Study of the seasonal distribution of Phlebotomus species found in Crete and their role in the transmission of Leishmania protozoan parasite, and study of the maintenance of acquired parasite drug resistance in its hosts: phlebotomus sp. and mice

Phlebotomine sandfly is the host of protozoan Leishmania that transmits leishmaniases. It was discovered in Greece in 1910, almost a decade since the term “Leishmania” was started to be used. Distribution of Phlebotomes covers all five continents and it includes tropical and sub-tropical regions, while some species can also be found in more temperate climates. The continuously increasing mean earth temperature is a cause of concern as more regions will have favourable conditions for sandfly reproduction and survival. Apart from mountains, climatological conditions are the only barrier that prevents their distribution in inland. In general, sandflies rest and reproduce in small cracks of rocks or walls, in caves and tree hollows, and in habitats with high vegetation and animal faeces. Out of the 31 Phlebotomus species that serve as host of Leishmania, those of major medical importance in Greece are Phlebotomus (Larroussius) tobbi, Phlebotomus (Paraphlebotomus) sergenti, Phlebotomus (Paraphlebotomus) papatasi, Phlebotomus (Larroussius) perniciosus, Phlebotomus (Larroussius) neglectus, as well as the suspect host Phlebotomus (Paraphlebotomus) similis. In Crete, Phlebotomus neglectus is vector of Leihsmania infantum that causes canine leishmaniasis and visceral leishmaniasis in humans. Phlebotomus similis is a suspected vector of Leishmania tropica, that causes anthroponotic cutaneous leishmaniasis, because it is a sister species with Phlebotomus sergenti, a verified vector of Leishmania tropica. Given the increasing leishmania cases in Greeece and especially in Crete, it is important to build the knowledge on the ecology of phlebotomes that transmit leishmaniases. Leishmaniasis is a disease caused by the protozoan – parasite of genus Leishmania and infects both mammals and reptiles. Phlebotomine sandflies are the main hosts that transmit leishmaniasis. There are three types of leishmaniasis: visceral, cutaneous and mucocutaneous. Every year 0.7 – 1 million new cases are recorded in 98 countries, while mortality occurs in 3.3% of cases. In Greece, symptoms of the illness are known even before the name “leishmaniasis” was given. An increase of the cases in Greece, especially in Crete has been observed over the last few years. The most important feature of Leishmania is the acquired multidrug resistance that develops, which is induced by the resistance that develops from a single drug (MDR phenotype). Leishmania presents exceptional plasticity in genome level and is mostly characterized by aneuploidy. The population of the parasite has the ability to transform in a very short time either in gene level, or in chromosomal level. Although new drugs are added in the medical arsenal against leishmaniases, it is very important to know if acquired MDR phenotype is inherited as the parasite transforms between amastigote and promastigote form. The aim of this dissertation was: 1. the study of seasonal distribution of Phlebotomus species that exist in Crete in order to collect important data for the transmission of protozoan – parasite Leishmania. 2. investigating if the acquired resistance, using Glucantime, is inherited or not, as the parasite continues its life cycle through different hosts (mammal – phlebotomine sandfly). Fodele, a village near Heraklion in Crete, where leishmaniasis has been reported in the past, was chosen for this research. Trapping of phlebotomes was held using CDC Miniature Light Traps (John W. Hock Co., Gainesville, FL, USA) and Sticky Traps for three days in the middle of every month for three continuous years. Sampling period covered the whole period that phlebotomes remain active. The first year, sampling was not possible to begin before June. The collection of environmental data was held by ten dataloggers that measured temperature, relative humidity and dew point, as well as an anemometer. Other environmental factors, like precipitations and moon phase, were recorded too. For the research of resistance, five “resistant” and five “susceptible” Leishmania infantum isolates were chosen, that were characterized in our laboratory using Flow Cytometry. The rate of efflux of the drug in 120 minutes gives the magnitude of resistance of Leishmania (slope α). All ten isolates where measured again after they were defrost and they injected in mice. Fifteen days later, mice were euthanized and their liver and spleen were cultured, in order to recover parasites. These parasites were measured again using flow cytometry. A single isolate out of these ten, was chosen to become “resistant” and to be used for the second part of the experiment. The induction of resistance was applied, using Glucantime®, in promastigotes that were kept in culture medium and in amastigotes that were grown and multiplied in the human monocytic cell line, THP-1 and in Rag1- deficient mice. No phlebotomine sandflies were used in this stage because it was already done by another research group. Induction of resistance in promastigote was not succeeded eventually. From almost 6,000 phlebotomes (60% males, 40% females) six species were captured: Phlebotomus neglectus (57.8%), Phlebotomus similis (30.4%), Phlebotomus papatasi (2.5%), Phlebotomus simici (0.1%), Sergentomyia minuta (8.8%) and the novel species Phlebotomus (Adlerius) creticus n. sp. (0,4%). Phlebotomus neglectus and Phlebotomus similis were the most dominant species. Relatice abundance of Phlebotomus similis was higher than Phlebotomus neglectus only in the first sampling year. These two species, as well as Sergentomyia minuta, were active the whole sampling period. Seasonal dynamics of Phlebotomus neglectus is characterized by two distinct peaks (May and August / Τ = 20,3οC – 27ο C, RH = 57,5% – 63,1%), while Phlebotomus similis shows two confluent peaks (June and July / Τ = 24,3οC – 27,2ο C, RH = 53,9% – 59,1%). Male and female individuals show similar trend. The peaks of both species together, clearly indicate that the highest potential risk for Leishmania infantum transmission occurs during the summer months. Sergentomyia minuta shows similar temperature and relative humidity preferences, but different seasonal dynamics pattern. Temperature ≥ 17 – 18οC signals the beginning of Phlebotomus neglectus activation period, while temperature ≥ 20 – 22οC signals the beginning of Phlebotomus similis and Sergentomyia minuta activation period. The abundance of phlebotomes was related with temperature increase (r = 0.776, p < 0.05) and relative humidity decrease (r = 0.644, p < 0.05). Both dominant species were present in all habitats and where captured in both CDC and ST traps. However, the majority was captured in CDC and mostly those that were placed in the two habitats where domesticated animals were present (hencoops and rabbits). Intense vegetation, animal faeces and rotten fruits and vegetables were present in those habitats. The majority of Phlebotomus papatasi was collected from one of those two habitats, as well as from ST traps. Sergentomyia minuta was captured mostly from ST traps (78%). 29.9% of female Phlebotomus papatasi, 23.2% of female Phlebotomus similis, 17.1% of female Phlebotomus neglectus and 15.6% of female Sergentomyia minuta were captured in different stages of bloodmeal digestion, as well as with eggs. 50% of parasitized individuals belong to Phlebotomus similis and 41% belong to Phlebotomus neglectus, while the most intense parasitism was observed to Phlebotomus papatasi (more than 20 scars in one individual). A female Phlebotomus neglectus was parasitized by a nematode.Regarding the resistance experiment, 70% of isolates showed increased resistance after defrosting, compared to that prior to frosting. 20% of isolates showed decreased resistance, whereas 10% showed the same resistance before and after they were frosted. All the “susceptible” isolates showed increased resistance. The same isolates infected Rag1- mice. Almost 100% of splene cultures showed parasites in three to fourteen days (average six). When those isolates were measured again, 70% showed reduced resistance in contrast with those before the infection, while 30% showed increased resistance. All “resistant” isolates showed decreased levels of resistance. The next step was the induction of resistance of one Leishmania isolate, using Glucantime®. The isolate that was chosen, was characterized as “susceptible” when it was cultured from the host. Following the procedure that was described above, this isolate became “resistant”. All the parasites (treated with or without Glucantime® treatment) that had infected THP-1 cells, showed reduced resistance in contrast with the primary isolate. Verapamil blocked the pumps of those that were treated with Glucantime®, so they showed reduced slope (slope β). Parasites that were not treated with Glucantime® (THP-1_C) influxed lower amount of Rhod-123, in contrast with the primary isolate in all cases (p < 0.05). Parasites that had infected Rag1- mice, were more “susceptible” than the primary without Glucantime® treatment (Rag1_NGAI), but more “resistant” than the primary with Glucantime® treatment (Rag1_GAI). FVI differed statistically between those that were treated and those that were not treated with Glucantime® (p < 0.05). Distribution of phlebotomine sandflies is going to expand in Europe the next fifty years, towards all directions. The activation period of the adult individuals is going to increase as well. The seasonal and geographical overlap of Phlebotomus species that transmit leishmaniases in Crete, should taking into consideration by public health services, since leishmania cases are increasing. Their activation period is one of the longest in Mediterranean. Phlebotomus papatasi, even if it does not transmit leishmaniasis because of the absence of Leishmania major natural host, it has medical importance because it transmits phleboviruses. The village of Fodele is an example of rural area, like many others in Crete that offers many habitats for phlebotomine resting and reproduction. Prevention measures should include insecticides or repellents, protection dogs, given they are the natural reservoir of the parasite and appropriate information to local communities about phlebotomes and the disease. Prevention action should take place before population outbreak at the spots that phlebotomes prefer for resting and reproducing. Dogs should be vaccinated and serologically tested both in the beginning and at the end of the activation period of phlebotomes. The use of drugs against leishmaniasis should take place in an effective way. This means that if treatment is started, it should be completed. Leishmania reveals a great genetic plasticity. Aneuploidy is very common in natural populations. Isolates in laboratories, undergo such mutations that can even alter their karyotype. Freeze – defreeze cycle, as well as the completion of their life cycle through mammal host, can alter their drug resistance. Every substance that is used against leishmaniasis, causes genetic change that allows gene – targets to be found. However, these gene – targets vary between Leishmania populations, as well as between different drugs. This plasticity of Leishmania genome, reveals the reason that most drugs aim the protein pump system that is responsible for their influx and efflux of the parasite, because this system is universal. Two factors should be considered in resistance experiments. The efflux rate (slope α) that is driven by efflux protein pumps and the amount that the parasite influxes the zero time (FVI), that is driven by influx protein pumps. Their combined action will determine how much drug will remain in the parasite and for how long, which is very important, since the drug is cytotoxic. The best model organism to be used is the mammal host, because it reflects the life cycle of the parasite in nature. The results of the present dissertation show that Leishmania reacted differently in mice and this has significant implications in administering the drugs effectively. The results of this dissertation suggest that temperature plays the primary role for the beginning of the activation period of phlebotomes and is species specific, while the relative humidity is more related to their abundance. It was found that there is a specific pattern with distinct population outbreaks for each of the species of medical interest, while a novel species was discovered that is related to Phlebotomus balcanicus, which is a known transmitter of Leishmania infantum. It was also made clear that domestic animals (rabbits, poultry) should be placed at a greater distance from people's homes. On the other hand, it appeared that the parasites that show greater resistance, influx more drug than the sensitive ones. Both the freeze – defreeze cycle and the passage through the host (THP-1 cells, Rag1- mice) can affect the resistance of the parasite. The best model organism in resistance experiments is the mammal. Inside mammals, the parasites gained resistance in contrast with the primary isolate, answering the thesis question in the affirmative: the acquired resistance in the mammalian host will be maintained when Leishmania continues its life cycle.Η φλεβοτόμος, είναι ο διαβιβαστής του πρωτοζώου Leishmania που προκαλεί λεϊσμανίαση. Η ανακάλυψη αυτή έγινε στην Ελλάδα το 1910, σχεδόν μια δεκαετία από τη χρήση της ονομασίας Leishmania. Η εξάπλωση των φλεβοτόμων καλύπτει και τις πέντε ηπείρους και καλύπτει τροπικές και υποτροπικές περιοχές, ενώ κάποια είδη τα βρίσκουμε και σε πιο εύκρατα κλίματα. Η συνεχώς αυξανόμενη μέση θερμοκρασία της γης προκαλεί ανησυχία γιατί νέες περιοχές θα γίνουν φιλόξενες για τις φλεβοτόμους. Εκτός από τους ορεινούς όγκους, οι κλιματολογικές συνθήκες είναι οι μόνες που περιορίζουν την εξάπλωσή τους στη χέρσο. Ενδεικτικά σημεία στα οποία μπορούν να ξεκουραστούν και να αναπαραχθούν, είναι μικρές σχισμές σε βράχους ή τοίχους, σπηλιές ή και κουφάλες δέντρων, αλλά και περιοχές με έντονη βλάστηση, περιττώματα ζώων και φυλλοστρωμνή. Από τα 31 είδη του γένους Phlebotomus που παίζουν το ρόλο του διαβιβαστή της Leishmania, μεγαλύτερο υγειονομικό ενδιαφέρον στην Ελλάδα παρουσιάζουν τα είδη Phlebotomus (Larroussius) tobbi, Phlebotomus (Paraphlebotomus) sergenti, Phlebotomus (Paraphlebotomus) papatasi, Phlebotomus (Larroussius) perniciosus, Phlebotomus (Larroussius) neglectus καθώς και ο πιθανός αλλά όχι εξακριβωμένος διαβιβαστής Phlebotomus (Paraphlebotomus) similis. Στην Κρήτη το Phlebotomus neglectus μεταδίδει τη Leihsmania infantum, η οποία προκαλεί σπλαχνική λεϊσμανίαση στον άνθρωπο και στο σκύλο. Το Phlebotomus similis ενοχοποιείται ως ο διαβιβαστής της Leishmania tropica, που προκαλεί τη δερματική λεϊσμανίαση, λόγω της υψηλής του συγγένειας με το Phlebotomus sergenti που είναι ο γνωστός διαβιβαστής της Leishmania tropica. Δεδομένης της συνεχόμενης αύξησης των κρουσμάτων στη χώρα μας και ειδικά στην Κρήτη, καθίσταται αναγκαία η εμπεριστατωμένη γνώση πάνω στην οικολογία των φλεβοτόμων που μεταδίδουν λεϊσμανίαση. Η λεϊσμανίαση είναι μια ασθένεια που προκαλείται από το πρωτόζωο παράσιτο του γένους Leishmania και προσβάλλει τόσο τα θηλαστικά, όσο και τα ερπετά. Η φλεβοτόμος είναι ο κύριος διαβιβαστής της. Συναντάται σε τρεις μορφές, τη σπλαχνική, τη δερματική και τη βλεννοδερματική λεϊσμανίαση. Κάθε χρόνο καταγράφονται περίπου 0,7 - 1 εκατομμύριο νέα περιστατικά λεϊσμανίασης σε 98 χώρες με το ποσοστό θνησιμότητας να αγγίζει το 3,3%. Στην Ελλάδα τα συμπτώματά της είναι γνωστά πριν ακόμη της δοθεί το όνομα «λεϊσμανίαση». Τα τελευταία χρόνια έχει παρουσιαστεί αύξηση των κρουσμάτων στη χώρα μας και ειδικά στην Κρήτη. Η ανάπτυξη επίκτητης αντοχής της Leishmania σε μία ομάδα φαρμάκων, που επάγεται από την ανάπτυξη αντοχής σε ένα μόνο φάρμακο (φαινότυπος MDR), είναι το πιο σημαντικό χαρακτηριστικό της. Είναι ένα παράσιτο με εξαιρετική πλαστικότητα στο γονιδίωμα και χαρακτηρίζεται κυρίως από ανευπλοειδία. Πληθυσμιακά μπορεί να μεταλλάσσεται σε πολύ σύντομο χρονικό διάστημα είτε σε επίπεδο γονιδίου, είτε σε επίπεδο χρωμοσώματος. Παρ’ ότι στη φαρέτρα έναντι της νόσου προστίθενται συνεχώς νέα φάρμακα, είναι πολύ σημαντικό να γνωρίζουμε εάν ο MDR φαινότυπος που αποκτά το παράσιτο παραμένει στις εναλλαγές του κύκλου ζωής του (αμαστιγωτή – προμαστιγωτή μορφή), ή χάνεται. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη της εποχιακής κατανομής των ειδών Phlebotomus που απαντώνται στην Κρήτη, ώστε να συλλεχθούν σημαντικά δεδομένα σε σχέση με τον ρόλο τους στη μετάδοση του πρωτόζωου παράσιτου Leishmania. Έπειτα, η διερεύνηση της διατήρησης ή μη, της αντοχής των παρασίτων μετά την ολοκλήρωση του κύκλου ζωής τους μέσα από τον κάθε ξενιστή (θηλαστικό – φλεβοτόμος). Επίσης, η δημιουργία ανθεκτικών παρασίτων με τη χρήση του σκευάσματος Glucantime® και στις δύο μορφές του κύκλου ζωής τους, μέσα και έξω από τον ξενιστή και η διερεύνηση της διατήρησης ή μη, της επίκτητης αντοχής που θα έχουν αποκτήσει. Το μέρος που επιλέχθηκε για τη διεξαγωγή της μελέτης της εποχιακής κατανομής των ειδών Phlebotomus που απαντώνται στην Κρήτη, ήταν το χωριό Φόδελε, στο οποίο είχαν παρουσιαστεί κρούσματα λεϊσμανίασης στο παρελθόν. Η συλλογή των φλεβοτόμων έγινε με τη χρήση CDC Miniature Light Traps (John W. Hock Co., Gainesville, FL, USA) και Sticky Traps για τρεις συνεχόμενες ημέρες στα μέσα κάθε μήνα επί τρία συναπτά έτη. Η περίοδος δειγματοληψίας κάλυπτε όλη τη χρονική περίοδο που οι φλεβοτόμοι παρέμεναν ενεργές. Το πρώτο έτος, οι δειγματοληψίες δεν ήταν δυνατό να ξεκινήσουν πριν τον Ιούνιο. Για τη συλλογή των περιβαλλοντικών δεδομένων χρησιμοποιήθηκαν δέκα καταμετρητές θερμοκρασίας, σχετικής υγρασίας και σημείου δρόσου, καθώς και ένα ανεμόμετρο. Λοιποί περιβαλλοντικοί παράγοντες, όπως υετός και φάση σελήνης, καταγράφονταν επίσης. Για τη μελέτη της διατήρησης της επίκτητης αντοχής του παράσιτου στα φάρμακα, επιλέχθηκαν πέντε ανθεκτικά και πέντε ευαίσθητα στελέχη Leishmania infantum που είχαν χαρακτηριστεί ως προς την ανθεκτικότητά τους στο εργαστήριό μας στο παρελθόν με τη χρήση της κυτταρομετρίας ροής. Το μέγεθος που ορίζει την αντοχή τους ονομάζεται «slope α» και μετράει το ρυθμό εκροής του φαρμάκου σε χρονικά διάστημα δύο ωρών. Η αντοχή των δέκα στελεχών μετρήθηκε ξανά όταν αποψύχθηκαν. Στη συνέχεια ενοφθαλμίστηκαν σε ποντίκια και με το πέρας 15 ημερών, αυτά θανατώθηκαν και καλλιεργήθηκε ο σπλήνας και το ήπαρ τους, ώστε να απομονωθούν ξανά τα παράσιτα. Αυτά μετρήθηκαν ξανά και στη συνέχεια ένα από αυτά επιλέχθηκε για την επαγωγή της αντοχής και την ολοκλήρωση του δεύτερου μέρους του πειράματος. Η επαγωγή της αντοχής εφαρμόστηκε στην προμαστιγωτή μορφή σε καλλιέργεια παρασίτων, καθώς και στην αμαστιγωτή μορφή μέσα στην ανθρώπινη κυτταρική σειρά THP-1 και ποντίκια τύπου Rag1-, με τη χρήση του σκευάσματος Glucantime®. Χρήση της φλεβοτόμου στο συγκεκριμένο πείραμα δεν έγινε γιατί πραγματοποιήθηκε στην πορεία από άλλη ερευνητική ομάδα. Η προσπάθεια επαγωγής της αντοχής στην προμαστιγωτή φάση στην καλλιέργεια δεν επιτεύχθηκε.Από το σύνολο των 6.000 περίπου φλεβοτόμων που συλλέχθηκαν (60% αρσενικά, 40% θηλυκά), αναγνωρίστηκαν τα είδη Phlebotomus neglectus (57,8%), Phlebotomus similis (30,4%), Phlebotomus papatasi (2,5%), Phlebotomus simici (0,1%), Sergentomyia minuta (8,8%), καθώς και το νέο-αναγνωρισθέν Phlebotomus (Adlerius) creticus n. sp. (0,4%). Το Phlebotomus neglectus ήταν το κυρίαρχο είδος, με το Phlebotomus similis να ακολουθεί, εκτός από την πρώτη χρονιά δειγματοληψίας. Τα δύο κυρίαρχα, καθώς και το Sergentomyia minuta, εμφανίστηκαν ολόκληρη τη δειγματοληπτική περίοδο. Η μελέτη της εποχιακής διακύμανσης των δύο κυρίαρχων δείχνει πως το Phlebotomus neglectus εμφάνισε δύο διακριτές πληθυσμιακές εκρήξεις (Μάιο και Αύγουστο), ενώ το Phlebotomus similis εμφάνισε μια διακριτή με μια λιγότερη διακριτή πληθυσμιακή έκρηξη να έπεται ή να προηγείται (Ιούνιο και Ιούλιο). Αρσενικά και θηλυκά παρουσίασαν παρόμοιο πρότυπο. Οι πληθυσμιακές τους εκρήξεις κουμπώνουν σαν παζλ, καθιστώντας όλους τους καλοκαιρινούς μήνες υψηλού υγειονομικού ενδιαφέροντος. Οι τιμές θερμοκρασίας και σχετικής υγρασίας που εμφάνισαν κορυφή τα δύο είδη ήταν Τ = 20,3οC – 27ο C, RH = 57,5% – 63,1% για το Phlebotomus neglectus και Τ = 24,3οC – 27,2ο C, RH = 53,9% – 59,1% για το Phlebotomus similis. Το εύρος θερμοκρασίας και υγρασίας που παρέμειναν ενεργά είναι σχεδόν το ίδιο με το Sergentomyia minuta, αλλά πολύ πιο διευρυμένο από τα υπόλοιπα είδη. Σε αυτό το είδος αρσενικά και θηλυκά παρουσίασαν διαφορετικό πρότυπο εποχιακής διακύμανσης. Η θερμοκρασία ήταν αυτή που σηματοδότησε την έναρξη της περιόδου ενεργοποίησης των φλεβοτόμων και ήταν ≥ 17 – 18οC για το Phlebotomus neglectus, ενώ ≥ 20 – 22οC για τα Phlebotomus similis και Sergentomyia minuta. Η αύξηση της θερμοκρασίας (r = 0,776, p < 0,05) και η μείωση της σχετικής υγρασίας (r = 0,644, p < 0,05), επηρέασαν θετικά μέχρι κάποιο βαθμό την αφθονία των φλεβοτόμων. Τα Phlebotomus neglectus και Phlebotomus similis επέδειξαν παρόμοιες οικολογικές προτιμήσεις, καλύπτοντας ολόκληρη την περιοχή μελέτης και συλλέχθηκαν και από τους δύο τύπους παγίδων. Η πλειονότητα των ατόμων όμως συλλέχθηκε από τις τύπου CDC και κυρίως από αυτές που ήταν στημένες κοντά σε περιοχές που λειτουργούσαν ως κοτέτσι και κουνελώνας και χαρακτηρίζονταν από έντονη βλάστηση. Στο έδαφος υπήρχαν περιττώματα αυτών των ζώων, καθώς και σάπια φρούτα και λαχανικά. Το Phlebotomus papatasi συλλέχθηκε σε μεγαλύτερο ποσοστό από τη μία από τις περιοχές που λειτουργούσαν ως κοτέτσι και κουνελώνας, αλλά και από τις παγίδες ST. Το Sergentomyia minuta συλλέχθηκε κυρίως από τις παγίδες ST (78%). Ένα μεγάλο ποσοστό θηλυκών Phlebotomus papatasi (29,9%) και Phlebotomus similis (23,2%) και ένα λίγο μικρότερο Phlebotomus neglectus (17,1%) και Sergentomyia minuta (15,6%) βρέθηκαν σε διάφορες φάσεις πέψης του γεύματός τους, καθώς και με αυγά. Τα πιο συχνά παρασιτούμενα ε

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