Diversity and distribution of sand flies (Diptera, Phlebotominae) in Serbia

Flebotomine (Diptera, Psychodidae, Phlebotominae) su veoma mali insekti nežne građe, koji su kod nas poznati kao peščane mušice ili papatači. Ženke ovih insekata imaju hematofagni režim ishrane i od davnina su poznate kao vektori različitih oboljenja. Najveći epidemiološki značaj flebotomine imaju kao vektori parazitskih protozoa iz roda Leishmania (Protozoa, Kinetoplastida) uzročnika lajšmanioza. Pored lajšmanioza, peščane mušice su vektori više vrsta arbovirusa, od kojih su svakako najznačajniji napuljski i sicilijanski serotipovi (Phlebovirus, Bunyaviridae) koji kod ljudi izazivaju flebotomsku tj. papatačijevu/papatačku groznicu. Peščane mušice su u Srbiji igrale veoma značajnu ulogu u drugoj polovini XX veka u period velikih epidemija lajšmanioze i papatačijeve groznice. Istraživanja peščanih mušica na teritoriji Republike Srbije sprovedena su u periodu od 1947. do 1990. godine. Tom prilikom registrovano je prisustvo 7 vrsta: Phlebotomus papatasi, P. neglectus, P. tobbi, P. perfiliewi s.l., P. sergenti s.l., P. simici i P. balcanicus. Nakon što je uočeno da broj peščanih mušica iz godine u godinu opada kao i da nema novih prijavljenih slučajeva lajšmanioze, istraživanja peščanih mušica u Srbiji su obustavljena. Pored toga što se u našoj državi lajšmanioza smatra davno iskorenjenom bolesti, u protekloj deceniji prisustvo sporadičnih slučajeva lajšmanioze kod ljudi, povećan broj registrovanih slučajeva autohtone lajšmanioze kod pasa kao i sumnja da je prisutan određeni broj nedijagnostikovanih tj. asimptomatskih slučajeva ukazali su na mogućnost cirkulacije bolesti u Srbiji. Obzirom da je lajšmanioza oboljenje čiji se prouzrokovač prenosi isključivo uz pomoć vektora, a da su za sada jedini potvrđeni vektori peščane mušice, počevši od 2013. godine, nakon više decenijske pauze, u okviru ove disertacije ponovo je uspostavljen entomološki nadzor ovih insekata. Entomološki nadzor otpočeo je 2013. godine prvo na teritoriji Autonomne Pokrajine Vojvodine, a već 2014. godine je proširen na jugoistočnu Srbiju i 2015. godine na zapadnu Srbiju sa Šumadijom. U periodu od 2013. do 2016. godine registrovano je prisustvo 10 vrsta peščanih mušica od kojih su 7 predhodno registrovane vrste a 3 vrste su nove za faunu Srbije. Dobijeni rezultati daju uvid u faunistički sastav flebotomina nakon višedecenijskog jaza u istraživanjima. U okviru ove disertacije dati su morfološki prikazi adulta, pregled distribucije i brojnosti za svih deset vrsta prisutnih u Srbiji. Pored toga izrađen je prvi ključ na srpskom jeziku za morfološku determinaciju odraslih mužjaka i ženki vrsta prisutnih u Srbiji, kao i vrsta koje se potencijalno mogu naći na teritoriji Republike Srbije. Determinacija na osnovu morfoloških karakteristika prvi put je potvrđena sekvencioniranjem COI regiona mitohondrijalnog gena i dobijene sekvence za vrste P. papatasi, P. neglectus, P. perfiliewi, P. tobbi, P. mascittii i P. balcanicus iz Srbije priključene su banci podataka GenBank. Prvi put je vršena analiza proteinskog spektra MALDI-TOF metodom i dobijeni rezultati za vrste P. neglectus, P. perfiliewi, P. sergenti, P. mascittii, P. balcanicus biće uvršteni u jedinstvenu bazu podataka koja je trenutno u osnivanju.Phlebotomines (Diptera, Psychodidae, Phlebotominae) are small fragile insects commonly known as sand flies (in Serbian: peščane mušice, papatači). Females are feeding on blood and they are capable to transmit parasitic protozoans from genus Leishmania (causative agent of leishmaniasis) and several phleboviruses (causative agents of sand fly fevers). Intensive sand fly research in Serbia was conducted during the years of the leishmaniasis and sand fly fever outbreaks, mainly in the infested areas. Sand fly research in Serbia was initiated in 1947 and ended in 1990, soon after the sand fly population density significantly diminished and leishmaniasis has been considered eradicated. During this period presence of seven species from Phlebotomus genus were detected – Phlebotomus papatasi, P. perfiliewi, P. tobbi, P. neglectus, P. simici, P. sergenti and P. balcanicus. Although leishmaniasis in Serbia is considered to be eradicated, for the past two decades sporadic human and dog cases were reported. It is suspected that the number of reported cases is higher, but real number remains unknown due to the underreporting and misdiagnosing especially among canine populations. This information suggested that both parasite and vector species are present and implicated the possibility of autochthonous transmission. Regarding these information, sand fly surveillance is resumed in 2013. Entomological surveillance of sand flies started in 2013 in Vojvodina Province, in 2014 it was extended to south-east Serbia and in 2015 to west Serbia. In period 2013-2016 in total 10 species of sand flies were recorded. Seven species were previously detected and 3 are new for sand fly fauna of Serbia. Obtained data provide insight in composition of sand fly fauna of Serbia after a gap of several decades. Within this thesis, detailed morphological descriptions of each recorded species, their distribution around the world and Serbia are given and elaborated. For the first time, the key for morphological identification of species present in Serbia was designed in Serbian language (for males and for females, separately). Morphological identification of samples was for the first time confirmed by molecular methods and by MALDI-TOF method of protein profiling. Obtained sequences for P. papatasi, P. neglectus, P. perfiliewi, P. tobbi, P. mascittii and P. balcanicus are deposited in the GenBank, while obtained protein spectra for P. neglectus, P. perfiliewi, P. sergenti, P. mascittii and P. balcanicus will be attributed to special data bank (currently under establishment). Accepted on Senate on: 18.09.2016

Dirofilariosis and Leishmaniasis in the Northern Region of Serbia

Research in the field of vector borne diseases and zoonozes became a topic of interest in Serbia, during the last decade. Climate changes as well as higher frequency of human and animal movement and travel, especially of dogs, is rising a threat of dirofilariosis and leismaniasis. The presence of native mosquito and sandfly vectors has already been confirmed in the country and some invasive/previously not detected were found. Dirofilariosis and leishmaniasis, which are found more or less often in dogs, cause clinical symptoms which are not obvious and therefore they represent a danger for public health with dogs acting as reservoirs of the infection

SPIDERS (ARANEAE) OF THE DELIBLATO SANDS (SERBIA)

The Special Nature Reserve “Deliblato Sands” in Serbia is the largest European continental sand area situated between the Danube River and western Carpathian slopes. Forestation, agriculture and fire are the main threats to this landscape. While plants and vertebrates are well known in this area, spiders were researched for the first time. Collecting with pitfall traps, sweep nets and beating in four different habitat types during a whole vegetation period yielded 1120 adult specimens and 96 species. Eight species represent first records for the Serbian fauna: Atypus muralis Bertkau, 1890, Gnaphosa muscorum (L. Koch, 1866), Zelotes segrex (Simon, 1878), Mecopisthes peusi Wunderlich, 1992, Oxyopes heterophthalmus (Latreille, 1804), Heliophanus lineiventris Simon, 1868, Simitidion simile (C. L. Koch, 1836) and Xysticus mongolicus Schenkel, 1963. In our study, six of these species were restricted to the open sandy habitat, highlighting the special importance of this unique habitat type

Effect of high-volume insecticide spraying on sand fly vectors in household gardens in Spain

This study investigated the efficacy of high-volume spraying with the adulticide α-cypermethrin alone and in combination with the larvicide diflubenzuron on the density of sand flies in gardens of three detached households in periurban areas in southeast Spain. Treatments were applied four times between June and August 2016, and four nearby sites, two households and two non-urbanized sites, were untreated controls. The number of sand flies collected between May and October 2016 using sticky interception and light attraction traps, was 4446 specimens. Species identified morphologically included Sergentomyia minuta (n = 2101; 48%), Phlebotomus perniciosus (n = 1922; 44%), Phlebotomus papatasi (n = 173; 4%), Phlebotomus sergenti (n = 161; 4%) and Phlebotomus ariasi (n = 36; 1%). Sand flies were detected in both treated and untreated sites. The proportion of positive sticky traps and the median (range) density of sand flies in positive traps were 61% traps and 7 (2-172) sand flies/m2 /day in untreated sites, and 43% traps and 4 (1-56) sand flies/m2 /day in treated sites (p < 0.05). Similarly, for light traps, it was 96% traps and 30 (3-168) flies/trap/day, and 83% traps and 3 (1-12) sand flies/trap/day, respectively (p < 0.05). However, sand fly density followed a comparable seasonal pattern in untreated and treated sites and did not consistently decrease following insecticide applications. These results were confirmed with mixed negative binomial modelling of sand fly density adjusted for time since application, month, environmental setting and site. The limited efficacy of the treatments, added to their cost, the impact of insecticides on non-target organisms and human health, and the risk of development of insecticide resistance, should dissuade similar outdoor applications to control sand fly vector populations in residential areas.Open Access funding was provided, thanks to the CRUE-CSIC UM agreement with Wiley.The study was funded by the Spanish Ministry of Science and Innovation (Grant number: AGL2013-46981-R) and received support from VectorNet, a European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). CMH held a PhD grant from the University of Murcia (Contrato predoctoral FPU). EB, MO, CMH, JR and SV were beneficiaries of a COST TD1303 short scientific mission grant to develop sand fly sampling and identification techniques. CMH is now supported by Margarita Salas Postdoctoral Grant in University of Murcia from the Program of Requalification of the Spanish University System (Spanish Ministry of Universities), founded by the European Union—NextGenerationEU.Peer reviewe

Evidence of West Nile virus (WNV) circulation in wild birds and WNV RNA negativity in mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016

Species diversity, host preference and arbovirus detection of Culicoides (Diptera: Ceratopogonidae) in south-eastern Serbia

BackgroundCulicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as no-see ums) that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia.ResultsIn total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, Others group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative.ConclusionsThe biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges funded by the programme SCOPES of the Swiss National Science Foundation

Protein methyltransferase 7 deficiency in Leishmania major increases neutrophil associated pathology in murine model

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology

Colonization and genetic diversification processes of Leishmania infantum in the Americas

Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control