Morphological keys for the identification of Italian phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae)

Background Phlebotomine sand flies are small blood-feeding insects of great medical and veterinary significance. Their identification relies basically on the microscopic examination of key morphological characters. Therefore, identification keys are fundamental to any researcher dealing with these insects. The Italian fauna of phlebotomine sand flies consists of eight species (Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus neglectus, Phlebotomus papatasi, Phlebotomus mascittii, Phlebotomus sergenti and Sergentomyia minuta), whose morphological delineation may be troublesome for non-taxonomists. Methods A total of 8,757 pictures were taken from the 419 selected phlebotomine sand fly specimens collected on different occasions. Twenty-eight characters for the males and 23 for the females were examined, resulting in a database containing over 10,000 entries. Representative phlebotomine sand fly specimens for each species available were selected and relevant characters were drawn with the aid of a camera lucida. Results After detailed morphological study of representative specimens, comprehensive identification keys based on key characters (e.g., pharynx and spermathecae of females and male terminalia) were elaborated. Conclusions The identification keys provided herein allow the identification of genera and species of phlebotomine sand flies of Italy and they will facilitate future studies on these medically important insects.


Background
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects of great medicoveterinary significance. Indeed, they are vectors of numerous pathogens to animals and humans, including protozoa, bacteria and viruses [1,2]. For instance, species of the genus Phlebotomus are vectors of phleboviruses (e.g., sand fly fever Naples virus, and sand fly fever Sicilian virus) causing the sand fly fever, which is a transient febrile illness that is mainly prevalent in the Mediterranean region [2,3]. Most importantly, phlebotomine sand flies are the biological vectors of Leishmania parasites which still cause disfiguring lesions and claim the lives of thousands of dogs and humans each year in more than 90 endemic countries [3].
Both cutaneous and visceral forms of leishmaniasis are quite prevalent in southern Europe [4]. Among other factors, the high prevalence of human and animal leishmaniasis in southern Europe is a consequence of the wide distribution and density of phlebotomine sand fly vectors. Indeed, they are spread throughout southern Europe, particularly in countries such as Portugal, Spain, France, Italy and Greece [4] [9], on the number of Newstead's spines (=hyaline sensilla) on the third palpal segment [10] and, most frequently, on the morphology of the spermathecae (receptacula seminis) of the female [11]. Nonetheless, comprehensive and illustrated identification keys for Italian sand flies are not available in the international literature. Indeed, recent Italian studies have adopted different taxonomic sources. For instance, Rossi et al. [12] and Morosetti et al. [13] have based their identification on French [14] and German [15] works, whereas Tarallo et al. [6] have adopted a paper on the identification of female sand flies of the subgenus Larroussius [16] and an Italian book with illustrations for the identification of acari and insects of medical and veterinary significance [17].
Recent studies have demonstrated the usefulness of different genetic markers (e.g., ITS2 and cytb-nd1 regions) for the molecular identification of phlebotomine sand flies [18][19][20]. In the same way, protein profiling using the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has also be proposed as a promising tool for the identification of phlebotomine sand flies [21]. Nonetheless, the identification of these insects is still primarily achieved through the microscopic examination of key morphological characters, including pharynx, spermathecae and cibarium of females as well as male terminalia [22,23]. Thus, morphological keys for the identification of phlebotomine sand flies are pivotal for studies dealing with these insects. In this context, we propose herein identification keys for genera and species of Phlebotominae of Italy.

Methods
Phlebotomine sand fly specimens used herein were collected at different occasions, in studies conducted in Apulia, Sicily and Basilicata regions, southern Italy [6][7][8]18]. As a rule, collection sites were selected based on their characteristics, including presence of animals, type of vegetation, and degree of urbanization. Phlebotomine sand flies were collected using ordinary collection methods, such as sticky traps (white paper sheets coated with Castor oil), light traps (model IMT, Byblos per l'Igiene Ambientale di Wehbe Nasser, Cantù, CO, Italy) or mouth aspirators. Phlebotomine sand flies collected with light traps and mouth aspirators were directly preserved in 70% ethanol. Those caught with sticky traps, however, were firstly washed with 90% ethanol, Table 1 Key to the genera of Phlebotominae of Italy 1. Cibarial teeth in a transverse row and pigment patch usually present ( Figure 1A; red circle). Hind end of abdominal tergites 2-6 with all or most setae recumbent, which arise from small sockets as compared with those on tergite 1 ( Figure 1B). Male style with 3 apical and 1 sub-apical long spines developed and 1 more basal setiform spine (accessory seta) (Figure 2A). Female spermatechae smooth ( Figure 3A)

Sergentomyia a
Cibarial teeth and pigment patch usually absent ( Figure 1C). Hind end of abdominal tergites 2-6 with many erect setae, which arise from large sockets of the same size as those on tergite 1 ( Figure 1D). Male style with 2 long apical spines ( Figure 2B,D-H) or 3 short apical spines ( Figure 2C) and setiform spine absent. Female spermatechae with superficial striation ( Figure 3B) or annulated ( Figure 3C-H)   Aedeagus with colourless plate (transparent process) on terminal portion P. perfiliewi ( Figure 2E) Aedeagus without colourless plate on terminal portion 5

5.
Aedeagus bifid at the end P. perniciosus ( Figure 2F) Aedeagus not bifid at the end 6 6. Adeagus clapper-like, with moderate subapical expansion P. ariasi ( Figure 2G) Adeagus with drumstick-like tip P. neglectus ( Figure 2H) in order to remove excess of oil [5] and then kept in labelled vials containing 70% ethanol. Before proceeding with species identification, phlebotomine sand flies were examined using a stereomicroscope (Leica Microsystems, MS5, Germany), separated from other insects and according to sex. For mounting on slides, specimens were cleared with 10% potassium hydroxide solution at room temperature for 2 h. The material was then washed with water for 1-2 min, immersed in 10% aqueous solution of glacial acetic acid for 30 min, washed again with water for 30 min and, finally, slide-mounted in Hoyer's solution as described by Lewis [24]. Species identification was made according to different morphological keys, species descriptions and other identification resources [14,16,17,25].
Out of about 16,500 phlebotomine sand flies examined over the past 10 years, representative specimens of each species were selected and further studied morphologically. Specimens of both sexes (i.e., 233 males and 186 females) were selected based on conservation status and quality of the clarification. In some cases, all insects of a given species (e.g., P. sergenti) or of a specific sex (e.g., P. neglectus female) were used, due to the limited number of specimens available. Several morphological characters were examined, but only key characters (e.g., pharynx and spermathecae of females and terminalia of males) were considered during the preparation of the identification keys. Incidentally, these characters were those reported in the keys proposed by Lewis [24].
Representative phlebotomine sand fly specimens for each species available were selected and relevant characters were drawn with the aid of a camera lucida (Leica Microsystems, L 3/20, Germany). The pencil drawings were scanned, the resulting files were imported into Adobe Illustrator C6 and the line drawings were made using a digitiser board (WACOM Intuous 5 touch PTH-650, Wacom Europe GmbH, Germany). Voucher phlebotomine sand fly specimens are deposited in the Laboratory

Results and discussion
After detailed morphological study of representative specimens, comprehensive identification keys for genera and species of phlebotomine sand flies of Italy were elaborated (Tables 1, 2  Identification keys are fundamental for anyone dealing with insects of medical and veterinary significance, such as phlebotomine sand flies. They are intended to provide a guide for those interested to identify field-collected specimens obtained for distinct purposes and different kind of studies (e.g., seasonality, vectorial role and taxonomy). Indeed, identification keys, especially those accompanied by line drawings illustrating taxonomically  relevant characters, are useful for species identification of phlebotomine sand flies [26].
Filippo Bonanni, an Italian Jesuit scholar, published in 1691 the first illustration of a phlebotomine sand fly. Later on, in 1786, the Italian naturalist Giovanni Antonio Scopoli described the species Bibio papatasi (later replaced in the genus Phlebotomus), the first phlebotomine sand fly ever described [25]. In the same way, at the end of the 19th and beginning of the 20th centuries, respectively, Rondani and Grassi described new species based on material collected in Italy [25]. Interestingly, in spite of the long tradition of Italy in the field of phlebotomine sand fly and leishmaniasis research, the identification of these insects in Italian studies has mostly been based on old keys and/or on an Italian book for the identification of acari and insects of medical and veterinary significance [17]. To the authors' knowledge, before the present work, no keys for the identification of genera and species of phlebotomine sand flies of Italy were available in the international literature.

Conclusions
In conclusion, the present paper provides identification keys for genera and species of phlebotomine sand flies found in Italy, which will facilitate future studies on these medically important insects. These simplified keys, along with the line drawings provided herein are intended for anyone dealing with sand fly identification in Italy and may also be useful for those working in other Mediterranean countries, as most of the species found in Italy are also prevalent in countries such as Spain, Portugal and Greece [4].