Phlebotomus (Euphlebotomus) barguesae n. sp. from Thailand (Diptera – Psychodidae)

Background A few studies have been carried out on the Phlebotomine sandflies from Thailand. Within the Phlebotomine sandflies, the genus Phlebotomus Rondani & Berté, 1840 contains the vectors of leishmaniases in Europe, Africa and Asia. It includes several subgenera. Among them the subgenus Euphlebotomus Theodor, 1948 contains at the present time 12 taxa. The type-species of this subgenus is P. argentipes Annandale & Brunetti, 1908, the vector of Leishmania donovani (Laveran & Mesnil, 1903) in India. Results A new species of sandfly, P. barguesae n. sp. is described from limestone caves in Thailand. The male-female gathering in the same species is based on ecological, morphological and molecular criteria (homology of mtDNA cytochrome c oxidase I sequences). The inclusion of P. barguesae n. sp. in the subgenus Euphlebotomus is justified on the basis of characters of the male genitalia (five spines on the style, bifurcated paramere, and no basal lobe on the coxite) and of female pharyngeal armature (two kinds of teeth). It well differenciated from another sympatric species: P. mascomai. Conclusion The new species described in the present study has smooth spermathecae. This original morphology opens a discussion on the heterogeneity of this subgenus.

All the specimens were caught in July 2004 and February 2005 (Barbazan recoltavit) by CDC miniature light traps between 5 p.m. and 9 a.m. They were kept in 96° ethanol, and mounted in toto for morphological study according to Abonnenc's method [6]: 4-8 hours in 10% KOH solution followed by eight baths (20 minutes each) in water, then at least 1 hour in Berlese's medium. Females were directly mounted in this liquid with their spermathecae dissected, then re-mounted in chloral gum when possible. Some males were dehydrated in ethanol of growing concentrations (from 70% to absolute ethanol) then in beech creosote and finally mounted in Canada balsam. 23 specimens have been studied among them 16 (10 males and 6 females) were prepared for morphological study and seven specimens (5 males and 2 females) were prepared for both morphological and molecular studies. Four topotypes of P. mascomai (two males and two females) coming from the same cave have also been prepared for both morphological and molecular studies and processed like P. barguesae n. sp. specimens. The selected gene was cytochrome c oxidase I of the mtDNA. The head and genitalia of each sandfly were cut off in a drop of ethanol, cleared in boiling Marc-André solution, and mounted under a cover slip in gum chloral for identification. These slides are available upon request to the corresponding author. Genomic DNA was extracted with the QIAmp DNA Mini Kit (Qiagen, Germany) by following the manufacturer's instructions, except for the crushing of sandfly tissues with a piston pellet (Treff, Switzerland). Polymerase chain reactions (PCR) were performed on a 50 μl volume using 5 μl of extracted DNA solution and 50 pmol of each of the two primers LepF (5'-ATTCAACCAAT-CATAAAGATATTGG-3) and LepR (5'-AAACTTCTGGAT-GTCCAAAAAATCA-3') [7] were used under the following thermal profile [8]: an initial denaturation step at 94°C for 3 min, followed by 5 cycles of (denaturation at 94°C for 30 s, annealing at 45°C for 90 s and extension at 86°C for 60 s), then 35 cycles of (denaturation at 94°C for 30 s, annealing at 51°C for 90 s and extension at 86°C for 60 s) and a final extension at 68°C for 10 min. using 0.25 μl of Taq DNA (5 prime, Germany). Sequencing was performed on both strands by the dideoxy chain-termination method with the Taq dye-terminator chemistry kit for ABI 373A (Perkin-Elmer, Foster City, CA, USA), using PCR primers. Direct sequencing of both DNA strands was performed using the primers used for DNA amplification. The correction of sequences was done using pregap and gap software [9,10]. Their alignment has been performed using Bioedit software [11].
Specimens were observed using a BX 50 microscope (Olympus, Japan). Measurements were collected using the Perfect Image software (ARIES Company, Chatillon, France) by means of a video camera connected to the microscope.     -Cibarium with denticles toward centre and back. No pigmented patch.

Description
-Pharynx: length = 170 to 240 μm. Typical armature of the subgenus Euphlebotomus with two kinds of teeth: medio-anterior long, dark, and pointing toward the front; latero-posterior short, in a semi-circular disposition.

Discussion
The male-female gathering in the same taxon is based on ecological (cave dwellers caught the same night, in the same place), morphological and molecular criteria. Length of male genital filaments is in agreement with length of females individual sperm ducts, and the width of males aedeagus with width of females common duct. The sequence length is 689 bp for each specimen. Molecular analysis showed 100% homology between cytochrome c oxidase I sequences of males and females [Genbank accession numbers FJ348734-FJ348740 for P. barguesae n. sp. and FJ493545 to FJ493548 for P. mascomai]. Despite the fact that this marker is commonly sequenced for the "barcode of life" program [13], it has been surprisingly used only once for Phlebotomine sand flies when Arrivillaga et al. [14] have characterised several cryptic species within the neotropical complex Lutzomyia longipalpis. It has also be used successfully to distinguish two closely related species from North Africa: P.  [15]. This marker strongly supports that the specimens sequenced belong to the same species: P. barguesae n. sp. The figure 3 shows the high variability between sympatric P. barguesae n. sp. and P. mascomai topotypes.
The differential diagnosis with other species of Euphlebotomus is easy for both sexes. The females have exclusive smooth spermathecae and the males have a blunt-end aedeagus not previously observed in the subgenus Euphlebotomus.
sequence alignment showing the 86 variable sites between sequences of P. barguesae n. sp topotypes and P. mascomai topotypes  In 1948, Theodor created within the genus Phlebotomus, the sub-genus Euphlebotomus, with P. argentipes as typespecies, that he individualized on the following states of characters [16]: for males: -style with 5 spines, similar to that of Larroussius -paramere trilobed, with or without an accessory spine, -aedeagus short and conical.
for females: -pharynx with median armature of small teeth and posterior parallel ridges, -spermathecae segmented or striated with apical segment defined and enlarged.  Leng. If we consider i) the inclusion of P. lengi within the subgenus Euphlebotomus needs further studies, ii) the sharing of the remarkable character "presence of a paramere spine" with some Anaphlebotomus species, iii) the definition doesn't covering the morphological patterns of spermathecae, and iv) the variable number of appendices of the paramere, a major revision of the "Euphlebotomus-Anaphlebotomus" group has to be done at the light of both molecular and morphological studies. Waiting this study, we consider P. barguesae n. sp. as belonging to the subgenus Euphlebotomus.

Conclusion
P. barguesae n. sp. is a new species of Phlebotomine sandflies. The male-female gathering in the same species is strongly supported by ecological, morphological and molecular criteria. We consider this new species belongs to the subgenus Euphlebotomus (male having 5 spines on the style and a trifurcated paramere, female pharyngeal armature). However, the original smooth spermathecae opens a discussion on the heterogeneity of this subgenus.