Phlebotomine sand flies from Madagascar (Diptera: Psychodidae). VII. An identification key for Phlebotomus with the description of Phlebotomus (Anaphlebotomus) vaomalalae n. sp.

An identification key of the Phlebotomus in Madagascar is proposed as well as the description of the male and female Phlebotomus (Anaphlebotomus) vaomalalae n. sp. from Mikea Forest in the south-west of Madagascar. The assignation of this new species to the genus Phlebotomus is based on the presence of mesanepisternal setae. Its inclusion in the subgenus Anaphlebotomus is based on the males on the presence of four spines on the style, the lack of a coxite basal process and the existence of a bifurcated paramere. The female has cibarial and pharyngeal armature and spermathecal architecture similar to Phlebotomus fertei and Phlebotomus berentiensis, two other Malagasy species which belong to Anaphlebotomus. Male and female are held to belong to the same species because of their morphological characters, the homology (100%) of their partial cytochrome b mtDNA sequences and their capture in the same trap. P. vaomalalae n. sp. is a small species compared to the other Phlebotomus species of Madagascar. The cibarium of the male and the female of P. vaomalalae n. sp. is armed with teeth, like those of other Malagasy Phlebotomus. However, it differs in the arrangement and shape of the respective teeth and denticles. The male of P. vaomalalae n. sp. looks like that of P. fontenillei due to its tuft of coxal setae (lacking in P. berentiensis and P. fertei) but differs from this species by the location of this tuft. As P. fertei and P. berentiensis, there is no spermathecal common duct in P. vaomalalae n. sp.


Introduction
The first record of Phlebotomus Rondani & Berté in Madagascar was reported in 2002 when Depaquit et al. [1] described the male of Phlebotomus fertei Depaquit, Léger & Robert and the female of Phlebotomus huberti Depaquit, Léger & Robert. Subsequently, the same authors described the female of P. fertei and the male of Phlebotomus berentiensis (Léger & Rodhain, 1978) [2]. They also included the latter species [3] in the genus Phlebotomus [2]. Later, they described P. fontenillei Depaquit, Léger & Robert whose female remains unknown [4]. Therefore, before the present study, the fauna of Phlebotomus from Madagascar included four species: P. berentiensis, P. fertei, P. huberti and P. fontenillei. In the archipelago of Comoros, no species of the genus Phlebotomus has been recorded [5].
Here, we describe the male and the female of a new species from the Southwest of Madagascar: Phlebotomus (Anaphlebotomus) vaomalalae n. sp. According to the increasing number of species, an identification key is provided for the identification of Phlebotomus from Madagascar (males and females).

Study site and collection method
The two sand flies examined (one male and one female) were collected in the southwestern administrative region (exprovince of Toliara), in the forest of Mikea, a dry deciduous forest belonging to the western phytogeographical region [6].
Sand flies were caught using CDC miniature light traps at the site named Abrahama -Jiloriaky, 7.5 km north-east of Tsifota (22°48.0 0 S -43°26.0 0 E) and 60 m a.s.l., over five consecutive nights from February 21 to 25, 2003. These collections were carried out in lowland forest, dominated by trees (Didieraceae, baobab trees and lianas) reaching 15 m 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.

Morphological analysis
The sand flies collected were 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 À20°C before DNA extraction. The specimens were 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 camera lucida installed on the microscope. To allow long-term preservation of the specimens, they were remounted on slides in Canada balsam, after complete processing by washing, dehydration in baths of ethanol 70-100 and immersion in creosote.

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 lL, as detailed in Depaquit et al. [2]. All the mtDNA amplifications were performed in a 50 lL volume using 5 lL 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 MgCl 2 , 50 mM KCl, 0.01% Triton X 100, 200 lM 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 cytochrome B gene was undertaken using the primers N1N-PDR and C3B-PDR [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 was done using the Pregap and Gap software included in the Staden Package [8]. Authorship: Note that the authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of International Code of Zoological Nomenclature [10].

Phlebotomus vaomalalae
Description (male: Figure 1; female: Figure 2) The terminology used in the description below is that of Abonnenc [9].

Holotype.
Head. Interocular suture: incomplete. Cibarial armature with some posterior teeth directed backwards and some anterior denticles. Pharynx quite narrow, with a discrete armature composed of very small aligned teeth, forming ripples. Some welldeveloped lateral teeth in the anterior part of the pharynx. Spermathecae. The body of each spermatheca consists of two successive parts. The apical section is larger (diameter = 34 lm), and is bordered by a thin and irregular wall. The smaller (diameter = 5 lm) is sclerotized and bounded by a thick rigid wall (diameter = 5 lm). The neck is 30 lm long and carries the head of the spermatheca. Absence of common duct is noticed. Individual ducts are approximately 180 lm long and tapered. In their apical part, they are narrow and their walls thicken irregularly and reveal discreet rings. Furca: observation difficult on our specimen.

Discussion
The presence of lower mesanepisternal setae justifies the inclusion of the new species in the genus Phlebotomus.
Until the revision of the subgenus Anaphlebotomus, suggested by Depaquit et al. [2,4] is undertaken, we have classified P. vaomalalae n. sp. in this subgenus due to the presence of the male characters listed by Theodor [11] when creating the subgenus: style bearing four spines absence of basal process on the coxite, and presence of a forked paramere. The female characters listed by that author for Anaphlebotomus do not justify the inclusion of P. vaomalalae n. sp. in this subgenus, although P. vaomalalae n. sp. should obviously be grouped with two other Phlebotomus females of the subgenus Anaphlebotomus already described from Madagascar: P. fertei and P. berentiensis. The females of these three species share a similar architecture of the spermathecae and similar pharyngeal and cibarial armatures. They differ markedly from the female of P. huberti that presents ringed frame spermathecae and a highly developed pharyngeal armature. The status of the latter species deserves special attention in light of the future description of the male.
We associate the male and female specimens described here with the following arguments. They were captured in the same location and in the same set of capture in light traps. The two specimens taken are the only Phlebotomus collected (along with 40 sand flies of the genus Grassomyia) in these series of catches. The two sexes are both close to P. fertei and P. berentiensis. Sequences of the Cytochrome b gene are identical (100% homology).
To date, four species belonging to the subgenus Anaphlebotomus have been reported in Madagascar.

Differential diagnosis
P. vaomalalae n. sp. is smaller than other Phlebotomus species of Madagascar.
In males (Table 1) and females (Table 2), the third antennal segment is much shorter in P. vaomalalae n. sp. than in P. fertei and slightly shorter than other Malagasy Phlebotomus, although the small sample size (only one specimen observed for each sex) does not allow us to assert it categorically. AIII is shorter than the length of the labrum in P. vaomalalae n. sp. whereas in the other species of Phlebotomus from Madagascar, AIII is longer than the labrum.
The wings of P. vaomalalae n. sp. are shorter than those of P. fertei. Their measurements are similar to those of other species    of Phlebotomus from Madagascar. The p (pi) of P. vaomalalae n. sp. is much lower than that of the other Phlebotomus, in both sexes (Tables 1 and 2). The cibarium of the male and the female of P. vaomalalae n. sp. is armed with teeth, like those of other Malagasy Phlebotomus. However, it differs in the arrangement and shape of the respective teeth and denticles. The cibarial armature of the male of P. vaomalalae n. sp. presents eight well-marked teeth with a few denticles. It is more developed in the male of P. fontenillei [2,4]. In contrast, the female of P. vaomalalae n. sp. carries only four teeth and thirty denticles. It is also clearly marked in P. fertei female and P. berentiensis [2,4].
The pharyngeal armature of P. vaomalalae n. sp. consists of small teeth and denticles irregularly organized in multiple rows.The pharyngeal armature of the male differs from that of P. fertei, which is formed of small teeth disposed on seven or eight concentric circular arcs, from that of P. berentiensis, which is narrow and lined with small pointed teeth and lateral teeth, and from that of P. fontenillei, which is composed of tapered corrugations and is well defined with the presence of some front lateral teeth [2,4].
The female of P. vaomalalae n. sp. differs from that of P. huberti by the armature of the pharynx and its spermathecal body with no ring. Its pharyngeal and cibarial armatures are comparable with those of P. berentiensis and P. fertei. They differ in the number, shape and arrangement of teeth and denticles. The pharyngeal armature of P. fertei and P. fontenillei comprises lateral teeth not present in P. berentiensis and P. vaomalalae n. sp.
The coxite and aedeagus of P. vaomalalae n. sp. are shorter than those of other Phlebotomus (Table 1).
The male of P. vaomalalae n. sp. looks like that of P. fontenillei due to its tuft of coxal setae, a tuft which is lacking in P. berentiensis and P. fertei. This tuft is implanted in the middle part of the inner face of coxite in P. vaomalalae n. sp. while it is located on the bottom part of the inner face of the coxite in P. fontenillei.
The spermathecae of P. vaomalalae n. sp. present no common duct, as in P. fertei and P. berentiensis. These species differ by spermathecal duct length, the structure of the tip, the basal sclerotization observed in P. fertei and the structure and thickness of the successive chambers, especially the largest one (Table 2).