Biologia

https://doi.org/10.1007/s11756-021-00822-w

SHORT COMMUNICATION

Morphological anomalies found in female Culicoides midges

(Diptera: Ceratopogonidae)
David W. Ramilo 1,2 & Ana Maria Filipe 3 & Javier Lucientes 4 & Maria Teresa Rebelo 3 & Luis Cardoso 5 &
Isabel Pereira da Fonseca 1

Received: 12 February 2021 / Accepted: 12 June 2021

# Institute of Zoology, Slovak Academy of Sciences 2021

Abstract
Females from genus Culicoides are responsible for transmitting important pathogenic agents to animals and humans, including
bluetongue, Schmallenberg and African horse sickness viruses and filarids. Species identification in laboratory is usually
performed by visualization of their wing pattern and/or using other morphological characters. During the National
Entomological Surveillance Program for Bluetongue Disease performed in Portugal (2005–2013) several Culicoides midges
were captured near domestic ruminants and, more recently, others were collected in a zoo from the Metropolitan Area of Lisbon
near sylvatic animals (2018–2019). From a total of 5,364 analysed specimens, some morphological alterations were observed in
154 specimens (2.9 %) from three different Culicoides spp. (Culicoides obsoletus, Culicoides scoticus and Culicoides dewulfi) on
palpi (e.g. defective sensorial pits), antennae (e.g. antennae with defective or fused articles), abdomen (e.g. abdomen with
aberrant structures) and spermathecae (e.g. specimens with one and four spermathecae). These types of anatomical alterations
probably occur due to genetic or morphogenetic malformation. Also, they are compatible with midge survival and thus main-
tained in adult specimens. The report of different types of anatomical anomalies is important, since these morphological
alterations can lead to incorrect species identification during sample analysis. Thus, this work gives essential information
concerning some morphological anomalies that can be observed in Culicoides biting midges, being their first reference in
C. scoticus and C. dewulfi species.

Keywords Culicoides . Morphology . Anomalies . Sylvatic animals . Domestic ruminants

Introduction African horse sickness) and humans (e.g. mansonellosis)

(Purse et al. 2015; Ta-Tang et al. 2018).
Adult females of some Culicoides spp. (Diptera: Bluetongue virus (BTV) causes bluetongue disease (BT),
Ceratopogonidae) are vectors of pathogenic agents that cause affecting both domestic and wild ruminants (Pérez et al.
disease in animals (e.g. Bluetongue, Schmallenberg and 2012). Some BT outbreaks have occurred in southern

David W. Ramilo and Ana Maria Filipe contributed equally for this work.

* Luis Cardoso 3
CESAM – Centre for Environmental and Marine Studies, Department
lcardoso@utad.pt of Animal Biology, Faculty of Sciences, University of Lisbon,
Lisbon, Portugal
4
1
CIISA – Centro de Investigação Interdisciplinar em Sanidade Parasitology and Parasitic Diseases, Department of Animal Pathology
Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, (Animal Health), Veterinary Faculty, University of Zaragoza,
Avenida da Universidade Técnica, Lisboa 1300-477, Portugal Zaragoza, Spain
5
2
Faculty of Veterinary Medicine, Lusófona University of Humanities Department of Veterinary Sciences, and Animal and Veterinary
and Technologies, Lisbon, Portugal Research Centre (CECAV), University of Trás-os-Montes e Alto
Douro (UTAD), Vila Real 5000 − 801, Portugal
 Biologia

Table 1 Identification of Culicoides spp. based on the 3rd palpus

segment length/width ratio and on the spermatecae size (Ramilo 2016)

Culicoides spp. Lenght/width ratio Spermathecae size (µm)

Culicoides obsoletus <2.7 37.5–62.5

Culicoides scoticus >2.7 57–95

Europe since 1998, including Portugal in 2004 (Pérez et al.

2012). Several programs aimed at monitoring Culicoides pop-
ulations have been established in BT-affected regions
(Mehlhorn et al. 2007). In Portugal, the National
Entomological Surveillance Program (NESP) for BT was im-
plemented for eight years (2005–2013) (Ramilo et al. 2017).
New Culicoides spp., such as Culicoides imicola Kieffer,
1913, Culicoides obsoletus (Meigen, 1818), Culicoides
scoticus Downes & Kettle, 1952, and new wild reservoir hosts
have been implicated in the transmission of arboviruses (Purse
et al. 2015). However, the information regarding Culicoides
spp. associated with natural environments, which can possibly
maintain BTV in sylvatic cycles involving only wild rumi-
nants, is scarce (Rossi et al. 2019).
In Europe, some Culicoides spp. are known as vectors of
BTV, including C. imicola, C. obsoletus, C. scoticus
(Hoffmann et al. 2009; Carpenter et al. 2008), Culicoides
chiopterus (Meigen, 1830) (Dijkstra et al. 2008; Venail et al.
2012), Culicoides dewulfi Goetghebuer, 1936 (Meiswinkel
et al. 2008), Culicoides pulicaris (Linnaeus, 1758) and
Culicoides punctatus (Meigen, 1804) (Purse et al. 2015).
Several Culicoides spp. are easily identified by observing
their wing pattern. However, some of them (e.g. C. obsoletus,
C. scoticus) have very similar wing pattern and other morpho-
Fig. 2 Morphological anomalies observed on the palpus. a, b Fused
articles (black arrows); c-e defective articles (black arrows). Scale bars
18.29 μm (a, e); 14.29 μm (b) and 19.81 μm (c, d)
Fig. 1 Morphological anomalies
observed in the 3rd palpus
segment. a Unequal length (black
and yellow arrows); b, d double
sensorial pit (black arrows); c
defective sensorial pit (black
arrow). Scale bars 20.87 μm (a, b)
and 11.34 μm (c, d)
Biologia
Fig. 3 Morphological anomalies
on the antenna. a Defective (black
arrow) and fused (blue arrows)
articles; b fused articles (blue
arrow). Scale bars 28.8 μm (a)
and 14.4 μm (b)
logical aspects must be observed to clearly identify these
midges.
Morphological anomalies have already been referred in
Culicoides spp. from Central and South America (Felippe-
Bauer and Silva 2006) and others from Nearctic and
Palearctic ecozones (Callot and Kremer 1963; Smith and
Perry 1967; McKeever et al. 1997), including C. obsoletus
(Callot and Kremer 1963; De Heredia and Lafuente 2011).
These anomalies in structures with specific functions can pos-
sibly affect insects’ life activities (Felippe-Bauer and Silva
2006). However, works concerning these morphological alter-
ations in Culicoides specimens are scarce.
The aim of this work is to describe morphological alter-
ations found in some Culicoides spp. captured during the
NESP near domestic ruminants and in a zoo from
Metropolitan Area of Lisbon near sylvatic animals, taking into
account the anatomical features considered to be normal in
those arthropods.
Materials and methods
Culicoides specimens were collected with CDC light traps (CDC
miniature black light model 1212, John Hock, USA) fitted with
4 W UV bulbs, suction fans and LCS-2 Photoswitch systems.
Traps were placed near animal enclosures (up to 30 m), approx-
imately 1.70 m above ground and operated from dusk to dawn,
once a week, throughout the year. Specimens were collected into
flasks containing 300 ml of 70 % ethanol and a few drops of
commercial liquid soap.
Fig. 4 Aberrant abdominal
structures (black arrows). Scale
bars 16.67 μm (a) and 25 μm (b)
Observation of Culicoides wing pattern was performed
using stereoscope microscopy (Olympus SZ51). Specimens
with similar/indistinct wing patterns were dissected into dif-
ferent body parts (head, thorax, abdomen and wings) using 26
Gauge (0.404 mm diameter) needles, mounted in glass slides
using Hoyer’s medium and dried in an incubator at 37 °C for
3–4 days. Specimens were identified to species using optical
microscopy (Olympus BX50 microscope) and different iden-
tification keys (Ramilo 2016; Mathieu et al. 2012). According
to these identification keys, the format and ratio length/width
(L/W) of the 3rd palpus segment and spermathecae size are
useful characteristics to distinguish Obsoletus complex spe-
cies (e.g. C. obsoletus, C. scoticus), as shown in Table 1.
Photos of morphological anomalies were obtained with an
Olympus DP10 camera.
Results
From 5,364 analysed specimens, the following anatomical
anomalies were observed in 154 biting midges (2.9 %): un-
equal lengths (Fig. 1a), doubled sensorial pits (Fig. 1b and d)
and defective sensorial pits (Fig. 1c) on the 3rd palpus seg-
ment; palpi (Fig. 2) and antennae (Fig. 3) with defective or
fused articles; abdomen with aberrant structures (Fig. 4); and
presence of one (Fig. 5a-c), three (Fig. 5d-h) and four (Fig. 5i)
functional spermathecae, instead of two functional and one
rudimentary spermatheca.
Morphological anomalies were observed in three different
Culicoides spp.: C. obsoletus, C. scoticus and C. dewulfi. In
 Biologia

Fig. 5 Irregular number of spermathecae. a-c One spermatheca; d-h three spermathecae; i four spermathecae. Scale bars 20 μm (a), 13.33 μm (b),
53.33 μm (c), 26.67 μm (d), 40 μm (e), 27.83 μm (f), 24.44 μm (g), 21.5 μm (h) and 37.1 μm (i)

only one specimen of C. obsoletus two anatomical alterations Discussion

(palpus and antenna with defective articles) were observed.
The distribution of morphological anomalies per species is The present report provides essential information about mor-
presented in Table 2. phological anomalies observed in three different Culicoides
spp.: C. obsoletus, C. scoticus and C. dewulfi.
Biologia

Table 2 Morphological
anomalies per Culicoides spp Morphological anomaly Culicoides Culicoides Culicoides Total
obsoletus scoticus dewulfi

3rd palpus segment Defective 9 1 0 10

sensorial pit
Double sensorial 10 0 0 10
pit
Unequal length 24 11 0 35
Palpus Defective articles 20 4 0 24
Fused articles 15 2 0 17
Antenna Defective articles 3 0 0 3
Fused articles 2 1 0 3
Abdomen Abnormal 8 2 0 10
structures
Number of One 4 1 0 5
spermathecae Three 31 5 1 37
Four 1 0 0 1

Previous works reported some morphological anomalies in
Culicoides specimens (Callot and Kremer 1963; Smith and
Perry 1967; McKeever et al. 1997; De Heredia and Lafuente
2011; Korneva 1965), including C. obsoletus specimens with
three spermathecae (De Heredia and Lafuente 2011).
To the best of our knowledge, this work describes for the
first time the following morphological alterations in
Culicoides specimens: defective sensorial pits and unequal
length of the 3rd palpus segment, palpus with defective arti-
cles, and specimens with one and four spermathecae.
Furthermore, this is the first report of anatomical anomalies
in C. scoticus and C. dewulfi.
The unequal length of the 3rd palpus segment in the same
specimen, the presence of three functional spermathecae in-
stead of the common two functional spermathecae and a rudi-
mentary one were the most common morphological alter-
ations observed in all three species.
Since each Culicoides female can deposit 10 to 675 eggs,
depending on species, there is a high probability for anatom-
ical alterations to occur due to genetic or morphogenetic mal-
formation (Felippe-Bauer and Silva 2006; De Heredia and
Lafuente 2011). The referred anomalies were observed be-
cause they are compatible with midge survival and thus main-
tained in adult specimens. On the other hand, Culicoides with
anatomical alterations that are not compatible with survival do
not reach the adult phase or do not live long enough to be
captured.
Culicoides specimens and the first reference of anatomical
anomalies for C. scoticus and C. dewulfi.
Acknowledgements We would like to express our sincere gratitude to all
the people who have collaborated both directly and indirectly in the
Portuguese National Entomological Surveillance Programme (DGV/
FMV, 2005–2013) and to Dr Lídia Gomes for laboratorial assistance.
Also, thank the financial support to CESAM (UID/AMB/50017/2019),
to FCT/MCTES through national funds, and the co-funding by the
FEDER, within the PT2020 Partnership Agreement and Compete 2020.
Authors’ contributions DWR and IPdF conceptualized the study. DWR
and AMF performed sample analysis and wrote the manuscript. JL,
MTR, LC and IPdF co-supervised the study and revised the manuscript.
All authors read and approved the final manuscript.
Funding This work was financed by national funds through FCT –
Foundation for Science and Technology, I.P., within the scope of the
project UIDB/00276/2020. This study was also funded by the project
UIDB/CVT/00772/2020 supported by the Portuguese Science and
Technology Foundation (FCT). DWR held post-doctoral grant
SFRH/BPD/115202/2016.
Data availability All data generated or analysed during this study are
included in this published article.
Code availability Not applicable.
Declarations
Conflict of interest The authors declare that they have no competing
interests.

Conclusions Consent to participate Not applicable.

This work gives essential information concerning morpholog- Consent for publication Not applicable.
ical anomalies that can be observed in Culicoides biting
Ethics approval Not applicable.
midges, being the first reference of some alterations in

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