MEDICAL ENTOMOLOGY /ENTOMOLOGIE MÉDICALE
Phlebotomine sand flies from Madagascar (Diptera: Psychodidae).
VIII – Phlebotomus ( Anaphlebotomus ) vincenti n. sp.
Les phlébotomes de Madagascar (Diptera: Psychodidae).
VIII– Phlebotomus (Anaphlebotomus) vincenti n. sp.
F. J. Randrianambinintsoa · J. Depaquit
Received: 6 June 2013; Accepted: 9 July 2013
© Société de pathologie exotique et Springer-Verlag France 2013
AbstractA new species of Phlebotomine sand fly (Diptera:
Psychodidae) is described from Mikea Forest in the south- west of Madagascar:Phlebotomus vincentin. sp. It is tem- porarily classified in the subgenusAnaphlebotomusaccord- ing to its spermathecal organization, similar to those of P. berentiensis,P. ferteiandP. vaomalalae, previously clas- sified in the subgenusAnaphlebotomusreferring to on male genitalia morphology. An original trumpet-like dilatation on the distal part of the spermathecal ducts individualizes P. vincentin. sp. from the other Malagasy species. Moreover, the second internal transcribed spacer (ITS2) sequences strongly individualisesP. vincentin. sp. from the other Mal- agasyPhlebotomus.
KeywordsPhlebotomine sand flies ·Phlebotomus vincenti n. sp. ·Anaphlebotomus· Taxonomy · Molecular systematic · Mikea · Madagascar · Indian Ocean
RésuméUne espèce nouvelle de phlébotome (Diptera : Psy- chodidae) est présentée, découverte dans la forêt de Mikea, dans le sud-ouest de Madagascar :Phlebotomus vincentin.
sp. Elle est provisoirement classée dans le sous-genreAna- phlebotomusen attendant sa révision en raison de l’architec- ture particulière de sa spermathèque dont l’organisation est
comparable à celle d’autres femelles malgaches du même sous-genre :P. berentiensis,P. ferteietP. vaomalalae. Elle se différencie de ces espèces par l’originalité d’une dilatation en trompette observée dans la partie distale des conduits des spermathèques et par des différences morphométriques. Les données moléculaires (séquence du second espace interne transcrit) excluent qu’il puisse s’agir de la femelle deP. fon- tenilleiqui demeure inconnue.
Mots clésPhlébotomes ·Anaphlebotomus·Phlebotomus vincentin. sp. · Taxinomie · Systématique moléculaire · Mikea · Madagascar · Océan Indien
Introduction
In 2002, the first phlebotomine sandflies of the genusPhle- botomuswere recorded from Madagascar with the descrip- tion of the male of Phlebotomus fertei and the female of P. huberti[8]. Later, the same authors described theP. fertei female and theP. berentiensismale, firstly described asSer- gentomyia berentiensis[12] and included the latter species in the genusPhlebotomus[9]. Then, they describedP. fonte- nilleiof which the female remains unknown [10]. The fifth and last described Malagasy species belonging to the genus Phlebotomus wasP. vaomalalae [16]. All these Phleboto- mus are endemic of Madagascar and have never been recorded in neighbouring countries [5,15]. In a batch of sand flies caught in the Mikea forest, we have observed a female belonging to the same group that we describe in the present study as a new species for Science.
Material and methods
The female examined was collected in the south-western administrative region (ex-province of Toliara), in the forest
F. J. Randrianambinintsoa (*) · J. Depaquit Université de Reims Champagne-Ardenne, ANSES, SFR Cap-Santé, EA4688–USC « transmission vectorielle et épidémiosurveillance de maladies parasitaires (VECPAR) », 51, rue Cognacq-Jay, 51096 Reims Cedex, France
e-mail : fanoujo@yahoo.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,
34394 Montpellier cedex 5, France DOI 10.1007/s13149-013-0305-x
of Mikea, a dry deciduous forest belonging to the western phytogeographical region [11].
Sand flies were caught ten years ago using CDC minia- ture light traps at the site named Abrahama – Jiloriaky 7.5 km north-east of Tsifota (22 ° 48.0’S–43 ° 26.0’E) and 60 m a.s.l., over five consecutive nights from February 21–25, 2003.
These collections were carried out in lowland forest, dom- inated by trees (Didieraceae, baobab trees and lianas) reaching 15 m in height, in high thickets on red to whitish rich alluvial, sandy soil. The forest is partly affected by anthropogenic pres- sure except where the undergrowth is impenetrable.
The specimen described here was stored in 96% etha- nol. The head and genitalia were cut off in a drop of etha- nol, cleared in boiling Marc-André solution, and mounted between slide and cover slide for species identification.
The body related to the specimen was dried and stored in a vial at -20°C in order to be available for molecular studies.
The specimen was observed under a BX50 microscope and measured using the Perfect Image software (Aries Company, Chatillon, France) and a video camera connected to the micro- scope. Drawings were made using the drawing tube installed on the microscope. To allow long-term preservation of the specimens, they were remounted in Canada balsam after complete processing by washing, dehydration in baths of eth- anol 70% to 100%, and immersion in creosote. Unfortunately, the basal part of the spermathecae has been lost during this last step.
Molecular analysis
Genomic DNA was extracted from the thorax, wings, legs and abdomen of individual sand flies using the QIAmp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions, modified by crushing the sand fly tissues with a piston pellet (Treff, Switzerland), and using an elution vol- ume of 200μl, as detailed in Depaquit et al. [8].
rDNA amplifications were performed in a 50μl volume using 5μl of extracted DNA solution and 50 pmol of each of the primers. The PCR mix contained (final concentrations) 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% Triton X 100, 200μM dNTP each base, and 1.25 units of 5 prime Taq polymerase (Eppendorf, Germany).
The cycle begins with an initial denaturation step at 94°C for 3 min and finishes with a final extension at 68°C for 10 min. Amplification of a fragment of ITS2 was undertaken using the primers C1a and JTS3 [7].
Amplicons were analysed by electrophoresis in 1.5%
agarose gel containing ethidium bromide. Direct sequenc- ing in both directions was performed using the primers used for DNA amplification. The correction of sequences
was done using the Pregap and Gap software included in the Staden Package [10]. Estimates of evolutionary diver- gence between sequences have been calculated using MEGA 4.0 software [19].
Results: Description of the holotype of Phlebotomus vincenti n. sp.
GenusPhlebotomusRondani & Berté, in Rondani, 1840 SubgenusAnaphlebotomusTheodor, 1948
SpeciesPhlebotomus vincentin. sp.
The terminology used in the description below is that of Abonnenc [1].
Holotype female (Fig. 1)
* Head
Interocular suture incomplete.
Cibarium armed with three vertical teeth oriented back- wards and 26 anterior denticles.
Pharynx widens evenly towards the rear. Small pharyn- geal armature containing small dots-like teeth at the back and short anterior teeth.
Palpal formula: 1, 4, 3, 2, 5. A few grouped Newstead’s scales on the third segment.
Antennal formula: 2/III-XV with long ascoids exceeding the next articulation.
A III = 171μm, little bit longer than A IV + A V.
Labrum = 295μm. AIII / E = 0.55.
* Thorax
Five setae on the antero-inferior part of the mesanepis- ternum.
Wing: length = 2055μm, width = 601μm,α= 464μm, β= 335μm,δ= 92μm (short),γ= 273μm,π= 154μm.
Width /γ= 2.20
* Spermathecae
The ducts (total length = 240μm) are joined at their base.
Their basal part is wide and the walls are thin. At the distal third, ducts gradually shrink and we notice a thickening of the wall, more important before the body of the spermatheca.
This body is spherical and discrete ill-defined (diameter = 25 to 50 μm). The ducts end with a characteristic trumpet- shaped expansion that arises in the head of the spermatheca carried by a long neck.
Furca very thick, without anterior lateral expansions.
Genital armature atrium well developed with many groups of several spines.
Genbank accession number of P. vincenti n. sp. ITS2 sequence female specimen processed during the present study: KF186427.
Derivatio nominis
We dedicate this species to our colleague Vincent Robert (Institut de Recherche pour le Développement), who
initiated the recent studies of Phlebotomine sandflies from Madagascar.
The holotype has been deposited at the Laboratory of Entomology of the“Muséum National d’Histoire Naturelle” of Paris.
Type locality: Abrahama–Jiloriaky in the Mikea forest.
Discussion
Seven genera of sand flies are recorded in the Old World at the present time [18]:PhlebotomusRondani, 1843;Sergentomyia (França & Parrot, 1921);SpelaeophlebotomusTheodor 1948;
Spelaeomyia Theodor, 1948; Idiophlebotomus Quate &
Fig. 1 P. vincentin. sp. holotype. A: pharynx and cibarium; B: palp; C: antennal segments III, IV and V; D: spermathecae and furca ; E:
wing / P. vincentin. sp. holotype. A: pharynx et cibarium; B: palpe labial; C: segments antennaires III, IV et V; D: spermathèques et furca ; E: aile.
Fairchild, 1961; Australophlebotomus Theodor, 1948 and ChiniusLeng, 1987. Moreover, the taxaParvidensTheodor
& Mesghali, 1964 is considered asincertae sedis[18].Phle- botomus vincentin. sp. possesses a group of antero-inferior mesanepisternal setae. Consequently, it belongs to the genus Phlebotomus sensuLewis [14].
The genusPhlebotomusincludes a total of nine subge- nera [18]:PhlebotomusRondani, 1843;Paraphlebotomus Theodor, 1948;SynphlebotomusTheodor, 1948;Larrous- siusTheodor, 1948 ;AdleriusNitzulescu, 1931;Transphle- botomusArtemiev, 1984; EuphlebotomusTheodor, 1948;
Anaphlebotomus Theodor, 1948; Kasaulius Lewis, 1982.
According to the recognized standards, mainly based on the male terminalia morphology [20], it is not possible to includeP. vincenti n. sp. in a subgenus. However, there is a strong homology of spermathecal morphology of P. vincenti n. sp. with those of P. fertei, P. berentiensis andP. vaomalalae[9,16]. Consequently, we have classified P. vincentin. sp. in the subgenusAnaphlebotomusdespite its probable heterogeneity [17] which requires an urgent revision, especially at the light of the new species described from Madagascar.
An alignment of 616 bases pairs (bp) has been ana- lysed. The ITS2 evolutionary divergence between ITS2 sequences has been calculated without all positions con- taining indels (complete deletion option). There were a total of 362 bp in the final dataset.P. vincentin. sp. shows 9.79% divergence with P. fontenillei and cannot be the female of the later species. The divergence calculated with other species is 10.1%, 11.67%, 11.70% and 37%
withP. vaomalalae,P. berentiensis,P. fertei, andP. huberti, respectively. These divergence levels are in agreement with the specific status of these taxa [6].
From an ecological point of view,P. vincentin. sp. has been caught in sympatry withP. vaomalalae. This biotope is very different from those where P. ferteiandP. fontenillei are recorded (tsingys) but similar to the P. berentiensis biotope.
The Mikea forest, the private reserves of Berenty, the Tsingys are all on the Morondava Basin in South-west part of Madagascar. P. vincenti n. sp. andP. vaomalalae have been captured in the center of the Mikea forest on a sedi- mentary substrate covered with sands. This coverage sander hides the deep geological formations of tertiary age: Eocene limestone karst and veneers Pliocene red sand. The vegetation cover of the Mikea forest is domi- nated by Euphorbiaceae and Didieraceae of the southern and south-west domain and characterisitc of dense dry for- est area [11].P. berentiensiswas also collected in the same type of plant formation in the southern and south-west domain in a small private reserve of Berenty consisting of a gallery forest in the thorny thickets in the extreme south of Madagascar on sedimentary and volcanic soils
dominated by the Pliocene continental sediment. By con- trast, P. fertei and P. fontenillei were captured in caves and excavations of Bemaraha and Namoroka tsingys which are both karst formations of the Middle Jurassic [2].
The differential diagnosis between P. vincentin. sp. and the other Malagasy species belonging to the subgenusAna- phlebotomusis based on the original trumpet-like structure on the distal part of the spermathecal ducts, and on several morphometric data (Table 1).
Identification key of the females of Phlebotomus from Madagascar:
•
1 - Spermathecae ringed:……….P. huberti Spermathecae not ringed:………. 2•
2 - AIII long 300μm:……….P. fertei AIII shorter than 290μm:………. 3•
3 - Largest spermathecal chamber with thick wall:……….P. berentiensis
Largest spermathecal chamber with thin:………. 4
•
4 - Distal part of the spermathecal ducts including a trumpet-like dilatation:……….P. vincentin. sp.Distal part of the spermathecal ducts including without a trumpet-like dilatation:……….P. vaomalalae
Conclusion
The description of the fifth species ofPhlebotomusin Mada- gascar shows the high biodiversity of this genus in this coun- try. The homogeneity of the subgenusAnaphlebotomushas to be tested and the potential vectorial role of the Malagasy species in the transmission ofLeishmania spp. needs to be evaluated, especially at the light of the anthropophily of some MalagasyPhlebotomus[4,13].
Acknowledgements The authors thank Steve Goodman (Field Museum of Natural History of Chicago, NGO Vaha- tra) for logistical support in the field, Vaomalala Rahari- manga who collected the holotype, and Sylvette Gobert for proofreading the manuscript. They are grateful to the Ministry of Forests of Madagascar and Madagascar National Park for providing a research permit. The field- work has received funds from the Institute of Research for Development, Institut Pasteur de Madagascar, and Volkswagen Stiftung. Laboratory work has received funds from the Program of“Institut Français de la biodiversité/
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 Mada- gascar”and The Robert S. McNamara Fellowship Program of the Worldbank.
References
1. Abonnenc E (1972) Les phlébotomes de la région éthiopienne (Diptera: Psychodidae). Cah ORSTOM, Sér Ent Méd Parasitol 55, 239 pp
2. Besairie H, Collignon M (1972) Géologie de Madagascar. 1- Les terrains sédimentaires. Annales Géologiques de Madagascar 35:1–463
3. Bonfield J, Staden R (1996) Experiment files and their applica- tion during large-scale sequencing projects. DNA Seq 6(2):
109–117
4. Clerc Y, Rodhain F, Digoutte JP et al (1982/1983) Le virus Péri- net, Rhabdoviridae, du genreVesiculovirusisolé à Madagascar de Culicidés. Archives de l’Institut Pasteur de Madagascar 49 (1):119–25
5. Davidson IH (1981) The subgenus Anaphlebotomus ofPhleboto- mus (Diptera: Psychodidae, Phlebotominae) in southern Africa.
J Ent Soc sth Afr 44(2):259-64
6. Depaquit J, Ferté H, Léger N, et al (2000) Molecular systemat- ics of the phlebotomine sandflies of the subgenusParaphlebo- tomus (Diptera, Psychodidae, Phlebotomus) based on ITS2 rDNA sequences. Hypotheses Of dispersion and speciation.
Insect Mol Biol 9(3):293–300
7. Depaquit J, Ferté H, Léger N, et al (2002) ITS 2 sequences heterogeneity inPhlebotomus sergentiandPhlebotomus similis (Diptera, Psychodidae): possible consequences in their ability to transmitLeishmania tropica. Int J Parasitol 32(9):1123–31 8. Depaquit J, Léger N, Robert V (2002) Première mention dePhle-
botomus à Madagascar (Diptera: Psychodidae). Description de Phlebotomus (Anaphlebotomus) fertein. sp. et de Phlebotomus (Anaphlebotomus)hubertin. sp. Parasite 9(4):325–31
9. Depaquit J, Léger N, Ferté H, Robert V (2004) Les phlébotomes de Madagascar (Diptera: Psychodidae). II. Description de la femelle de Phlebotomus (Anaphlebotomus) fertei Depaquit, Léger &
Robert, 2002; description du mâle et redescription de la femelle dePhlebotomus(Anaphlebotomus)berentiensis(Léger & Rodhain, 1978) comb. nov. Parasite 11(2):201–9
10. Depaquit J, Léger N, Robert V (2004) Les Phlébotomes de Madagascar (Diptera: Psychodidae). III. Description dePhleboto- mus(Anaphlebotomus)fontenillein. sp. Parasite 11(3): 261–5 11. Humbert H (1954) Les Territoires Phytogéographiques de Mada-
gascar. Leur Cartographie. Colloque sur les Régions Ecologiques du Globe. Annales de Biologie (Paris) 31:195–204
12. Léger N, Rodhain F (1978)Sergentomyia berentiensisn. sp. (Dip- tera: Psychodidae). Description à partir d’un exemplaire femelle récolté à Madagascar. Bull Soc Pathol Exot Filiales 71(6):476–9 13. Léger N, Rodhain F, Coulanges P, et al (1978/1979) Présence de
Phlébotomes anthropophiles à Madagascar. Arch Inst Pasteur Madagascar 47(1):249–50
14. Lewis D (1982) A taxonomic review of the genusPhlebotomus.
Bull Brit Mus Nat Hist (Ent) 45(2):121–209
15. Randrianambinintsoa FJ, Depaquit J, Brengues C, et al (2012) First record of Phlebotomine sandflies (Diptera: Psychodidae) in the Comoros Archipelago with description of Sergentomyia Table 1 Malagasy Anaphlebotomus female measurements (in μm) / Mensuration des femelles Anaphlebotomus de Madagascar (inμm).
P. vincentin. sp. P. vaomalalae [16] P. fertei[9] P. berentiensis[9] P. huberti[8]
(n = 1) (n = 1) (n = 20) (n = 1) (n = 2)
Head
AIII 171 221 401 (332-483) 227 260
AIV 85 106 173 (146-224) 104 125
AV 85 102 171 (145-216) 98 125
labrum 295 304 447 (379-540) 153 190 (180-200)
AIV+AV 170 208 344 202 250
antennal formula 2/III-XV 2/III-XV 2/III-XV 2/III-XV 2/III-XV
palpal formula 1, 4, 3, 2, 5 1, 4, 3, 2, 5 1, 4, 3, 2, 5 1, 4, 3, 2, 5 1, 2, 3, 4, 5 Wings
length 2055 1939 2396 (2022-3017) 2041 1730
width 601 699 676 (544-902) 645 450
α 464 535 658 (480-994) 548 370
β 335 241 303 (232-405) 279 330
δ 92 108 218 (88-418) 164 220
γ 273 260 240 (191-307 282 250
π 154 64 99 (42-165) 95 120
w/γ 2.20 2.69 2.8 2.29 1.83
Spermathecae
Duct 240 182 323 (237-480) 162 470
(Vattieromyia) pessoni n. sp. and S. (Rondanomyia) goodmani comorensisn. ssp. Parasite 19(3):195–206
16. Randrianambinintsoa FJ, Léger N, Robert V, Depaquit J (2013) Phlebotomine sand flies from Madagascar (Diptera: Psychodidae).
VII. An identification key for Phlebotomus with the description of Phlebotomus(Anaphlebotomus)vaomalalaen. sp. Parasite 20:6 17. Rispail P, Léger N (1998) Numerical taxonomy of Old World Phle-
botominae (Diptera: Psychodidae). 1. Considerations of morpho- logical characters in the genus Phlebotomus Rondani & Berté 1840. Mem Inst Oswaldo Cruz 93(6):773–85
18. Rispail P, Léger N (1998) Numerical taxonomy of Old World Phlebotominae (Diptera: Psychodidae). 2. Restatement of classi- fication upon subgeneric morphological characters. Mem Inst Oswaldo Cruz 93(6):787–93
19. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecu- lar Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–9
20. Theodor O (1948) Classification of the Old World species of the subfamily Phlebotominae (Diptera, Psychodidae). Bull Entomol Res 39(Pt 1):85–115
