Wings’ Descriptive and Geometric Morphometry Analyses Support Distinct Phlebotomus sergenti Parrot Populations in Central Morocco

Phlebotomus (Paraphlebotomus) sergenti Parrot, 1917, the proven vector of Leishmania tropica Wright, 1903, the causative agent of anthroponotic cutaneous leishmaniasis, is widely distributed in Morocco. Previous works using molecular markers (ITS2 rDNA and Cyt b mtDNA) have hy-pothesized the existence of multiple closely related populations of sandfly species (cryptic species) that would exhibit distinct vectorial capacities. In this work, descriptive characteristics of wings (size and shape of the right and left wings) were measured in samples collected from fourteen sta-tions in central Morocco. These analyses support the existence of distinct P. sergenti populations, enlightening significant phenotypic variations of P. sergenti’s wings, regarding their size and shape, depending on geographic origin. In addition, geomorphometric analyses of wing’s length, centroid size, alpha, and beta distances allowed clear discrimination of P. sergenti sub-populations. These data pinpoint the adaptative ability of P. sergenti to local environmental conditions. Additional studies are now required to further shed light on the genetic structure of P. sergenti populations in Morocco.

Comparative Testing of Susceptibility Levels of Phlebotomus sergenti, the Main Vector of Anthroponotic Cutaneous Leishmaniasis, to Conventional Insecticides Using Two Capture Methods in Kerman City, Southeastern Iran

Background: Collecting live sand flies from indoor sites is a major challenge for researchers in large cities due to the reluctance of families to survey their homes. This study was conducted to assess the efficacy of two methods for col-lecting sand flies for use in susceptibility tests in the urban area of Kerman, southeastern Iran. Methods: Sandflies were mainly collected using both baited traps and hand catch methods from outdoor and indoor sites. Susceptibility tests were separately done according to the standard World Health Organization testing protocol on Phlebotomus sergenti, including 60-minute exposure to DDT 4.0%, propoxur 0.1%, deltamethrin 0.05%, and malathion 5.0%. Results: During this research, the natural habitats and suitable indoor sites were selected to predict the density of live sand fly with perfect accuracy. The number of live Ph. sergenti caught by hand catch and baited traps methods was 42 and 361 in indoor and outdoor sites, respectively. The mortality rate of Ph. sergenti exposed to DDT 4%, deltamethrin 0.05, malathion 5%, and propoxur was 100%. Conclusion: The baited traps showed a significant efficiency compared to hand catch for collecting live Ph. sergenti for use in susceptibility tests in urban areas. The Ph. sergenti collected from both indoor and outdoor sites were susceptible to all insecticides.

Palaeoclimatic models - predicted changes in the potential Neogene distribution patterns of Phlebotomus similis and Phlebotomus sergenti (Insecta: Diptera: Psychodidae)

Former studies proposed that the speciation of the subgenus Paraphlebotomus happened in the Neogene Epoch in the circum-Mediterranean region due to the geographical segregation effect of the former Paratethys Sea. It was aimed to study whether the modelled Neogene ranges of Phlebotomus sergenti and Phlebotomus similis support or contradict this barrier role of the Paratethys in the speciation of Paraphlebotomus sandfly. For this purpose, the potential Neogene geographical ranges of Phlebotomus sergenti and Phlebotomus similis were modelled based on the present climatic requirements of the taxa. The Miocene models do not support the circum-Paratethyan migration of the ancestor of Phlebotomus similis. In general, Phlebotomus similis shows a low affinity to the North Paratethyan shorelines during the entire Miocene epoch. The only exceptions are the Tortonian and early Messinian periods when the climatic conditions could be suitable for Phlebotomus similis in the North Paratethyan shorelines. It was found that neither the modelled late Miocene, Pliocene nor the mid-Pleistocene period distributions of Phlebotomus sergenti and Phlebotomus similis shows notable differences in the suitability values in the Balkans and the Middle East. It is most plausible that the divergence of the Phlebotomus similis and its relatives was related to the tectonic subsidence of the Hellene Orogenic Belt and Phlebotomus similis specialised in the Balkan Peninsula and the present-day North Pontic area during the middle-late Miocene epoch. The Messinian desiccations of the Mediterranean Basin and the Zanclean re-flood caused the migration, but not the speciation of Phlebotomus similis and its sister taxa.

Understanding the factors that determine the emergence of anthroponotic cutaneous leishmaniasis due to Leishmania tropica: Comparison of the density and mitochondrial lineage of Phlebotomus sergenti between endemic and free areas in Morocco

Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is spreading to new areas. Exposure to the vector, Phlebotomus sergenti, is the only proven risk factor. Our objective was to compare the densities and genetic characteristics of P. sergenti populations in two nearby localities in Morocco, one within an ACL endemic area (El Borouj) and another undamaged (Sidi Hajjaj). Statistically significant differences were detected between P. sergenti densities with a higher density of P. sergenti in the endemic town (p≤ 0.032). A different main P. sergenti mitochondrial lineage was evidenced in each one of the 2 localities, and for the first time, the lineage of P. sergenti specimens that are acting as a vector of L. tropica has been identified. Bioclimatic differences were detected between both localities. In conclusion, between an ACL endemic locality and another ACL free there are differences in both the density of P. sergenti and the mitochondrial lineage that may explain the different epidemiological situation. Given that the density of P. sergenti in the locality without ACL cases seems sufficient to allow transmission, the main factor that would justify its ACL undamaged character could be the absence of P. sergenti Lineage IV, which seems to prefer warmer and drier climates.

Molecular Detection of Virulence Factor Glycoprotein (Gp63) of Leishmania spp. in Phlebotomus Sand Flies

Introduction: Gp63 is the major surface glycoprotein of Leishmania which is prevalent in the promastigote stage of Phlebotomus sergenti. Glycoprotein 63 (gp63) or leishmanolysin is a zinc-dependent metalloprotease found on the surface of Leishmania. It was initially discovered in 1980and described biochemically and genetically as a surface antigen expressed in promastigotes of Leishmania species, having a range of substrates including casein, albumin, fibrinogen, haemoglobin, and gelatin. On the surface of amastigote, GP63 is present at a very low level. Glycoprotein 63 is a zinc-dependent metalloprotease that is active in the pH range of 7–10, which is neutral to alkaline. Materials and Methods: During the study, which was carried out in Al-Muthanna province from July 2017 to August 2018, about 2550 sand fly samples including 719 males and 1633 females were collected. Three species of sand flies were recorded according to morphological features which included Phlebotomus papatasi, Phlebotomus sergenti, and Sergentomyia sintoni. The caught specimens showed that P. papatasi was the most prevalent species. Two primers were used for the diagnosis of Leishmania spp. using a nested PCR technique, which was designed in a previous study for 1250 samples of sand flies from 25 locations in the study area. Results: Results showed that 13 samples were positive, containing DNA for the Leishmania parasite, and 12 samples were negative. Positive samples include 11 samples of L. major species and only 2 samples of L. tropica species. Additionally, the results showed that the L. major species was the dominant species in the study area. Positive samples (13) of sand flies had Leishmania parasite based on molecular diagnosis and virulence factor gene (Gp63) of Leishmania parasite was detected in sand flies using PCR method The results showed that virulence factor gene (Gp63) was detected on the surface of promastigote. This refers to the presence of Leishmania parasite in sand fly. The study is the first one which detected the presence of the virulence factors gene (GP63) of Leishmania in the body of sand flies. Conclusion: This study revealed the possibility of diagnosing leishmaniasis by virulence factor gene (Gp63) in leishmania promastigotes.

Emerging of Cutaneous Leishmaniais due to Leishmania major in a New Focus in Esfahan Province, Central Iran

Background: Esfahan Province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Recently, ZCL distribution is expanding through this province leading to report of new cases in non-endemic areas. In the current study epidemiological aspects of ZCL has been investigated in Naein County in Esfahan Province. Methods: Adult sand flies were collected from beginning to the end of their seasonal activity. Rodents were caught by Sherman live traps once a month for one year. To active case detection, a hundred households in each selected village were visited in November and December 2016. Nested-PCR was employed to detect Leishmania parasite in the vector, reservoir and human. Results: Totally 1562 sand flies including Phlebotomus sergenti, Phlebotomus papatasi, Sergentomyia sintoni and Sergentomyia mervinae were collected and identified. No Leishmania infection was detected in the collected sand flies. All of the 30 collected rodents were identified as Rhombomys opimus, and of these 3.3% and 26.7% were infected by Leishmania major using microscopic and molecular technique respectively. Totally, 914 individuals were investigated and the ulcer and scar rates of ZCL calculated to be at 1.1 and 15.3 per 1000 population, respectively. Molecular results confirmed L. major infection in human and reservoir samples. Conclusion: It is concluded that ZCL is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in the near future in this region.

Comparative Performance of Different Traps for Collection of Phlebotominae Sand Flies and Estimation of Biodiversity Indices in Three Endemic Leishmaniasis Foci in North Khorasan Province, Northeast of Iran

Background: Phlebotominae sand flies (Diptera: Psychodidae) are the vectors of leishmaniasis. There are different methods for sand fly collection with different performance. The purpose of the current study was to compare the effect of different traps for collection of Phlebotominae sand flies in three endemic leishmaniasis foci in North Khorasan Prov-ince, northeast of Iran. Methods: Sand flies were collected using seven different traps from three villages, three times each twenty days during peak periods of seasonal activity in 2016. Results: A total of 7253 sand flies were collected. The specimens belonged to19 species. Phlebotomus sergenti was the most predominant species in the study area. Light trap baited with Carbon dioxide (CLT) and sticky paper trap (SPT) caught 22.6% and 22.3% of sand flies respectively. Animal baited trap (ABT) and white Shannon trap (WST) caught significantly fewer sand flies than the other traps. The sex ratio was different by phlebotominae sand fly species and collection methods. The sex ratio was highest in SPT and lowest in black Shannon trap (BST). Species diversity and species richness in SPT were more than other traps. Conclusion: Our findings confirm that CLT and SPT are the most efficient sand fly collection methods. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus ge-nus during surveillance. SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and provide a more realistic estimation of sand flies biodiversity.