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First report of 13 species of Culicoides (Diptera: Ceratopogonidae) in mainland Portugal and Azores by morphological and molecular characterization

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First report of 13 species of Culicoides (Diptera: Ceratopogonidae) in mainland Portugal and Azores by morphological and molecular characterization
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/224847785 First Report of 13 Species of Culicoides (Diptera:Ceratopogonidae) in Mainland Portugal andAzores by Morphological...  Article   in  PLoS ONE · April 2012 DOI: 10.1371/journal.pone.0034896 · Source: PubMed CITATIONS 14 READS 98 9 authors , including: Some of the authors of this publication are also working on these related projects: Kids Fellows Project   View projectVectors distribution /Distribución de vectores   View projectIsabel Pereira Da FonsecaTechnical University of Lisbon 59   PUBLICATIONS   282   CITATIONS   SEE PROFILE Anthony James WilsonThe Pirbright Institute 55   PUBLICATIONS   1,078   CITATIONS   SEE PROFILE José MeirelesTechnical University of Lisbon 31   PUBLICATIONS   100   CITATIONS   SEE PROFILE Javier LucientesUniversity of Zaragoza 124   PUBLICATIONS   2,201   CITATIONS   SEE PROFILE All content following this page was uploaded by Isabel Pereira Da Fonseca on 01 December 2016. The user has requested enhancement of the downloaded file.  First Report of 13 Species of   Culicoides   (Diptera:Ceratopogonidae) in Mainland Portugal and Azores byMorphological and Molecular Characterization David W.Ramilo 1 . ,SurayaDiaz 1 . ,IsabelPereiradaFonseca 1 * ,Jean-ClaudeDele´ colle 2 ,AnthonyWilson 3 ,Jose´  Meireles 1 , Javier Lucientes 4 , Rita Ribeiro 1 , Fernando Boinas 1 1 Interdisciplinary Centre of Research in Animal Health (CIISA), Veterinary Medicine Faculty, Technical University of Lisbon, Lisbon, Portugal,  2 Laboratory of Entomology,Institute of Parasitology and Tropical Pathology, Faculty of Medicine, Strasbourg, France,  3 Pirbright Laboratory, Institute for Animal Health, Pirbright, Woking, Surrey,United Kingdom,  4 Parasitology and Parasitic Diseases, Department of Animal Pathology (Animal Health), Veterinary Faculty, University of Zaragoza, Zaragoza, Spain Abstract The genus  Culicoides  (Diptera: Ceratopogonidae) contains important vectors of animal and human diseases, includingbluetongue, African horse sickness and filariosis. A major outbreak of bluetongue occurred in mainland Portugal in 2004,forty eight years after the last recorded case. A national Entomological Surveillance Plan was initiated in mainland Portugal,Azores and the Madeira archipelagos in 2005 in order to better understand the disease and facilitate policy decisions.During the survey, the most prevalent  Culicoides  species in mainland Portugal was  C. imicola  (75.3%) and species belongingto the Obsoletus group (6.5%). The latter were the most prevalent in Azores archipelago, accounting for 96.7% of the totalspecies identified. The Obsoletus group was further characterized by multiplex Polymerase Chain Reaction to species levelshowing that only two species of this group were present:  C. obsoletus sensu strictu  (69.6%) and  C. scoticus  (30.4%). Ninespecies of   Culicoides  were detected for the first time in mainland Portugal:  C. alazanicus ,  C. bahrainensis ,  C. deltus ,  C. lupicaris , C. picturatus ,  C. santonicus ,  C. semimaculatus ,  C. simulator   and  C. subfagineus . In the Azores,  C. newsteadi   and  C.circumscriptus  were identified for the first time from some islands, and bluetongue vectors belonging to the Obsoletusgroup ( C. obsoletus  and  C. scoticus ) were found to be widespread. Citation:  Ramilo DW, Diaz S, Pereira da Fonseca I, Dele´colle J-C, Wilson A, et al. (2012) First Report of 13 Species of   Culicoides  (Diptera: Ceratopogonidae) inMainland Portugal and Azores by Morphological and Molecular Characterization. PLoS ONE 7(4): e34896. doi:10.1371/journal.pone.0034896 Editor:  Ulrike Gertrud Munderloh, University of Minnesota, United States of America Received  August 23, 2011;  Accepted  March 8, 2012;  Published  April 19, 2012 Copyright:    2012 Ramilo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  Sources of funding include Direcc¸a˜o-Geral de Veterina´ria of Portugal and Faculty of Veterinary Medicine of the Technical University of Lisbon. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: ifonseca@fmv.utl.pt .  These authors contributed equally to this work. Introduction The genus  Culicoides   (Diptera: Ceratopogonidae) includes 1.316recognised species [1]. Adult female  Culicoides   are haematophagousand feed on a wide range of hosts including humans, livestock andother mammals, amphibians, and birds.  Culicoides   can transmitpathogens responsible for several diseases with veterinary andpublic health significance, including bluetongue (BT) in ruminants, African horse sickness (AHS) in equids, epizootic hemorrhagicdisease (EHD) in deer, and filarial diseases such as onchocercosisand mansonellosis, which affect various species including humans[2,3]. Bites may also cause hypersensitivity reactions in equines, acondition known as ‘‘sweet itch’’.  Culicoides   larval habitats areaquatic or semi-aquatic, and different species utilize habitats witha broad range of salinity and acidity, for example salt marshes andpeat bogs. Species of veterinary interest generally feed on livestock and horses, and breed in associated habitats such as leaf litter,rotting manure and organically-enriched mud. An outbreak of AHS occurred in mainland Portugal in 1989[4], and BT transmission resumed in mainland Portugal in 2004[5,6], 48 years after the last recorded case. This occurred during aperiod in which bluetongue virus (BTV) activity was increasing across Europe as a whole.  Culicoides  -borne viruses are introducedinto new regions through windborne transportation of   Culicoides   orby accidental importation of infected hosts [5,6]. Climate changewas thought to have facilitated spread into Europe [7,8], whichmay have allowed species such as  C. imicola  , which prefer hot, dryconditions, to expand their range in southern Europe as well asincreasing vector density, their seasonal activity period and/ortheir susceptibility to infection with viruses. As a result of increasing global trade [9] and the proximity of the IberianPeninsula to the North of Africa, the likelihood of new  Culicoides  -borne diseases being introduced into Portugal is likely to remainhigh for the foreseeable future.In recognition of this, the Portuguese authorities established aNational Entomological Surveillance Program (ESP) in 2005. Theobjective was to collect and identify  Culicoides   biting midges inorder to better understand the distribution of different species,thereby assisting policy formulation. The Program covers all partsof Portugal including mainland Portugal, the Azores (Sa˜o Miguel,Santa Maria, Terceira, Graciosa, Sa˜o Jorge, Pico, Faial, Floresand Corvo islands) and Madeira (Madeira and Porto Santoislands) archipelagos. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34896  Figure 1. Geographical Units in mainland Portugal and Azores archipelago: 1 GU=50 km 6 50 km.  Azores archipelago GU – 46: SantaMaria island; 47 and 48: Sa˜o Miguel island; 49: Terceira island; 50: Graciosa island; 51: Sa˜o Jorge island; 52: Pico island; 53: Faial island; 54: Flores island;55: Corvo island.doi:10.1371/journal.pone.0034896.g001 Table 1.  Proportion of   Culicoides  species collected on mainland Portugal from 2005 to 2010 and identified solely from wingpattern. Species % of total (both sexes) Presence in Geographical Units 1 C. circumscriptus  (Kieffer, 1918) 0.2 3–5; 7; 10; 12; 13; 16–18; 20; 22–40; 42; 44 C. imicola  (Kieffer, 1913) 75.3 16–18; 20–40; 42; 44 C. maritimus  (Kieffer, 1924) 0.1 7; 12; 17; 23; 26; 27; 29; 30; 31; 34–36; 38–40; 42; 44 C. newsteadi   (Austen, 1921) 2.1 1; 3–7; 10; 12–18; 20–40; 42; 44 C. pulicaris  (Linne´, 1758) 0.1 1; 3–10; 12; 16–18; 20–26; 28; 29; 31; 40; 42; 44 C. punctatus  (Meigen, 1804) 6.4 1; 3–10; 12–18; 20–40; 42; 44 C. univittatus  (Vimmer, 1932)  , 0.1 5; 7; 10; 12; 13; 17; 18; 20; 22–27; 29–31; 34–36; 38–40; 42; 44Obsoletus group * 1 6.5 1; 3–10; 13–18; 20–31; 33–40; 42; 44Other  Culicoides  species 9.3 1; 3–10; 12; 13; 15–18; 20–40; 42; 44 Total 100 1 See Figure 1.*Includes males and females from  C. obsoletus sensu stricto ,  C. scoticus ,  C. chiopterus  and  C. dewulfi   species (these cannot easily be differentiated by morphology). 1 280 females were identified to species level via multiplex PCR:  C. obsoletus s.s. -  69.6%;  C. scoticus  - 30.4%;  C. chiopterus  – 0%;  C. dewulfi   – 0%.doi:10.1371/journal.pone.0034896.t001 Report of Thirteen  Culicoides  Species in PortugalPLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34896  C. imicola   and the Obsoletus and Pulicaris groups are easilyidentified based on their wing patterns. However, identification of species in these groups to species level, or identification of otherspecies, requires more detailed morphological study or the use of molecular techniques. This paper describes the use of morpho-logical and molecular techniques to identify collections to specieslevel during the ESP from 2005 to 2010 including thirteen Culicoides   species reported for the first time in mainland Portugal orfrom Azores archipelago. Materials and Methods Insect collection Culicoides   were collected using miniature CDC light traps (CDCminiature blacklight model 1212, John Hock, USA) fitted with 4WUV bulbs, suction fans and LCS-2 Photoswitch systems. AlthoughOnderstepoort light traps may be more effective at collecting  Culicoides   [10], the miniature CDC light trap is considerably lighter Table 2.  Frequency and distribution of   Culicoides  species identified in a subsample of 5% of specimens collected in mainlandPortugal which could not be identified using the wing pattern keys or were not priority for ESP for bluetongue. Species % of total (both sexes) Presence in Geographical Units 1 C. achrayi   (Kettle & Lawson, 1955) 47.3 3; 5; 7; 9; 17; 18; 25; 39 C. alazanicus  X (Dzhafarov, 1961) 1.2 40 C. bahrainensis  X (Boorman, 1989) 0.2 36; 38 C. corsicus  (Kremer, Leberre & Beaucournu-S., 1971) 0.1 23; 35; 36 C. deltus  X (Edwards, 1979) 0.2 9 C. fascipennis  (Staeger, 1839) 3.8 5; 7; 9; 12; 18; 25; 26; 30; 39 C. festivipennis  (Kieffer, 1914) 5.8 7; 12; 18; 23; 25; 30; 35; 40 C. gejgelensis  (Dzhafarov, 1964) 6.7 7; 12; 18; 20; 22; 23; 26; 30; 36; 44 C. heliophilus  (Edwards, 1921) 0.2 18 C. heteroclitus  (Kremer & Callot, 1965) 3.3 7; 9; 25 C. impunctatus  (Goetghebuer, 1920) 0.1 4; 20 C. indistinctus  (Khalaf, 1961) 0.6 7; 9; 18; 25; 32; 35 C. jumineri   (Callot & Kremer, 1969) 2.6 5; 7; 17; 18; 23; 32; 35; 38; 39 C. jurensis  (Callot, Kremer & De´duit, 1962) 0.2 30; 36; 39 C. kibunensis  (Tokunaga, 1937) 0.4 23 C. longipennis  (Khalaf, 1957) 5.1 7; 23; 32; 35; 39 C. lupicaris  X (Downes & Kettle, 1952) 0.1 25 C. nubeculosus  (Meigen, 1930) 0.4 23; 27; 29; 40 C. odiatus  (Austen, 1921) 0.1 9; 18; 23; 30 C. parroti   (Kieffer, 1922) 1.9 23; 39 C. picturatus  X (Kremer & De´duit, 1961) 1.7 23 C. pseudopallidus  (Khalaf, 1961) 1.0 7; 23; 35; 38 C. puncticollis  (Becker, 1903) 0.9 23; 32 C. sahariensis  (Kieffer, 1923) 0.1 39 C. santonicus  X (Callot, Kremer, Rault & Bach, 1968) 11.5 7; 18; 22–24; 40 C. semimaculatus  X (Clastrier, 1958) 0.1 40 C. simulator   X (Edwards, 1939) 0.2 7 C. subfagineus  X (Dele´colle & Ortega, 1998) 0.7 7; 18; 30; 31; 40 C. subfascipennis  (Kieffer, 1919) 2.1 7; 23; 25; 30; 32; 39 C. vexans  (Staeger, 1839) 1.8 7 Total 100 X Species reported for the first time in mainland Portugal.doi:10.1371/journal.pone.0034896.t002 Table 3.  Total  Culicoides  species reported in the islands of Azores Archipelago (N=30879 specimens). Species % of total (both sexes) Geographical Units 1 C. circumscriptus  2.5 46–53 C. newsteadi   0.8 46–52Obsoletus group * 1 96.7 46–55 Total 100 *Includes males and females from  C. obsoletus s. s. ,  C. scoticus ,  C. chiopterus  and C. dewulfi   species (these cannot easily be differentiated by morphology). 1 8 females were identified to species level by multiplex PCR:  C. obsoletus s.s. - 0%;  C. scoticus  - 100%;  C. chiopterus -  0%;  C. dewulfi   – 0%.doi:10.1371/journal.pone.0034896.t003 Report of Thirteen  Culicoides  Species in PortugalPLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34896  and can be powered by a battery pack rather than requiring amains electrical connection, making it easier to deploy in remoteareas. CDC traps are also effective at attracting mosquitoes,allowing the Surveillance Program to be adapted for mosquitosurveillance if required. CDC traps are also used by other national Culicoides   surveillance schemes in Europe, such as Spain andFrance, and the use of a consistent trapping protocol facilitatescomparison of results across different schemes.To ensure systematic coverage, Portugal was divided into 57geographical units (GUs), each 50 km 6 50 km (Figure 1). Four of these were not considered to be of epidemiological interest due tolow livestock densities and were not sampled. Within eachremaining GU, two sheep, goat, cattle or mixed farms with atleast five animals were selected. These farms were at least 2.5 kmfrom the coast and separated by at least 10 km. The farms werenot allowed to use insecticides during the study.CDC traps were placed within 30 m of animal enclosures,1.70 m above ground. The LCS-2 Photoswitch system automat-ically switched the trap on at dusk and off after dawn. Each trapwas operated for one night per week throughout the year. Insectswere collected into flasks containing 75% of 70% ethanol and 25%of antifreeze, in a final volume of 500 ml, and were identified atthe Interdisciplinary Centre of Research in Animal Health (CIISA)at the Faculty of Veterinary Medicine, Technical University of Lisbon (FMV-UTL). Collection of environmental data Minimum and maximum temperatures and relative humiditywere obtained from the closest meteorological station to thetrapping site (Annex S1). Maximum temperature was recorded onthe day the trap was set and minimum temperature on the day thetrap was emptied, to reflect conditions corresponding to theevening/night of collection. Humidity was recorded at15.00 hours on the day the trap was set and 09.00 hours on theday of collection for the same reason. Wind direction and averagespeed were recorded on the site on the day of collection.The presence of livestock species and bodies of water on thefarm, human housing and other livestock farms within 10 km wasrecorded by state veterinary staff during the placement of traps, bycompletion of a standardized questionnaire. Land cover types inthe same parish were extracted from the cartographic farming database maintained by the Institute for Agricultural Financing of the Portuguese Ministry of Agriculture. The proximity of bodies of water, human housing, other farms and vegetation to the trapping site was later verified using aerial photography. Morphological identification Identification of collected  Culicoides   was first attempted using stereoscope microscopy (SM) and wing patterns [11,12,13,14],allowing the identification of   C. imicola   (Kieffer), species from theObsoletus group [  C. obsoletus   (Meigen),  C. scoticus   (Downes andKettle),  C. chiopterus   (Meigen) and  C. dewulfi   (Goetghebuer)],  C. pulicaris   (Linnaeus),  C. newsteadi   (Austen),  C. circumscriptus   (Kieffer), C. punctatus   (Meigen),  C. maritimus   (Kieffer) and  C. univittatus  (Vimmer). This information was supplied to the CentralVeterinary Services (CVS) on a weekly basis in order to supportpolicy decisions. Culicoides   spp. that could not be identified using keys based onwing pattern and those that were not priority for the ESP forbluetongue (‘‘other  Culicoides   species’’ in table 1) were preserved in96% alcohol. Five percent were randomly selected from GUswhere at least 65 ‘‘other  Culicoides   species’’ were collected(Figure 1). These specimens were dissected into different bodyparts (head, thorax, abdomen, wings and legs) using 26 Gauge     T   a    b    l   e    4 .      C    u     l     i    c    o     i     d    e    s    s    p    e    c     i    e    s    r    e    p    o    r    t    e     d     i    n    t     h    e    n     i    n    e     I    s     l    a    n     d    s    o     f     A    z    o    r    e    s     A    r    c     h     i    p    e     l    a    g    o     (     N   =     3     0     8     7     9    s    p    e    c     i    m    e    n    s     ) .     S   p   e   c    i   e   s    I   s    l   a   n    d   s   o    f    A   z   o   r   e   s    A   r   c    h    i   p   e    l   a   g   o    S   a   n    t   a    M   a   r    i   a    S   a    ˜   o    M    i   g   u   e    l    T   e   r   c   e    i   r   a    G   r   a   c    i   o   s   a    S   a    ˜   o    J   o   r   g   e    P    i   c   o    F   a    i   a    l    F    l   o   r   e   s    C   o   r   v   o    A    B    A    B    A    B    A    B    A    B    A    B    A    B    A    B    A    B      O     b    s    o     l    e    t    u    s     G    r    o    u    p         {       2      9   -      1      0   -      0      8      8     1 .     2     9      ,       0      9   -      0      5      8     9 .     0     7      ,       0      9   -      0      5      7     7 .     3     7       1      6   -      1      1   -      0      8      7     1 .     4     3      ,       0      9   -      0      5      9     9 .     6     5      ,       0      9   -      0      5      9     7 .     4     2      ,       0      9   -      0      5      7     0 .     5     9       1      3   -      0      8   -      0      8      9     9 .     8       2      9   -      0      9   -      0      9      8     3 .     3      C .    s    c    o    t     i    c    u    s           1       0      6   -      0      9   -      1      0      0 .     0     5     *       3      1   -      0      8   -      0      8      0 .     2     1       2      2   -      0      6   -      0      9      2 .     1     9       1      8   -      1      2   -      0      9      7 .     1     4     *       1      3   -      1      0   -      1      0      0 .     0     1     *       0      3   -      1      1   -      0      8      0 .     0     4     *     *       1      9   -      1      1   -      0      8      0 .     9       1      1   -      0      8   -      1      0      0 .     2     *       1      6   -      1      2   -      0      9      1     6 .     7     *      C .    n    e    w    s    t    e    a     d     i       2      8   -      1      0   -      0      8      1     1 .     4     8       1      9   -      1      0   -      0      5      1 .     4     4       0      1   -      0      7   -      0      8      5 .     8     4       0      3   -      1      2   -      0      9      7 .     1     4       2      0   -      0      7   -      1      0      0 .     0     1       0      6   -      0      7   -      1      1      0 .     0     1     N     R     N     R     N     R      C .    c     i    r    c    u    m    s    c    r     i    p    t    u    s       2      9   -      1      0   -      0      8      7 .     1     9       1      9   -      1      0   -      0      5      9 .     2     8       2      8   -      0      7   -      1      0      1     4 .     6       0      2   -      1      2   -      0      9      1     4 .     2     9       2      7   -      0      7   -      1      0      0 .     3     3       2      0   -      0      9   -      0      9      2 .     5     4       0      4   -      0      8   -      0      9      2     8 .     5     1     N     R     N     R     A  –     D    a    t    e    o     f    t     h    e     f     i    r    s    t    s    p    e    c     i    m    e    n     i     d    e    n    t     i     f     i    c    a    t     i    o    n   ;     B  –     P    e    r    c    e    n    t    a    g    e     (    o     f    t     h    e    t    o    t    a     l     )    o     f      C    u     l     i    c    o     i     d    e    s     s    p    e    c     i    m    e    n    s     f    o    u    n     d     i    n    t     h    e     i    s     l    a    n     d    u    n    t     i     l     N    o    v    e    m     b    e    r     2     0     1     0   ;     N     R  –     N    o    t    r    e    p    o    r    t    e     d   ;         {      O     b    s    o     l    e    t    u    s     G    r    o    u    p     i    n    c     l    u     d    e    t     h    e     f    e    m    a     l    e    s    o     f    t     h    e      C .    o     b    s    o     l    e    t    u    s    s .    s .  ,      C .    s    c    o    t     i    c    u    s  ,      C .    c     h     i    o    p    t    e    r    u    s     a    n     d      C .     d    e    w    u     l     f     i     s    p    e    c     i    e    s   ;           1      A     l     l    t     h    e    s    p    e    c     i    m    e    n    s     f    o    u    n     d    a    r    e    r    e     f    e    r    r    e     d    t    o    t     h    e    m    a     l    e    g    e    n    r    e ,    e    x    c    e    p    t     *    w     h    e    r    e     5     f    e    m    a     l    e    s    w    e    r    e     i     d    e    n    t     i     f     i    e     d     b    a    s    e     d    o    n    m    o    r    p     h    o     l    o    g     i    c    a     l     f    e    a    t    u    r    e    s    a    n     d     *     *    w     h    e    r    e     8     f    e    m    a     l    e    s    w     h    e    r    e     i     d    e    n    t     i     f     i    e     d     b    y    m    u     l    t     i    p     l    e    x     P     C     R     [     1     5     ] .     T     h    e     d    a    t    e    s     i    n    n    o    r    m    a     l    w    r     i    t     i    n    g    r    e     f    e    r    t    o    s    p    e    c     i    e    s    t     h    a    t    w    e    r    e     f     i    r    s    t     i     d    e    n    t     i     f     i    e     d     b    y    o    t     h    e    r    a    u    t     h    o    r    s    u    n    t     i     l     S    e    p    t    e    m     b    e    r     2     0     0     5     [     2     3     ] .     T     h    e     d    a    t    e    s     i    n     b    o     l     d    a    n     d     i    t    a     l     i    c    r    e     f    e    r    t    o    t     h    e     f     i    r    s    t    m    e    n    t     i    o    n    o     f    t     h    e    s    p    e    c     i    e    s     i    n    t     h    e     i    s     l    a    n     d     b    y    t     h    e     E     S     P .     d    o     i   :     1     0 .     1     3     7     1     /     j    o    u    r    n    a     l .    p    o    n    e .     0     0     3     4     8     9     6 .    t     0     0     4 Report of Thirteen  Culicoides  Species in PortugalPLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34896
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