MEDICAL ENTOMOLOGY /ENTOMOLOGIE MÉDICALE
Record of two species of Culicoides (Diptera, Ceratopogonidae) new for Madagascar and molecular study showing the paraphylies
of the subgenus Oecacta and the Schultzei group
Mention de deux espèces de
Culicoides(Diptera, Ceratopogonidae) nouvelles à Madagascar et étude moléculaire montrant les paraphylies du sous-genre Oecacta et du groupe Schultzei
D. Augot · F. J. Randrianambinintsoa · A. Gasser · J. Depaquit
Received: 25 May 2013; Accepted: 25 June 2013
© Société de pathologie exotique et Springer-Verlag France 2013
AbstractCulicoidesare vectors of diseases of Veterinary Med- icine importance (bluetongue, African horse sickness, Schmal- lenberg virus) all over the world. In the present study, we report two species new for Madagascar:C. nevilliandC. enderleini.
They belong to the Schultzei group which is sometimes classi- fied in the subgenusOecactaand sometimes in the subgenus Remmia, depending on authors. Consequently, we carried out a molecular cladistics of these groups based on cytochrome C oxidase subunit I mtDNA sequences. We processed the Mala- gasy specimens and someC. furens(theOecactatype-species) caught in Florida and we analyzed their sequences and those available in Genbank:C. schultzei,C. oxystoma, C. festivipen- nis,C. brunnicans, C. kibunensis, C. truncorumandC. vexans.
C. (Avaritia)imicolahave been selected as an outgroup. The maximum parsimony analysis showed the paraphylies of the Schultzei group (=Remmia) and of the subgenus Oecactaif the first group is excluded from the latter. Our results underline the doubtful current classification and need to be validated by other molecular markers in the future.
KeywordsCulicoides nevilli·Culicoides enderleini· Cytochrome C oxidase I · Schultzei group ·Oecacta· Florida · Madagascar · Indian Ocean
Résumé Les Culicoides sont les vecteurs de nombreuses maladies d’intérêt vétérinaire à travers le monde (maladie de la langue bleue, peste équine, virus de Schmallenberg…).
Dans cette étude, nous rapportons la mention de nouvelles espèces pour Madagascar: C. nevillietC. enderleini. Elles appartiennent au groupe Schultzei qui est, selon les auteurs, soit rangé dans le sous-genreOecacta, soit formant un sous- genre propre :Remmia. Pour tenter de résoudre ce problème, nous avons conduit une étude cladistique basée sur les séquences de l’ADN mitochondrial de la sous-unité I de la cytochrome C oxydase. Nous avons inclus dans cette étude les spécimens malgaches, quelquesC. furens(l’espèce-type du sous-genre Oecacta) capturés en Floride ainsi que des espèces dont les séquences homologues étaient disponibles dans Genbank:C. schultzei,C. oxystoma, C. festivipennis, C. brunnicans, C. kibunensis, C. truncorum et C. vexans.
Nous avons choisi C. (Avaritia) imicola comme extra- groupe. L’analyse, conduite selon la méthode de parcimonie, montre les paraphylies du groupe Schultzei (=Remmia) et du sous-genre Oecacta si le premier groupe est exclu du sec- ond. Nos résultats soulignent les limites de la classification actuelle qui nécessiterait d’être révisée à l’occasion de nou- velles études conduites sur plus de marqueurs moléculaires.
Mots clésCulicoides nevilli ·Culicoides enderleini· Cytochrome C oxydase I · Groupe Schultzei · Oecacta· Floride · Madagascar · Océan Indien
Introduction
Culicoides biting midges (Diptera: Ceratopogonidae) are among the smallest hematophagous flies. They occur throughout the world, except in New Zealand and Antarctica.
D. Augot (*) · F. J. Randrianambinintsoa · A. Gasser · J. Depaquit
ANSES-LSA, Université Champagne-Ardenne, Faculté de Pharmacie, EA4688, SFR Cap Santé, Usc Transmission Vectorielle et épidemiosurveillance de maladies Parasitaires (VECPAR), Reims, France e-mail : denis.augot@anses.fr
F. J. Randrianambinintsoa
Département de biologie animale, Faculté des sciences, Université d’Antananarivo, Madagascar
MIVEGEC, UMR IRD 224-CNRS 5290-UM1-UM2, 911, avenue Agropolis, BP 64501,
F-34394 Montpellier cedex 5, France DOI 10.1007/s13149-013-0302-4
They act as vectors of several arboviral veterinary diseases which affect ruminants and equines [21].
The identification based on morphological characters of Culicoidesis difficult [9]. Except for some subgenera, the taxonomy ofCulicoidesremains in disarray [14]. Their cur- rent classification is based on a classical systematics, without any phylogenetic evidence. It includes more than 1300 known species dispatched partly in 39 subgenera and partly ungrouped [3-5].
In the present study, we report two new species for Mada- gascar:C. nevilliandC. enderleini.
Recently, we have had access to some specimens of C. enderleiniandC. nevillifrom Madagascar andC. furens from Florida. The first ones belong to the Schlutzei group which is sometimes classified in the subgenusOecactaPoey, 1851 and sometimes in the subgenus Remmia Glukhova 1977, depending on authors.
We carried out a molecular systematics of these species and related ones whose sequences were available in Gen- bank, by comparison of the sequences of the mitochon- drial DNA cytochrome C oxidase I (COI), in order to know the phylogenetic relationships between them and to test the monophilies of the subgeneraOecactaandRemmia (=Schlutzei group).
Material and methods
Collection ofCulicoidesand identification
Biting midges from Madagascar were captured in the village of Andasibe near the Special Reserve of Analamazaotra in
Alaotra-Mangoro Region at the Fivondronana of Mora- manga. (18°55,412 S and 48°24,913 E; altitude: 858 m a.s.l.) in a peri-domestic area, in a zone of degraded forest and at Port Sainte-Lucie in Florida North America along the road (27°16 33 N; 80°21 18 O, altitude: 5 m) in 2010 and 2012 respectively using ultraviolet CDC traps and standard CDC miniature light traps (John W. Hock Company, Gaines- ville, FL, USA). Midges were stored in 96% ethanol before morphological and molecular analyses.
Wings were directly mounted whereas head and abdomen of individual midges were mounted in Chloral gum after clearing in boiling Marc-André solution on microscope slides for morphological identification [8,11,12,16,25,26].
Eight individuals were dissected and identified (Table 1).
DNA extraction, PCR amplification and sequencing
Total DNA was extracted for each dissected midges from thorax, legs and the anterior part of the abdomen using the QIAmp DNA Mini Kit (Qiagen®, GmbH, Hilden, Ger- many), following manufacturer’s instructions with a final elution volume of 100μl.
COI was amplified using the primers LepF and LepR as indicated by Augot et al [1].
Direct sequencing of both DNA strands was performed with the primers used for PCR. The DNA sequence-based analyses were performed using maximum parsimony (MP) method with MEGA software version 3.1 [18] using a Branch and Bound search option. Bootstrap values have been calculated after 1000 replicates. For comparison, we have included C. schultzei from Israel (Genbank under accession number JN54053),C. oxystoma from Japan and
Table 1 List of Culicoidesprocessed in the present study. *: photographied specimens / Liste des Culicoides utilisés dans notre étude.* : spécimens photographiés.
Taxa Subgenus or group N°(codification) Country Wings
C. furens Oecacta D710*
D711 D712 D713
USA
C. nevilli Schultzei group D357* Madagascar
C. enderleini D385
D378 D363*
Madagascar
Israel (AB360985 and JN54054 respectively) which have been found to be closely related according to molecular data [22], C. festivipennis from Switzerland (HQ824482), C. brunnicans from Sweden (JQ620042), C. kibunensis from Spain (HM241872), C. truncorum from Sweden (JQ620244) andC. vexansfrom Sweden (JQ62050). C. imi- colahave been processed as out group [22].
Results
The sequences obtained in the present study have been depos- ited in GenBank under the accession numbers:C. enderleini (KF186429- KF186431), C. nevilli (KF186428), C. furens (KF186432- KF186435). The alignment was restricted to 449 bp homologous positions including 174 variable sites out of which 121 were parsimony informative.
The genetic pairwise distance between species is indi- cated in table 2. The genetic distance pairwise within spe- cies showed 99.4%, 99.7% and 97.1% homology within C. enderleini, C. furensandC. oxystomarespectively.
The branch and bound search provided three trees equi- parcimonious, 443 steps long. Their strict consensus is given in Figure 1. Consistency index= 0.55. Retention index=
0.59.
Discussion
In Madagascar, the fauna of Culicoides species included 18 species:C. subflavesens[24];C. bisolis[17];C. mada- gascarensis [10]; C. schultzei, C. pallidicornis, C. ful- vithorax, C. africanusetC. moreli [6]; C. distinctipennis, C. fulvithorax, C. imicola, C. moreli, C. naevei, C. rhizo- phorensisandC. schultzei[12];C. imicola[23]; C. bolitinos [19];C. imicola[13]. The record ofC. schultzeiin Madagas-
car [6,12] was prior to the description ofC. enderleiniand C. nevilli[8]. Consequently, we don’t know if the specimens of the latter study referred toC. schultzeis. st. or toC. ender- leini and C. nevilli. However, the mention of two species new for Madagascar encourages new inventories.
The subgenusOecactaPoey, 1851 is a group of veterinary importance [2]. However, its systematics is still confused.
The subgenusOecactais considered as a catch fall subgenus for many species [8,14]. Its type-species is C. furensPoey, 1851. In 1954, Khalaf [15] divided the subgenus in three complexes, each including many groups: the complex Hae- matopotus included the groups Clavipalpis, Albicans, Furens, and Haematopotus; the complex Villosipennis included the groups Neavi, Heliophilus, Pictipennis, Villosipennis, and the Guttipennis complex included the groups Spino- sus, Biguttatus, Guttipennis, and Schultzei. In 1985 and 1988, Wirth et al [25,26] have reported only four (C. barbo- sai, C. furens, C. furensoidesand C. stellifer) and five spe- cies (C. alahialinus, C; barbosai, C. cancer, C. furensand C. gorgasi) in their nearctic and neotropical atlas of Culi- coides, respectively.
In 1977, Glukhova created the subgenusRemmiafor the members of the Schultzei group and designatedC. schultzei (Enderlein, 1908) as its type-species. Remmia was mainly characterized by a marked reduction of the radial cells of the wing. However, this reduction in venation was commonly observed in other ceratopogonids genera and was therefore not considered as a valid basis to propose a new subgenus [12]. Some authors still consideredRemmiaas a valid subge- nus [5,20]. Others continue to include the Schultzei group in the subgenusOecacta[7,8] and do not consider the subgenus Remmiaas a synonym of the subgenusOecacta.
In 1994, Cornet et Brunhes [8] revised the Schultzei group and concluded that Oecactasubgenus includes only Furens group (C. furens;C. stellifer;C. alahialinus,C. gor- gasi,C. furensoides,C. barbosai, andC. cancer) and maybe
Table 2 Estimation of pairwise distance between sampled species for COI of the mtDNA /Estimation de la distance génétique entre les espèces échantillonnées basée sur l’alignement des séquences de COI (ANDmt).
1 2 3 4 5 6 7 8 9 10
1.C. nevilli
2.C. enderleini 0,124
3.C. furens 0,132 0,149
4.C. oxystoma 0,080 0,124 0,122
5.C. schultzei 0,113 0,130 0,156 0,104
6.C. imicola 0,189 0,184 0,183 0,167 0,153
7.C. festivipennis 0,198 0,214 0,183 0,178 0,173 0,198
8.C. brunnicans 0,167 0,195 0,145 0,172 0,194 0,192 0,216
9.C. kibunensis 0,181 0,180 0,175 0,186 0,175 0,216 0,216 0,164
10.C. truncorum 0,161 0,188 0,168 0,170 0,181 0,198 0,243 0,195 0,204
11.C. vexans 0,228 0,222 0,207 0,201 0,205 0,231 0,242 0,183 0,202 0,214
Schultzei group (C. schultzei, C. kingi, C. rhizophorensis, C. enderleini, C. nevilli., C. subschultzei, C. neoschultzei and a few undescribed species). Lastly, Borkent [5] included more than 170 species in the subgenusOecactaand added C.
oxystoma to the Schultzei group within the subgenus Remmia.
The maximum parsimony analysis (Fig. 1) suggests the paraphyly of both the subgenera Oecacta and Remmia (=Schultzei group). Within the subgenusOecacta, one clade includesC. furens,C. truncorum,C. kibunensis,C. brunni- cansandC. vexanswhereasC. festivipennis diverged early and is the sister species of all the others in the ingroup. This paraphyly is supported by a bootstrap value of 60%. The para- phyly of the Schultzei group is not supported by bootstrap value, which is linked to the low CI meaning a high degree of homoplasy in the database. In the future, a study based on another molecular marker than COI will be necessary.
AcknowledgmentsWe thank Sylvette Gobert for proofread- ing this manuscript. Fieldwork in Madagascar have received funds from the Program of“Institut Français de la biodiver- sité/CNRS/AIRD “Biodiversité dans les îles de l’Océan Indien”’ itself included in the Regional Project “Insectes vecteurs (phlébotomes et moustiques) dans les îles de l’Océan Indien: Madagascar, Seychelles et Comores”, the GDRI “Biodiversité et Développement Durable à Madagascar.
Competing interests:The authors declare that they have no competing interests
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