Description of Sergentomyia phadangensis n. sp. (Diptera, Psychodidae) of Thailand

Since 1996, there are emerging autochthonous cases of leishmaniasis in Thailand due to Leishmania “siamensis” and to L. martiniquensis explaining a recent interest for the sand fly fauna where Sergentomyia gemmea and Se. barraudi have been considered possible vectors in the country. Field studies were undertaken in a cave of Phitsanulok Province, Thailand. Phlebotomine sandflies have been studied morphologically and some have been processed for molecular biology (sequencing of cytB rDNA). A new species of sand fly, belonging to the genus Sergentomyia: Se. phadangensis n. sp., is described. The association of the male and female is supported by the homology of the sequences of cytochrome b rDNA. The description of a new species in Thailand is of importance in view of the existence of autochthonous leishmaniases.


Background
As it is an area which has long been considered to be free of leishmaniasis, there are few data related to Phlebotomine sand flies in South-Eastern Asia. However, the first autochthonous South-Eastern Asian case of leishmaniasis was discovered in Thailand in 1996, followed by a total of 13 cases due to Leishmania "siamensis" and, surprisingly, to L. martiniquensis [1], a species of the French West Indies, recently described [2]. This new epidemiological datum explains a growing interest in the sand fly fauna of Thailand. A total of 26 species of phlebotomine sand flies have been recorded in this country: one Chinius, three Idiophlebotomus, eight Phlebotomus, one Grassomyia and 13 Sergentomyia [3][4][5][6], excluding Nemapalpus vietnamensis not considered to be a sand fly [7] and some doubtful records.
During field work carried out over one year in the Phadang limestone cave, the most abundant species caught -new to Science -is described in the present paper.

Sampling
Field studies were undertaken between February 2010 and January 2011 in Phadang cave in Noen Maprang, Phitsanulok Province, located in the lower northern part of Thailand at 16°30'58.8"N, 100°40'00.5"E. This limestone cave, situated in a mountainous area, is found in the foothills of a mountain and consists of two asymmetrical caverns. It is estimated to be 15-80 m wide, 80-100 m long, and 1-20 m high, and lies at 108 meters a.s.l. The cave may be wet or dry depending on the season and the amount of sunlight it receives. It is accessible to travelers, but is home to many bats.

Mounting
Phlebotomine sand flies were collected using CDC miniature light traps usually installed overnight from 6 p.m. to 6 a.m. Specimens collected were stored in 96 % ethanol. Some of them were mounted in toto in Hoyer's medium and others for the application of molecular biology techniques. Regarding the latter: head, thorax and genitalia were cut off in a drop of ethanol, cleared in boiling Marc-André solution and, after dehydration, mounted on slides in Canada Balsam. The anterior part of the abdomen of each specimen was dried and stored in a vial at −20°C, before DNA extraction.
The specimens were observed under a BX53 microscope equipped with a video camera and measured using Stream motion software (Olympus, Japan). The usual keys for the identification of the sand flies from the indo-chinese region have been consulted [8][9][10]. Drawings were made using a camera lucida. Morphological terminology is that commonly used for Old World sandflies [4,5,8,[10][11][12][13] and we add the corresponding nomenclature proposed for Diptera [14]. Abbreviations of generic names follow [15]. All measurements are in μm.

Molecular analysis
We decided to sequence a fragment of cytochrome b (Cyt b) which is the most used molecular marker for Phlebotomine sand fly systematic studies [16]. Genomic DNA was extracted individually from the part of the abdomen of sand flies (5 males and 5 females) 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 50 to 200 μl [12].
All the mtDNA and rDNA amplifications were performed in a 50 μl volume using 5 μl of extracted DNA solution and 50 pmol of each of the primers N1N-PDR and C3B-PDR [17]. 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 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. PCRs were done with the following temperature profile: 5 cycles with 30 sec 94°C, 40 sec 40°C, 1 min 68°C and 35 cycles with 30 sec 94°C, 30 sec 44°C, 1 min 68°C.
Amplicons were analyzed 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 Pregap and Gap softwares included in the Staden Package [18]. Consensus sequences were aligned by the Clustal W algorithm [19] from the BioEdit 4.8.10 sequence editor [20].

Molecular analysis
The sequences analysed in the present study have been deposited in Genbank under numbers KT266691 to KT266700. All of them are similar (100 % homology). Cibarium with a very important curved armature consisting of 6 to 8 strong and pointed teeth on each side and a few much smaller median and central ones. All these teeth are darkly pigmented in brown. Sometimes some accessory teeth in the upper and lateral parts of the cibarium. Pigment patch: wide, less pigmented than the teeth, triangular with rounded angles.

Description of
Palpal formula: 1, 2, (3, 4), 5. About forty Newstead's scales club-like in a patch on the basal face of the third segment.
*Spermathecae: smooth and wide. No limit between the body and individual duct. Presence of a common basal duct.
Furca with a long and thin anterior part.

*Head
Inter-ocular suture complete. Cibarium with an important curved armature consisting of three to five large pointed teeth on each side and twelve to twenty denticles along the curve and sometimes outside this line. The latter are observable using the phase contrast option of the microscope.
No trace of a pigmented area could be seen. Pharynx quite narrow, with a discrete armature composed of small teeth oriented backward.
*Genital Armature Coxite with a few internal setae not grouped in a tuft. Style narrow with four terminal spines. The accessory spine is implanted distally (between the distal quarter and third).
The holotype (female) and 10 paratypes (5 females and 5 males) have been deposited in the National Science Museum of Pathumthani (Thailand). Fourteen paratypes (ten females and four males) have been Etymology: the name Sergentomyia phadangensis n. sp. is related to the Phadang cave where it has been caught.

Discussion
For a long time, the species of the genus Sergentomyia were little studied as they were not regarded as vectors of any human leishmaniasis agent. However, some of them have recently been presented as possible vectors of Leishmania responsible for human infection [21], in Thailand too [22].
Sergentomyia is the genus with the greatest known diversity, surpassing the number of species of all other Old World genera (Phlebotomus, Idiophlebotomus, Chinius, Spelaeophlebotomus, Grassomyia, Parvidens, Spelaeomyia and Demeillonius) taken together [23,24]. The species of the genus Sergentomyia share the following characters: a mesanepisternum without setae, abdominal tergites 2-6 usually carrying all or most recumbent hairs, a usual 1/III-XV antennal formula in males and 2/III-XV in females with some exceptions, a cibarium with an armature of teeth and/or denticles more developed in females than in males (apart from exceptions), a single paramere, a style with four terminal spines (or often 2 terminal and 2 subterminal ones) and an accessory spine.
The species Se. phadangensis n. sp. exhibits the characters qualifying it for inclusion in the genus Sergentomyia.
Mainly based on the spermathecal morphology, the genus Sergentomyia is subdivided into eight subgenera: Se. phadangensis n. sp. exhibits wide and smooth spermathecae and can therefore be included in the subgenus Sergentomyia.
This new species can be easily differentiated from all the other species recorded in South-Eastern Asia by its highly developed cibarial armature in both females and males. The teeth are bigger and darker than those of the most closely related species, Se. dentata (Figs. 4 and 5). Moreover, the pharynx of Se. dentata is wide with a Se. antennata-like proeminent armature whereas that of Se. phadangensis n. sp. is narrow with a Se. schwetzi-like discret armature (Fig. 6). The record of Se. dentata in Thailand [26] should be checked in the light of the description of this new species. Initially described from Pakistan, Se. dentata is mainly distributed in the Middle-East and the Eastern Mediterranean basin [27].