Molecular phylogeny of Phlebotomus sp., vectors of protozoan parasite Leishmania sp., from Crete and Cyprus and development of molecular tools for their typing

Leishmaniasis is one of the most important parasitological diseases worldwide. The parasite, Leishmania, is found, among other areas, in all the Mediterranean countries and Portugal causing the visceral as well as the cutaneous form of the disease. Visceral Leishmaniasis (VL) is the most aggressive form and it is fatal, if left untreated, for the 95% of the cases. Clinical manifestations are fever, lymph and spleen enlargement. Cutaneous Leishmaniasis (CL) is mild and manifests as cutaneous ulcers.Sand flies, genus Phlebotomus, are the vectors of the parasite. Their diet consists of plant sugars but females feed on mammal blood (such as humans) to complete egg development. During those meals, sand flies transmit parasites and other pathogens, as Bartonella sp. and Phleboviruses to humans and other animals.In the Old World, there are 49 sand fly species of the genus Phlebotomus that act as Leishmania vectors. Different sand fly species transmit different Leishmania species. Thus, by studying the geographical distribution of sand fly species and knowing their Leishmania infection rates is possible to evaluate the risk of leishmaniasis in an area. Furthermore, the potential of disease emergence in a new area can be predicted if the sand fly species present in an area is known.Typing Phlebotomus species is done mainly by morphology using a microscope. In addition, due to the limitations of morphological identification, molecular typing methods are developed in order to save time and to increase specificity. Nevertheless, there is no universal molecular typing method used by researchers. Phylogenetic relationships among Phlebotomus species are not well studied and, as a result, morphological typing, on its own, often offers limited information. Given that, researchers tend to have conflicting opinions regarding the taxonomy of several sand fly species. It is clear that there is a need for quick and universal molecular typing tools to be applied together with morphotyping. Using only morphological typing can lead to incorrect conclusions regarding the geographical distribution of the Phlebotomus species found in an area and hence the risks posed to human health in relation to the pathogens the particular species may transmit. Phylogenetic relationships and species typing of Phlebotomus species from Crete and Cyprus have been remotely analyzed, therefore there is no detailed knowledge, on the species that reside and transmit Leishmania in these areas. Human and dog leishmaniasis cases in both these regions are increasing. Therefore, the knowledge on the sand fly vectors responsible for this is imperative.This work aimed at discriminating phylogenetic relationships of sand fly species from Crete and Cyprus and to develop new, reliable molecular typing methods for doing so. Sand fly sampling was done in years 2011 - 2014 during the EU, FP7, EDENext research project. Choosing the sand flies for the analyses was done carefully in order to have the right gender ratio and sample size, for a balanced representation of the sand fly populations circulating in these islands during that period.The DNA barcoding method was used to analyze the phylogenetic relationships of sand fly species. This process consists of sequencing a fragment of the mitochondrial gene encoding cytochrome oxidase 1 (COI). Sequences derived from that gene provided information on the molecular identity of the species studied. They also provided the ability to build, based on that information, the phylogenetic tree that defines the systematic status of each species and its relationship with others. Results, among others, revealed, for the first time, the COI barcodes for P. neglectus and P. similis, vectors of the parasites L. infantum (causing VL) and L. tropica (causing CL) in Greece, respectively.Resolving the phylogenetic position of the sand fly species studied has provided the way to develop a molecular typing tool for Larroussius subgenus using the PCR – RFLP method. This subgenus contains vectors of the parasite L. infantum, the VL causing agent. Identification of the Larroussius subgenus members is difficult, based on current methods especially that of the females. This tool proved to be quick and reliable in typing Larroussius species from the study area.Finally, this work helped to develop a new and promising sand fly molecular typing tool using the individual’s proteome information, i.e. the insect tissue protein composition profile. This method uses mass spectrometry (MALDI – TOF MS). The first analyses were conducted on laboratory reared sand flies and this was the first such study in the field, worldwide. This method, in the future, promises to be one of the top tools used in identification studies of sand flies and other insects.Η Λεïσμανίαση αποτελεί μια από τις σοβαρότερες ασθένειες, παρασιτολογικής αιτιολογίας, για τον άνθρωπο. Το παράσιτο Leishmania ενδημεί, μεταξύ άλλων, σε όλες τις χώρες της λεκάνης της Μεσογείου και στην Πορτογαλία προκαλώντας σπλαχνική και δερματική νόσο. Η σπλαχνική μορφή (ΣΛ) είναι η πιο επιθετική και είναι θανατηφόρα χωρίς θεραπεία στο 95% των περιπτώσεων. Οι κλινικές εκδηλώσεις, μεταξύ άλλων, είναι πυρετός, διόγκωση λεμφαδένων και σπλήνας. Η δερματική μορφή (ΔΛ) είναι ήπια και εκδηλώνεται με δερματικά έλκη.Οι σκνίπες, γένος Phlebotomus, αποτελούν τους διαβιβαστές ξενιστές στον κύκλο ζωής του παρασίτου. Η διατροφή τους αποτελείται από σάκχαρα προερχόμενα από φυσικές πηγές. Τα θηλυκά όμως, για να ολοκληρώσουν την ωοτοκία, τρέφονται και με αίμα θηλαστικών όπως ο άνθρωπος. Κατά τη διάρκεια τέτοιων γευμάτων, η σκνίπα μεταφέρει το παράσιτο και άλλα παθογόνα (όπως βακτήρια του γένους Bartonella και Phlevoviruses) στον άνθρωπο και σε άλλα ζώα.Στον Παλαιό Κόσμο απαντώνται 31 είδη σκνιπών, του γένους Phlebotomus, που παίζουν το ρόλο του διαβιβαστή της Leishmania. Διαφορετικά είδη σκνίπας διαβιβάζουν διαφορετικά είδη Leishmania και έτσι, μελετώντας τη γεωγραφική κατανομή των πληθυσμών των διάφορων ειδών του εντόμου και γνωρίζοντας τα επίπεδα μόλυνσης των σκνιπών από το παράσιτο Leishmania, είναι δυνατόν να αξιολογηθεί το μέγεθος του κινδύνου για τη δημόσια υγεία σε κάθε περιοχή αλλά και να γίνει πρόβλεψη της εξάπλωσης της νόσου σε νέες περιοχές.Όπως είναι κατανοητό, η ακριβής τυποποίηση των ειδών του γένους Phlebotomus είναι σημαντική για τη Δημόσια Υγεία. Παραδοσιακά, η τυποποίηση γίνεται ως επί το πλείστο με βάση τις μορφολογικές τους διαφορές, εφαρμόζοντας μικροσκοπικές μεθόδους. Ωστόσο, έχει γίνει φανερό ότι η μορφολογική τυποποίηση των σκνιπών δεν είναι πάντα ακριβής. Για το λόγο αυτό αναπτύσσονται μοριακές μέθοδοι τυποποίησης με στόχο τη μείωση του χρόνου αναγνώρισης των ειδών και την αύξηση της ακρίβειας της ταυτοποίησης. Εντούτοις, δεν χρησιμοποιείται μια κοινή μοριακή μέθοδος τυποποίησης από τους ερευνητές ώστε να εγκαταλειφθεί ή να παραμείνει συμβουλευτική η μορφολογική τυποποίηση. Πέρα από τη ταυτότητα των ειδών σκνίπας που ενδημούν σε κάθε περιοχή, είναι απαραίτητη και η γνώση των φυλογενετικών σχέσεων μεταξύ τους. Τα στενά συγγενικά είδη σκνιπών ενδέχεται να συμπεριφέρονται με παρόμοιο τρόπο ως διαβιβαστες ξενιστές του παρασίτου Leishmania. Παράλληλα, η έλλειψη βαθιάς γνώσης για τη συστηματική κατάσταση των σκνιπών οδηγεί σε διχογνωμίες μεταξύ των ειδικών σχετικά με τη ταξινόμηση πολλών ειδών.Οι φυλογενετικές σχέσεις και η τυποποίηση των ειδών Phlebotomus, από την Κρήτη και την Κύπρο έχουν αναλυθεί ακροθιγώς και δε γνωρίζουμε με ακρίβεια, πόσα και ποια είδη ενδημούν σε αυτές τις περιοχές. Το γεγονός ότι, τα τελευταία χρόνια, ο αριθμός των κρουσμάτων λεϊσμανίασης στα νησιά αυτά έχει αυξηθεί στον άνθρωπο και στο σκύλο επιβάλλει την, σε βάθος, μελέτη των σκνιπών, διαβιβαστών ξενιστών της νόσου που ενεργοποιούνται σε αυτές τις περιοχές.Η παρούσα διδακτορική διατριβή στόχευε στην εξακρίβωση των φυλογενετικών σχέσεων ειδών σκνιπών που απαντώνται στην Κρήτη και στην Κύπρο και περαιτέρω αποσκοπούσε στη δημιουργία νέων, αξιόπιστων, συστημάτων μοριακής αναγνώρισης των συγκεκριμένων ειδών. Η συγκέντρωση των ατόμων σκνίπας για τη μελέτη προήλθε από δειγματοληψίες κατά τα έτη 2011 – 2014 στα πλαίσια του προγράμματος της ΕΕ, FP7, EDENext. Η επιλογή των σκνιπών, τόσο σε αριθμό όσο και σε αναλογία φύλου, έγινε προσεκτικά με στόχο την πλήρη και ακριβώς σταθμισμένη αντιπροσώπευση των πληθυσμών των εντόμων που κυκλοφορούσαν την περίοδο εκείνη στα δύο νησιά.Η ανάλυση των φυλογενετικών σχέσεων των ειδών σκνίπας έγινε χρησιμοποιώντας τη μέθοδο DNA barcoding, μια διαδικασία αλληλούχισης ενός τμήματος του μιτοχονδριακού γονιδίου της κυτοχρωμικής οξειδάσης Ι (COI). Οι αλληλουχίες που προέκυψαν από το συγκεκριμένο γονίδιο μας έδωσαν τη δυνατότητα αφενός να αποκτήσουμε πληροφορίες για τη μοριακή ταυτότητα των ειδών που μελετήσαμε και αφετέρου να κατασκευάσουμε, βάσει των δεδομένων αυτών, το φυλογενετικό δέντρο που περιγράφει τη συστηματική κατάσταση κάθε είδους και τη σχέση του με τα υπόλοιπα είδη. Τα αποτελέσματα, μεταξύ άλλων, ανέδειξαν, για πρώτη φορά, τις barcode αλληλουχίες για τα είδη P. neglectus και P. similis, διαβιβαστές ξενιστές των παρασίτων L. infantum, υπεύθυνο για τη ΣΛ, και L. tropica υπεύθυνο για τη ΔΛ στην Ελλάδα, αντίστοιχα. Η αποσαφήνιση της φυλογενετικής θέσης των ειδών σκνίπας που ερευνήθηκαν, άνοιξε το δρόμο για να αναπτυχθεί ένα μοριακό εργαλείο τυποποίησης του υπογένους Larroussius χρησιμοποιώντας τη μέθοδο PCR – RFLP. Το υπογένος αυτό εμπεριέχει τους διαβιβαστές ξενιστές του παρασίτου L. infantum, αιτιολογικό παράγοντα της ΣΛ, και η διακριτική ικανότητα ταυτοποίησης των ειδών αυτών, με τις υπάρχουσες μεθόδους, είναι ελλιπής. Το εργαλείο που αναπτύχθηκε είναι γρήγορο και αξιόπιστο στην τυποποίηση ατόμων του συγκεκριμένου, σημαντικού, υπογένους στη περιοχή μελέτης.Τέλος, αναπτύχθηκε ένα νέο, και πολλά υποσχόμενο, εργαλείο μοριακής τυποποίησης σκνιπών χρησιμοποιώντας την πληροφορία του πρωτεώματος του ατόμου, δηλαδή την πρωτεϊνική σύσταση του ιστού του εντόμου. Η μέθοδος χρησιμοποιεί τη τεχνολογία τυποποίησης φασματοσκοπίας μάζας (MALDI – TOF MS). Η ανάλυση έγινε σε δείγματα σκνίπας εργαστηριακής καλλιέργειας και αποτελεί την πρώτη μελέτη στο πεδίο παγκοσμίως. Αυτή η μέθοδος θα διαδραματίσει στο μέλλον πρωταγωνιστικό ρόλο στο έργο της τυποποίησης και ταυτοποίησης σκνιπών

Identification of wild-caught phlebotomine sand flies from Crete and Cyprus using DNA barcoding

Abstract Background Phlebotomine sand flies (Diptera: Psychodidae) are vectors of Leishmania spp., protozoan parasites responsible for a group of neglected diseases called leishmaniases. Two sand fly genera, Phlebotomus and Sergentomyia, contain species that are present in the Mediterranean islands of Crete and Cyprus where the visceral (VL), cutaneous (CL) and canine (CanLei) leishmaniases are a public health concern. The risk of transmission of different Leishmania species can be studied in an area by monitoring their vectors. Sand fly species are traditionally identified using morphological characteristics but minute differences between individuals or populations could be overlooked leading to wrong epidemiological predictions. Molecular identification of these important vectors has become, therefore, an essential tool for research tasks concerning their geographical distribution which directly relates to leishmaniasis control efforts. DNA barcoding is a widely used molecular identification method for cataloguing animal species by sequencing a fragment of the mitochondrial gene encoding cytochrome oxidase I. Results DNA barcoding was used to identify individuals of five sand fly species (Phlebotomus papatasi, P. similis, P. killicki, Sergentomyia minuta, S. dentata) circulating in the islands of Crete and Cyprus during the years 2011–2014. Phlebotomus papatasi is a known vector of zoonotic CL in the Middle East and it is found in both islands. Phlebotomus similis is the suspected vector of Leishmania tropica in Greece causing anthroponotic CL. Phlebotomus killicki was collected in Cyprus for the first time. Sergentomyia minuta, found to present intraspecific diversity, is discussed for its potential as a Leishmania vector. Molecular identification was consistent with the morphological identification. It successfully identified males and females, which is difficult when using only morphological characters. A phylogenetic tree was constructed based on the barcodes acquired, representing their genetic relationships along with other species from the area studied. All individuals identified were clustered according to their species and subgenus. Conclusions Molecular identification of sand flies via DNA barcoding can accurately identify these medically important insects assisting traditional morphological tools, thus helping to assess their implication in Leishmania transmission

A molecular phylogeny and phylogeography of Greek Aegean Island sand flies of the genus Phlebotomus (Diptera: Psychodidae)

The genus Phlebotomus (Diptera: Psychodidae: Phlebotominae) comprises a group of small winged insect species of medical importance. To date, ten species of Phlebotomus are known to be present in Greece; yet their evolutionary history is poorly studied due to the lack of comprehensive phylogenetic and phylogeographic studies. Herein, we aim to clarify the phylogenetic relationships amongst the local species collected from 12 Aegean Islands, Cyprus and Turkey; and to identify which of the palaeogeographic events may have influenced their biogeographic history. Our analyses revealed for the first time the presence of P. cf. major and P. sergenti in the Aegean Islands. All studied local species were retrieved as monophyletic and the mtDNA and nDNA phylogenetic trees indicated a plausible mitochondrial introgression between the closely related species of the P. major complex. From a palaeogeographic viewpoint, the major driving force that shaped the biogeographic history of the studied Phlebotomus species seems to be the dispersal that started in the Oligocene epoch, followed by several speciation events that occurred at the end of Miocene and the Plio-Pleistocene, including multiple dispersal events of Asiatic origin. The Messinian Salinity Crisis, the bimodal Mediterranean climate, and the glacial and interglacial periods were identified as key drivers for the diversification of the local species of Phlebotomus

Phlebotomine Sand Flies (Diptera: Psychodidae) in the Greek Aegean Islands: Ecological Approaches

Background Blood-sucking phlebotomine sand flies are the vectors of the protozoan parasites Leishmania spp. Different Phlebotomus species transmit different Leishmania species causing leishmaniases which are neglected diseases emerging/reemerging in new regions. Thirteen sand fly species, ten belonging to the medically important genus Phlebotomus and three belonging to Sergentomyia are known in Greece. An increasing number of human and dog cases are reported each year from all parts of the country including the Aegean Islands. However, no previous study has been conducted on the sand fly fauna on the islands, except for Rhodes and Samos. The aim of this study was to investigate sand fly species in eleven small Aegean islands; to understand species-specific relationships with environmental and climatic factors and to compare sand fly community parameters among islands. A risk analysis was carried out for each species using climatic and environmental variables. Results Nine sand fly species: Phlebotomus neglectus, P. tobbi, P. similis, P. simici, P. perfiliewi, P. alexandri, P. papatasi, Sergentomyia minuta and S. dentata, were collected from the islands studied. Phlebotomus (Adlerius) sp. and Sergentomyia sp. specimens were also collected but not identified to the species level. There was a positive effect of distance from the sea on the abundance of P. neglectus, S. minuta and S. dentata, and a negative effect on the abundance of P. tobbi, P. simici and P. similis. In general, temperature preferences of sand fly populations were between 21 and 29 °C. Nevertheless, there were significant differences in terms of temperature and relative humidity preference ranges among species. The most important species found, P. neglectus, was indisputably the most adapted species in the study area with a very high reaction norm, favoring even the lower temperature and humidity ranges. Overall, the sand fly fauna in the islands was very rich but there were differences in species diversity, as indicated by the values of the Shannon-Wiener index, along with evenness and richness of the sand fly fauna between the islands and altitude ranges in the islands. Conclusions The study indicated that the Greek Aegean Islands, however small, maintain a rich sand fly fauna. This includes important vectors of Leishmania spp. representing a risk for parasite transmission to humans and dogs along with the danger of maintaining new Leishmania spp. if introduced to the area. Electronic supplementary material The online version of this article (10.1186/s13071-018-2680-4) contains supplementary material, which is available to authorized users.PubMedWoSScopu

Will the introduction of Leishmania tropica MON-58, in the island of Crete, lead to the settlement and spread of this rare zymodeme?

International audienceThe rare zymodeme, Leishmania tropica MON-58, was isolated from a young Afghan refugee with a facial cutaneous lesion who had come to live in Crete early 2008. The same zymodeme variant was isolated from a local dog that had never travelled outside the island, with symptoms of visceral leishmaniasis, which stayed in the area where the patient worked during the summer months. This is the first record of L. tropica in a host, other than human, in Greece and another example of introduction of a vector borne pathogen in a focus where local vector/s can sustain it, with the risk of initiation of new transmission cycle/s.Keywords

Sand fly fauna of Crete and the description of Phlebotomus (Adlerius) creticus n. sp. (Diptera: Psychodidae)

International audienceBackground: The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. Methods: All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. Results: Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. Conclusions: The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry

Additional file 1: Table S1. of Phlebotomine sand flies (Diptera: Psychodidae) in the Greek Aegean Islands: ecological approaches

Risk factors using the multivariate logistic regression model (using SPSS 22). (DOCX 21 kb