Phlebotomus

Phlebotomine sand fly specimens used herein were collected at different occasions, in studies conducted in Apulia, Sicily and Basilicata regions, southern Italy [6 (link)-8 (link),18 (link)]. 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, 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 (link),16 (link),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 of Parasitology and Parasitic Diseases at the Department of Veterinary Medicine, University of Bari, Italy.

We investigated the design of potential immunogenic peptides of the gSG6 protein using bio-informatic tools. The strategy was i) to identify potential immunogenic epitopes predicted by algorithms; and ii) to research the specificity of An. gambiae gSG6 peptide sequence compared to the genome/ Expressed Sequence Tag (EST) libraries of other organisms.
This analysis was based on the amino acid sequence of mature An. gambiae gSG6 (“UniProtKB/TrEMBL:Q9BIH5” and “gi:13537666”, [23] (link)).
The identification of putative linear B-cell epitopes of An. gambiae gSG6 was performed by computerized predictions of antigenicity based on physico-chemical properties of the amino-acid sequences with the BcePred database [24] and with the FIMM database [25] (link). We also identified the MHC class 2 binding regions using the ProPred-2 online service [26] (link).
Sequence alignments were done with the Tblastn program in Vectorbase database [27] (link) which enabled comparing a sequence of gSG6 peptides with known genomes or EST libraries of Aedes aegypti, Ixodes scapularis, Culex pipiens, Pediculus humanus, Glossina morsitans, Rhodnius prolixus, Lutzomia longipalpis and Phlebotomus papatasi. Concomitantly, we investigated sequence alignments with the Blast program to compare the gSG6 peptides sequence with all non-redundant GenBank CDS database [28] (link).
Peptides were synthesized and purified (>80%) with Genosys (Sigma-Genosys, Cambridge, UK) with an added N-terminal biotin. All peptides were shipped lyophilized and they were resuspended in 0.22 µm filtered milliQ water and stored in aliquots at −80°C.

The colony of P. papatasi was maintained at 26°C on 50% sucrose and 14 h light/10 h dark photoperiod as described previously (Benkova and Volf, 2007 (link)). Sand fly females were infected by feeding through a chick-skin membrane on heat-inactivated rabbit blood containing 10⁶ promastigotes ml⁻¹. Engorged sand flies were maintained in the same conditions as the colony and dissected 2, 5, 9 and 12 days PBM. The location of Leishmania infections in the sand fly digestive tract (foregut, SV, thoracic and abdominal midgut, and endoperitrophic and ectoperitrophic space) was determined by dissection and examination by light microscopy. Parasite loads were estimated by two methods: infections seen in the gut in situ were graded according to Myskova et al. (2008 (link)) as light (< 100 parasites per gut), moderate (100–1000 parasites per gut) and heavy (> 1000 parasites per gut). Alternatively, 30–40 guts from females with late infections (10–12 days PBM) were individually dissected into NET 50 and stored in −20°C for qPCR. Sand fly infection experiments were repeated four times for combinations of wild-type (FVI), KO and Kin lines and twice for combinations of FVI, +HASPB and +SHERP lines.

Descriptive statistical analyses were performed on data from the clinical examinations, daily health assessments, BW and hair length measures, safety observations (i.e. administration observations, local tolerance observations and adverse effects), infestation intensity for fleas and ticks, and sand fly counts and serological and molecular analyses. Median (Mdn), mean (M) and standard deviation (SD) values were calculated for genome copy values of L. infantum obtained by qPCR.

Samples were collected in June 2019 and September 2020 in Casalgrande locality (44°33′40.3″ N, 10°43′27.8″ E), Reggio Emilia province. The site was located in a hilly area of the Emilia-Romagna region at 257 m altitude (Table 1). Sandfly sampling was performed overnight using two types of traps: carbon-dioxide-baited traps and CDC light traps. In 2019, sandflies were morphologically identified with an optical microscope after clarification, while in 2020, the collected sandflies were stored in dry ice and used directly for virus isolation in cell culture with a maximum of 25 specimens per pool.

Six mice were anesthetized intraperitoneally with ketamine (100 mg/kg) mixed with xylazine (10 mg/kg). Ophthalmic ointment (Major Pharmaceuticals) was topically applied to the eyes to prevent corneal dryness. Three mice were exposed to ten uninfected sand flies and 3 mice were exposed to ten sand flies harboring mature infections. The sand flies were applied to both ears of each mouse using vials with a meshed surface held in place by custom-made clamps. Sham mice exposed to empty vials were used as negative controls. Sand flies were allowed to feed for 2–3 h in the dark. After exposure to mice, the sand flies were aspirated from vials, anesthetized with CO₂, washed in 5% soap solution, rinsed, and placed in a petri dish containing PBS. The number of blood-fed flies was counted under a stereomicroscope. Mice were euthanized 30 min, 6 h, 24 h, and 48 h after sand fly bites. For every experiment, both earlobes of 3 mice were used. Each earlobe was cut into 3 segments, and 2 replicates were made for every experimental condition (n = 36 earlobe segments analyzed for every condition).