.K., Lloyd, J.E., 2019. Louse ies, keds, and bat ies (Hippoboscoidea) Medical
and Veterinary Entomology, 3rd edn (ed. by G. Mullen & L. Durden), pp. 421–438.
Academic Press, London. https://doi.org/10.1016/B978-0-12-814043-7.00020-0.
Salvetti, M., Bianchi, A., Marangi, M., Barlaam, A., Giacomelli, S., Bertoletti, I., Roy, L.,
Giangaspero, A., 2020. Deer keds on wild ungulates in northern Italy, with a
taxonomic key for the identication of Lipoptena spp. of Europe. Med. Vet. Entomol.
34 (1), 74–85. https://doi.org/10.1111/mve.12411.
Skvarla, M.J., Machtinger, E.T., 2019. Deer keds (Diptera: Hippoboscidae: Lipoptena and
Neolipoptena) in the United States and Canada: new state and county records,
pathogen records, and an illustrated key to species. J. Med. Entomol. 56 (3),
744–760. https://doi.org/10.1093/jme/tjy238.
Tamura, K., Nei, M., Kumar, S., 2004. Prospects for inferring very large phylogenies by
using the neighbor-joining method. Proc. Natl. Acad. Sci. U. S. A. 101, 11030–11035.
https://doi.org/10.1073/pnas.0404206101.
Tamura, K., Stecher, G., Kumar, S., 2021. MEGA11: molecular evolutionary genetics
analysis version 11. Mol. Biol. Evol. 38, 3022–3027.
Tiawsirisup, S., Yurayart, N., Thongmeesee, K., Sri-in, C., Akarapas, C.,
Rittisornthanoo, G., Bunphungbaramee, N., Sipraya, N., Maikaew, U.,
Kongmakee, P., Saedan, A., 2023. Possible role of Lipoptena fortisetosa (Diptera:
Hippoboscidae) as a potential vector for Theileria spp. in captive Eld’s deer in Khao
Kheow open zoo, Thailand. Acta Trop. 237, 106737 https://doi.org/10.1016/j.
actatropica.2022.106737.
V´
azquez, L., 2010. Ectopar´
asitos presentes en corzos (Capreolus capreolus) de Galicia (NO
Espa˜
na). Galemys Span. J. Mammal. 22, 243–253. https://doi.org/10.7325/
galemys.2010.ne.a15.
Wechtaisong, W., Sri-in, C., Thongmeesee, K., Yurayart, N., Akarapas, C.,
Rittisornthanoo, G., Bunphungbaramee, N., Sipraya, N., Bartholomay, L.C.,
Maikaew, U., Kongmakee, P., Saedan, A., Tiawsirisup, S., 2023. Diversity of
Anaplasma and novel Bartonella species in Lipoptena fortisetosa collected from captive
Eld’s deer in Thailand. Front. Vet. Sci. 10, 1247552 https://doi.org/10.3389/
fvets.2023.1247552.
Werszko, J., Steiner-Bogdaszewska, Z., Je˙
zewski, W., Szewczyk, T., Kuryło, G.,
Wołkowycki, M., Wr´
oblewski, P., Karbowiak, G., 2020. Molecular detection of
Trypanosoma spp. in Lipoptena cervi and Lipoptena fortisetosa (Diptera:
Hippoboscidae) and their potential role in the transmission of pathogens.
Parasitology 147 (14), 1629–1635. https://doi.org/10.1017/S0031182020001584.
Yamauchi, T., Tsuda, Y., Sato, Y., Murata, K., 2011. Pigeon louse y, Pseudolynchia
canariensis (Diptera: Hippoboscidae), collected by dry-ice trap. J. Am. Mosq. Control
Assoc. 27 (4), 441–443. https://doi.org/10.2987/11-6183.1.
Zug, R., Hammerstein, P., 2012. Still a host of hosts for Wolbachia: analysis of recent data
suggests that 40% of terrestrial arthropod species are infected. PLoS One 7, 38544.
https://doi.org/10.1371/JOURNAL.PONE.0038544.
M.A. Gonz´
alez et al.
Veterinary Parasitology 332 (2024) 110300
11