Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.     

Prevalence of sand flies and Leishmania donovani infection in a natural population of female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.     

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.     

Multilocus molecular and phylogenetic analysis of phlebotomine sand flies (Diptera: Psychodidae) from southern Italy

This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of phlebotomine sand flies (Diptera: Psychodidae) from the Mediterranean region. A ～900 bp long fragment of the mitochondrial DNA encompassing regions within cytb and nd1 gene and the complete ITS2 ribosomal region (～500 bp) were sequenced and characterized for Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus papatasi, and Sergentomyia minuta, captured in two sites of southern Italy. From one to eight mitochondrial haplotypes and from one to three ITS2 sequence types were found for the examined specimens according to the different sand fly species. The mean interspecific difference in the mitochondrial sequences was of 16.1%, with an overall intraspecific nucleotide variation from 0.1 to 2.8%. A higher interspecific difference (mean 25.1%) was recorded for the ITS2 sequence, with an overall intraspecific nucleotide variation up to 4.9%. The sequence types alignment of ITS2 region showed that all phlebotomine specimens possessed a split 5.8S rRNA, consisting of a mature 5.8S rRNA and a 2S rRNA separated by a short transcribed spacer. Phylogenetic analysis of the Phlebotomus spp. sequences, herein determined and of those available in GenBank? were concordant in clustering P. neglectus, P. perfiliewi and P. papatasi with the same species collected from different geographic areas of the Mediterranean basin in four main clades for mtDNA and ITS2, respectively. This study demonstrates the utility of multilocus sequencing, provides a dataset for the molecular identification of the most prevalent phlebotomine sand flies in southern Europe and defines the phylogenetic relationships among species examined.     

DNA barcoding for identification of sand fly species (Diptera: Psychodidae) from leishmaniasis-endemic areas of Peru

Phlebotomine sand flies are the only proven vectors of leishmaniases, a group of human and animal diseases. Accurate knowledge of sand fly species identification is essential in understanding the epidemiology of leishmaniasis and vector control in endemic areas. Classical identification of sand fly species based on morphological characteristics often remains difficult and requires taxonomic expertise. Here, we generated DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene using 159 adult specimens morphologically identified to be 19 species of sand flies, belonging to 6 subgenera/species groups circulating in Peru, including the vector species. Neighbor-joining (NJ) analysis based on Kimura 2-Parameter genetic distances formed non-overlapping clusters for all species. The levels of intraspecific genetic divergence ranged from 0 to 5.96%, whereas interspecific genetic divergence among different species ranged from 8.39 to 19.08%. The generated COI barcodes could discriminate between all the sand fly taxa. Besides its success in separating known species, we found that DNA barcoding is useful in revealing population differentiation and cryptic diversity, and thus promises to be a valuable tool for epidemiological studies of leishmaniasis.     

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.     

Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.     

Natural Leishmania Infection of Lutzomyia auraensis in Madre de Dios, Peru, Detected by a Fluorescence Resonance Energy Transfer-Based Real-Time Polymerase Chain Reaction

Abstract. Leishmania species of the Viannia subgenus are responsible for most cases of New World tegumentary leishmaniasis. However, little is known about the vectors involved in disease transmission in the Amazon regions of Peru. We used a novel real-time polymerase chain reaction (PCR) to assess Leishmania infections in phlebotomines collected in rural areas of Madre de Dios, Peru. A total of 1,299 non-blood fed female sand flies from 33 species were captured by using miniature CDC light traps. Lutzomyia auraensis was the most abundant species (63%) in this area. Seven of 164 pools were positive by PCR for Leishmania by kinetoplast DNA. The real-time PCR identified four Lu. auraensis pools as positive for L. (Viannia) lainsoni and L. (V.) braziliensis. The minimum infection prevalence for Lu. auraensis was estimated to be 0.6% (95% confidence interval = 0.20-1.42%). Further studies are needed to assess the importance of Lu. auraensis in the transmission of New World tegumentary leishmaniasis in hyperendemic areas of Peru.     

Distribution and ecological aspects of sand flies(Diptera:Psychodidae) species in Northeastern Iran

Objective:To determine both the distribution and the ecological characteristics of sand flies in Golestan Province,northeast of Iran in 2016.Methods:In this study,34 villages were selected based on their geographical conditions.Sticky paper traps were used for collecting the sand flies.Sampling was carried out in each of villages from May to November.In each village,60 traps for indoors and 60 for outdoors were monthly installed.The species of all collected sand flies were determined using approved morphological keys.Pearson coefficient correlation was used to find the relationship between the number of collected Phlebotomus papatasi from different villages and incidence rate of zoonotic cutaneous leishmaniasis as well as the number of positive cases of the disease.The altitude of the studied villages was extracted from digital elevation model of the area using GIS and vegetation cover density index of the province was extracted from Modis satellite imagery and distribution map of sand flies drown up.Results:Overall,5 428 sand flies were collected and identified,belonging to 18 species.Phlebotomus wenyoni was reported for the first time from the area in this study.The frequency of sand flies in the villages located in northeast of the Golestan province(the plateau area,lower altitude,arid and semi-arid climates,and lower vegetation cover density),were more than other villages in this province.There was a significant correlation between the number of collected Phlebotomus papatasi and incidence rate of the zoonotic cutaneous leishmaniasis cases in different villages(r=0.837,P=0.019) as well as the number of positive cases of the disease(r=0.688,P0.001).Conclusions:In the northeaster areas of Golestan Province which is known as the endemic foci of zoonotic cutaneous leishmaniasis,the abundance of sand flies were more and the conditions for their growth and development were more appropriate.     

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) in urban rainforest fragments,Manaus – Amazonas State,Brazil

The non-flooded upland rainforest fragment in the Federal University of Amazonas Campus is considered one of the world's largest urban tropical woodland areas and Brazil's second largest one in an urban setting. It is located in the city of Manaus, State of Amazonas at 03° 04′ 34″ S, 59° 57′ 30″ W, in an area covering nearly 800 hectares. Forty-one (41) sand fly species belonging to genus Lutzomyia were found attaining a total of 4662 specimens collected. Lutzomyia umbratilis was the dominant species at all heights, followed by Lutzomyia anduzei and Lutzomyia claustrei. The fauna alpha diversity index showed to be 6.4, which is not much lower than that reported for areas of continuous forest in this Amazonian region. This data provides additional evidence on Phlebotomine sand flies found to transmit Leishmania and other trypanosomatids to humans and other animals circulating in this area. This is the first study being reported on sand flies collected in an urban rainforest fragment in Amazonia.     

Studies of phlebotomine sand fly (Diptera: Psychodidae) populations in limestone areas and caves of western Malaysia

Phlebotomine sand flies were collected using CO2 baited CDC light trap in 2000 and 2001 in limestone areas and caves of western Malaysia. A total of 1,548 specimens were collected comprising 18 species from two genera: Phlebotomus (6 spp) and Sergentomyia (12 spp). Phlebotomus major major (38.9%) was the predominant species followed by Sergentomyia perturbans (20.1%), P. stantoni (15.3%) and others. Biting activity of the sand flies at the Gua Senyum caves, Gua Kota Gelanggi, Batu caves and Gua Kelam were observed using the bare leg landing catch (BLC) technique. Four Phlebotomus spp at Gua Senyum were found to bite humans with a unimodal biting peak (between 01:00 and 04:00 AM). At Gua Kota Gelanggi P. major major was observed to bite humans, but at Batu Caves and Gua Kelam no sand flies were observed to bite humans. Sergentomyia spp did not feed on humans even though high numbers were caught in light traps. The populations of phleobotomine sand flies fluctuated, with several peaks especially among P. major major which peaked in December and was low in February and August. Phlebotomus stantoni was abundant throughout 2001. Most species populations were weakly related to rainfall because they inhabited caves.     

Genetic diversity of the mitochondrial cytochrome b gene in Lutzomyia spp., with special reference to Lutzomyia peruensis,a main vector of Leishmania (Viannia) peruviana in the Peruvian Andes

The genetic divergence caused by genetic drift and/or selection is suggested to affect the vectorial capacity and insecticide susceptibility of sand flies, as well as other arthropods. In the present study, cytochrome b (cyt b) gene sequences were determined in 13 species circulating in Peru to establish a basis for analysis of the genetic structure, and the intraspecific genetic diversity was assessed in the Lutzomyia (Lu.) peruensis, a main vector species of Leishmania (Viannia) peruviana in Peruvian Andes. Analysis of intraspecific genetic diversity in the cyt b gene sequences from 36 Lu. peruensis identified 3 highly polymorphic sites in the middle region of the gene. Haplotype and gene network analyses were performed on the cyt b gene sequences of 130 Lu. peruensis in 9 Andean areas from 3 Departments (Ancash, Lima and La Libertad). The results showed that the populations of La Libertad were highly polymorphic and that their haplotypes were distinct from those of Ancash and Lima, where dominant haplotypes were observed, suggesting that a population bottleneck may have occurred in Ancash and Lima, but not in La Libertad. The present study indicated that the middle region of the cyt b gene is useful for the analysis of genetic structure in sand fly populations.     

Molecular detection of the blood meal source of sand flies (Diptera: Psychodidae) in a transmission area of American cutaneous leishmaniasis,Paraná State,Brazil

The feeding behavior of sand flies provides valuable information about the vector/host interactions and elucidates the epidemiological patterns of American cutaneous leishmaniasis (ACL) transmission. The aim of this study was to identify the blood meal sources of sand flies in endemic areas of leishmaniasis in Paraná State through polymerase chain reaction (PCR) amplification of a prepronociceptin (PNOC) gene fragment and its subsequent DNA sequencing. Moreover, molecular assays were conducted to evaluate the sensitivity and reproducibility of the PNOC gene amplification. Besides that, a time-course digestion test of the blood using sand flies that fed artificially on BALB/c mice was performed. Of 1263 female sand flies collected in the field, 93 (3.6%) specimens were engorged and 27 allowed efficient amplification of the PNOC gene. These flies had fed on equine (Equus caballus), porcine (Sus scrofa) and canine (Canis lupus familiaris) species. The results also showed that the identification of the blood meal sources of the sand flies using the molecular method was directly linked to the level of digestion of the blood (time-course) and not to the amount of blood that had been ingested or to the presence of inhibitors in the blood.     

Study of sand flies (Diptera: Psychodidade) in visceral and cutaneous leishmaniasis areas in central western of Minas Gerais state – Brazil

The transmission of Leishmania involves several species of sand flies that are closely associated with various parasites and reservoirs, with differing transmission cycles in Brazil. A study on the phlebotomine species composition has been conducted in the municipality of Divinópolis, Minas Gerais, Brazil, an endemic area for cutaneous leishmaniasis (CL), which has intense occurrence of visceral leishmaniasis (VL) cases. In order to study the sand flies populations and their seasonality, CDC light traps (HP model) were distributed in 15 houses which presented at least one case of CL or VL and in five urban parks (green areas). Collections were carried out three nights monthly from September 2010 to August 2011. A total of 1064 phlebotomine specimens were collected belonging to two genera and seventeen species: Brumptomyia brumpti, Lutzomyia bacula, Lutzomyia cortelezzii, Lutzomyia lenti, Lutzomyia sallesi, Lutzomyia longipalpis, Lutzomyia migonei, Lutzomyia intermedia, Lutzomyia neivai, Lutzomyia whitmani, Lutzomyia christenseni, Lutzomyia monticola, Lutzomyia pessoai, Lutzomyia aragaoi, Lutzomyia brasiliensis, Lutzomyia lutziana, and Lutzomyia sordellii. L. longipalpis, the main vector of Leishmania infantum in Brazil, was the most frequent species, accounting for 76.9% of the total, followed by L. lenti with 8.3%, this species is not a proven vector. Green and urban areas had different sand flies species composition, whereas the high abundance of L. longipalpis in urban areas and the presence of various vector species in both green and urban areas were also observed. Our data point out to the requirement of control measures against phlebotomine sand flies in the municipality of Divinópolis and adoption of strategies aiming entomological surveillance.     

基于COI与18S rRNA基因序列的4种肉食螨DNA条形码研究

目的　基于线粒体细胞色素C氧化酶亚基I（COI）与核糖体18S小亚基（18S rRNA）基因序列,分析确定4种肉食螨合适的DNA条形码,从而进一步完善肉食螨DNA条形码数据库。方法　2018年5月—2019年7月于安徽省阜阳、芜湖、铜陵等3市小型面粉作坊采集分离肉食螨样本,经形态学鉴定后,提取单个螨基因组DNA,经PCR扩增、克隆和测序获得COI与18S rRNA基因序列,所获序列进行BLAST比对。结合已报道肉食螨序列,用ClustalX 1.83软件进行多序列比对,基于MEGA X软件进行序列分析并计算遗传距离,采用最大似然法构建系统发育树。结果　马六甲肉食螨、转开肉食螨、鳞翅触足螨分子鉴定结果与形态学一致,而GenBank中缺少网真扇毛螨相关序列。4种肉食螨COI与18S rRNA 基因序列（A + T）分别为69.6%和55.1%,碱基替换数分别为137对和46对。基于COI与18S rRNA基因序列变异位点分析,发现4种肉食螨种间变异位点分别为154 ~ 321个和58 ~ 99个。4种肉食螨COI与18S rRNA基因序列种内遗传距离均≤ 0.020,种间遗传距离分别为0.235 ~ 0.583和0.078 ~ 0.114。基于COI与18S rRNA基因序列的系统发育树显示,4种肉食螨均能各自聚为一支,支持率均达100%,与形态学鉴定结果一致。结论　线粒体COI基因序列作为4种肉食螨DNA条形码优于18S rRNA基因序列,更适用于探索肉食螨的属、种低阶元亲缘关系。     

Distribution of cave-dwelling phlebotomine sand flies and their nocturnal and diurnal activity in Phitsanulok Province, Thailand

An entomological survey of sand flies was conducted in Naresuan Cave in Noen Maprang District, Phitsanulok Province, during November 2009 to December 2010. A total of 10,115 cave-dwelling sand flies were collected with CDC light traps nocturnally (06:00 AM and 06:00 PM) and diurnally (06:00 PM and 06:00 AM). The ratio between male and female sand flies was 1:1.3 (4,363:5,752). The ratio between the number of sand flies caught nocturnally and diurnally was 2.6:1 (7,268:2,847). In this study, 13 species belonging to 4 genera were identified, of which 4 belonged to the genus Phlebotomus, 7 to Sergentomyia, 1 to Nemopalpus and 1 to Chinius. An abundance of species were observed: Nemopalpus vietnamensis (49.15%), P. argentipes (20.15%), C. barbazani (15.79%), P. teshi (9.53%), and S. anodontis (3.21%). Less common species (<1%) were S. barraudi (0.63%), P. stantoni (0.57%), S. dentata (0.49%), S.quatei (0.17%), P. philippinensis gouldi (0.12%), S.silvatica (0.10%), S. gemmea (0.05%), and S. iyengari (0.04%). The predominant species in the Naresuan Cave was Nemopalpus vietnamensis (49.15%). The data demonstrates variability in sand fly prevalence, species composition, and relative abundance in caves. P. argentipes was found throughout the day in the caves, which is important because it is believed to be the Leishmania spp vector. This study highlights the diurnal activity of the sand fly and the day-time risk of leishmaniasis. In conclusion, although leishmaniasis has not been reported in Phitsanulok, there should be heightened awareness of infection in these areas with vectors of the protozoa.     

The identification of sandfly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.     

Sand Fly Fauna (Diptera,Pcychodidae, Phlebotominae) in Different Leishmaniasis-Endemic Areas of Ecuador,Surveyed Using a Newly Named Mini-Shannon Trap

To study the sand fly fauna, surveys were performed at four different leishmaniasis-endemic sites in Ecuador from February 2013 to April 2014. A modified and simplified version of the conventional Shannon trap was named “mini-Shannon trap” and put to multiple uses at the different study sites in limited, forested and narrow spaces. The mini-Shannon, CDC light trap and protected human landing method were employed for sand fly collection. The species identification of sand flies was performed mainly based on the morphology of spermathecae and cibarium, after dissection of fresh samples. In this study, therefore, only female samples were used for analysis. A total of 1,480 female sand flies belonging to 25 Lutzomyia species were collected. The number of female sand flies collected was 417 (28.2%) using the mini-Shannon trap, 259 (17.5%) using the CDC light trap and 804 (54.3%) by human landing. The total number of sand flies per trap collected by the different methods was markedly affected by the study site, probably because of the various composition of species at each locality. Furthermore, as an additional study, the attraction of sand flies to mini-Shannon traps powered with LED white-light and LED black-light was investigated preliminarily, together with the CDC light trap and human landing. As a result, a total of 426 sand flies of nine Lutzomyia species, including seven man-biting and two non-biting species, were collected during three capture trials in May and June 2014 in an area endemic for leishmaniasis (La Ventura). The black-light proved relatively superior to the white-light with regard to capture numbers, but no significant statistical difference was observed between the two traps.     

Relationship between sand fly fauna and kala-azar endemicity in Bangladesh

An entomological survey was carried out in Mymensingh district which contributes the highest incidence of visceral leishmaniasis (kala-azar) in Bangladesh. For the first time in Bangladesh CDC miniature light trap was used for indoor collection of sand flies. A total of 726 sand fly specimens belonging to nine species, one species of the genus Phlebotomus and eight species Sergentomyia genus were collected. Phlebotomus argentipes Annaandale Brunetti made up 59.2% of the total collection. Among Sergentomyia species Sergentomyia shorttii Adler & Theodor contributed 14.4% of the total collection. Density of sand flies both vector and non-vector species were significantly higher in endemic areas than non-endemic areas.     

Effect of night time-intervals,height of traps and lunar phases on sand fly collection in a highly endemic area for canine leishmaniasis

The activity of phlebotomine sand flies was monitored in a sub-urban area of Sicily in order to acquire data on seasonality and to elucidate the effect of the night time-intervals, height of traps from ground and lunar phases on the abundance of the capture. The study was conducted in the farm of the University of Messina (Italy). Light traps were placed as in the following: biweekly, from dusk to dawn, and from May to November; for three consecutive nights from 18:00 to 6:00, with the net bag being changed every 2 h; for 30 days, at different heights from 18:00 to 6:00. A total of five species (i.e., Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus sergenti, Phlebotomus perfiliewi, and Sergentomyia minuta), three of which are proven vectors of Leishmania infantum, were captured. The most abundant species was P. perniciosus (73.3%) followed by S. minuta (23.3%). The highest number of phlebotomine sand flies was collected in August and September with a peak of collection recorded in the evening (i.e., from 20:01 to 22.00). The number of phlebotomine sand flies collected at 50 cm above the ground was significantly higher (P = 0.041) than that captured at 150 cm. Results of this study shed light on the ecology of main phlebotomine species in the Mediterranean area, and on the influence of some factors, such as time and height of traps, on the light trap capture efficiency.