Phlebotomine sand flies from Madagascar - Springer Link

Bull. Soc. Pathol. Exot. (2013) 106:206-211 DOI 10.1007/s13149-013-0305-x

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

Abstract A new species of Phlebotomine sand fly (Diptera: Psychodidae) is described from Mikea Forest in the southwest of Madagascar: Phlebotomus vincenti n. sp. It is temporarily classified in the subgenus Anaphlebotomus according to its spermathecal organization, similar to those of P. berentiensis, P. fertei and P. vaomalalae, previously classified in the subgenus Anaphlebotomus referring to on male genitalia morphology. An original trumpet-like dilatation on the distal part of the spermathecal ducts individualizes P. vincenti n. sp. from the other Malagasy species. Moreover, the second internal transcribed spacer (ITS2) sequences strongly individualises P. vincenti n. sp. from the other Malagasy Phlebotomus.

comparable à celle d’autres femelles malgaches du même sous-genre : P. berentiensis, P. fertei et P. 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 de P. fontenillei qui demeure inconnue.

Keywords Phlebotomine sand flies · Phlebotomus vincenti n. sp. · Anaphlebotomus · Taxonomy · Molecular systematic · Mikea · Madagascar · Indian Ocean

Introduction

Résumé Une espèce nouvelle de phlébotome (Diptera : Psychodidae) est présentée, découverte dans la forêt de Mikea, dans le sud-ouest de Madagascar : Phlebotomus vincenti n. sp. Elle est provisoirement classée dans le sous-genre Anaphlebotomus en attendant sa révision en raison de l’architecture particulière de sa spermathèque dont l’organisation est 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 : [email protected]

Mots clés Phlébotomes · Anaphlebotomus · Phlebotomus vincenti n. sp. · Taxinomie · Systématique moléculaire · Mikea · Madagascar · Océan Indien

In 2002, the first phlebotomine sandflies of the genus Phlebotomus were recorded from Madagascar with the description of the male of Phlebotomus fertei and the female of P. huberti [8]. Later, the same authors described the P. fertei female and the P. berentiensis male, firstly described as Sergentomyia berentiensis [12] and included the latter species in the genus Phlebotomus [9]. Then, they described P. fontenillei of which the female remains unknown [10]. The fifth and last described Malagasy species belonging to the genus Phlebotomus was P. vaomalalae [16]. All these Phlebotomus 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.

F. J. Randrianambinintsoa Département de Biologie Animale, Faculté des Sciences, Université d’Antananarivo, Madagascar

Material and methods

MIVEGEC, UMR IRD 224-CNRS 5290-UM1-UM2, 911, avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France

The female examined was collected in the south-western administrative region (ex-province of Toliara), in the forest

Bull. Soc. Pathol. Exot. (2013) 106:206-211

of Mikea, a dry deciduous forest belonging to the western phytogeographical region [11]. Sand flies were caught ten years ago using CDC miniature 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, dominated 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 pressure except where the undergrowth is impenetrable. The specimen described here was stored in 96% ethanol. The head and genitalia were cut off in a drop of ethanol, 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 microscope. 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 ethanol 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 volume of 200 μl, as detailed in Depaquit et al. [8]. rDNA amplifications were performed in a 50 μl volume using 5 μl 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, 200 μM 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 sequencing in both directions was performed using the primers used for DNA amplification. The correction of sequences

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was done using the Pregap and Gap software included in the Staden Package [10]. Estimates of evolutionary divergence between sequences have been calculated using MEGA 4.0 software [19].

Results: Description of the holotype of Phlebotomus vincenti n. sp. Genus Phlebotomus Rondani & Berté, in Rondani, 1840 Subgenus Anaphlebotomus Theodor, 1948 Species Phlebotomus vincenti n. 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 backwards and 26 anterior denticles. Pharynx widens evenly towards the rear. Small pharyngeal 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 = 171 μm, little bit longer than A IV + A V. Labrum = 295 μm. AIII / E = 0.55. * Thorax Five setae on the antero-inferior part of the mesanepisternum. Wing: length = 2055 μm, width = 601 μm, α = 464 μm, β = 335 μm, δ = 92 μm (short), γ = 273 μm, π = 154 μm. Width / γ = 2.20 * Spermathecae The ducts (total length = 240 μm) 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 trumpetshaped 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

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Fig. 1 P. vincenti n. sp. holotype. A: pharynx and cibarium; B: palp; C: antennal segments III, IV and V; D: spermathecae and furca ; E: wing / P. vincenti n. sp. holotype. A: pharynx et cibarium; B: palpe labial; C: segments antennaires III, IV et V; D: spermathèques et furca ; E: aile.

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]: Phlebotomus Rondani, 1843; Sergentomyia (França & Parrot, 1921); Spelaeophlebotomus Theodor 1948; Spelaeomyia Theodor, 1948; Idiophlebotomus Quate &


Fairchild, 1961; Australophlebotomus Theodor, 1948 and Chinius Leng, 1987. Moreover, the taxa Parvidens Theodor & Mesghali, 1964 is considered as incertae sedis [18]. Phlebotomus vincenti n. sp. possesses a group of antero-inferior mesanepisternal setae. Consequently, it belongs to the genus Phlebotomus sensu Lewis [14]. The genus Phlebotomus includes a total of nine subgenera [18]: Phlebotomus Rondani, 1843; Paraphlebotomus Theodor, 1948; Synphlebotomus Theodor, 1948; Larroussius Theodor, 1948 ; Adlerius Nitzulescu, 1931; Transphlebotomus Artemiev, 1984; Euphlebotomus Theodor, 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 include P. 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 and P. vaomalalae [9,16]. Consequently, we have classified P. vincenti n. sp. in the subgenus Anaphlebotomus despite 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 analysed. The ITS2 evolutionary divergence between ITS2 sequences has been calculated without all positions containing indels (complete deletion option). There were a total of 362 bp in the final dataset. P. vincenti n. 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% with P. vaomalalae, P. berentiensis, P. fertei, and P. huberti, respectively. These divergence levels are in agreement with the specific status of these taxa [6]. From an ecological point of view, P. vincenti n. sp. has been caught in sympatry with P. vaomalalae. This biotope is very different from those where P. fertei and P. 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. and P. vaomalalae have been captured in the center of the Mikea forest on a sedimentary 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 dominated by Euphorbiaceae and Didieraceae of the southern and south-west domain and characterisitc of dense dry forest area [11]. P. berentiensis was 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

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dominated by the Pliocene continental sediment. By contrast, 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. vincenti n. sp. and the other Malagasy species belonging to the subgenus Anaphlebotomus is 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 300 μm: ………. P. fertei AIII shorter than 290 μm: ………. 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. vincenti n. sp.

Distal part of the spermathecal ducts including without a trumpet-like dilatation: ………. P. vaomalalae

Conclusion The description of the fifth species of Phlebotomus in Madagascar shows the high biodiversity of this genus in this country. The homogeneity of the subgenus Anaphlebotomus has to be tested and the potential vectorial role of the Malagasy species in the transmission of Leishmania spp. needs to be evaluated, especially at the light of the anthropophily of some Malagasy Phlebotomus [4,13]. Acknowledgements The authors thank Steve Goodman (Field Museum of Natural History of Chicago, NGO Vahatra) for logistical support in the field, Vaomalala Raharimanga 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 fieldwork 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

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Table 1 Malagasy Anaphlebotomus female measurements (in μm) / Mensuration des femelles Anaphlebotomus de Madagascar (in μm).

Head AIII AIV AV labrum AIV+AV antennal formula palpal formula Wings length width α β δ γ π w/γ Spermathecae Duct

P. vincenti n. sp.

P. vaomalalae [16]

P. fertei [9]

P. berentiensis [9]

P. huberti [8]

(n = 1)

(n = 1)

(n = 20)

(n = 1)

(n = 2)

171 85 85 295 170 2/III-XV 1, 4, 3, 2, 5

221 106 102 304 208 2/III-XV 1, 4, 3, 2, 5

401 (332-483) 173 (146-224) 171 (145-216) 447 (379-540) 344 2/III-XV 1, 4, 3, 2, 5

227 104 98 153 202 2/III-XV 1, 4, 3, 2, 5

260 125 125 190 (180-200) 250 2/III-XV 1, 2, 3, 4, 5

2055 601 464 335 92 273 154 2.20

1939 699 535 241 108 260 64 2.69

2396 (2022-3017) 676 (544-902) 658 (480-994) 303 (232-405) 218 (88-418) 240 (191-307 99 (42-165) 2.8

2041 645 548 279 164 282 95 2.29

1730 450 370 330 220 250 120 1.83

240

182

323 (237-480)

162

470

GDRI “Biodiversité et Développement Durable à Madagascar” and The Robert S. McNamara Fellowship Program of the Worldbank.

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