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

BACKGROUND: 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.

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

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.

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.

DNA sequencing confirms PCR-RFLP identification of wild caught Larroussius sand flies from Crete and Cyprus.

Many Phlebotomine sand fly species (Diptera, Psychodidae) are vectors of the protozoan parasite Leishmania causing a group of diseases called the leishmaniases. The subgenus Larroussius includes sand fly vectors found in South East Mediterranean Basin responsible for Visceral (VL) and Cutaneous human leishmaniasis (CL). It is important to monitor these medically important insects in order to safely predict possible Leishmania transmission cycles. Leishmania infantum is endemic in the islands of Crete and Cyprus with increasing VL cases in humans and dogs and in Cyprus the newly introduced Leishmania donovani causes both VL and CL in humans. The morphological identification of the females of the subgenus Larroussius often presents difficulties. Morphology and COI PCR - RFLP were used to identify wild caught Larroussius sand flies belonging to Phlebotomus tobbi, P. perfiliewi, and P. neglectus species from Crete and Cyprus. The identification results were further confirmed by sequencing (DNA barcoding) and Bayesian phylogenetic analysis. COI PCR - RFLP, when correctly optimized and with respect to geographical origin, can serve as an initial patterning identification tool when large sand fly numbers need to be identified. It could accurately assign Larroussius females and males to their taxa overcoming the difficulties of morphological identification. Finally, DNA barcoding will contribute to a molecular identification database to be used for in-depth species studies.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Detection of Leishmania Infantum in red foxes (Vulpes vulpes) in Central Greece.

This is the first record of Leishmania detection in foxes in Greece. Spleen, lymph nodes, bone marrow and blood samples were collected from 47 red foxes (Vulpes vulpes) found dead or captured, narcotized and freed after bleeding, from November 2009 to 2011, in Fthiotida prefecture, central Greece. This is an endemic for canine leishmaniasis area with several human visceral leishmaniasis cases. The samples were tested for Leishmania infantum and Leishmania tropica by molecular methods (polymerase chain reaction (PCR) and restriction fragment length polymorphism) and serology (indirect immunofluorescent antibody test; when blood samples were available). Leishmania infantum DNA was detected in 28 animals (59·5%). PCR positivity was related to animal age, sex, weight, characteristics of the area trapped, presence of leishmaniasis symptoms and presence of endo- and ecto-parasites. The results were related to dog seropositivity obtained earlier in the area. The findings support the hypothesis that this wild canid may serve as a reservoir for Leishmania in areas where the sandfly vectors are found. In the prefectures of Larisa and Magnisia, adjacent to Fthiotida, Phlebotomus perfiliewi and Phlebotomus tobbi (known vectors of L. infantum) have been reported.

The proliferation potential of promastigotes of the main Leishmania species of the old world in NNN culture medium prepared using blood of four different mammals.

The efficacy of the in vitro cultivation of promastigotes of four Leishmania spp. was tested in the biphasic Novy-MacNeal-Nicolle (NNN) medium prepared using blood from different animals (horse, donkey, goat and sheep). The aim was to test which NNN preparation gave the best yield in the shortest time for different parasite species, in order to obtain a large crop of promastigotes for experimental work and for antigen preparation. Promastigotes of Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania major, the four main parasite species occurring in the old world, were defrosted from -80 °C and placed, at equal numbers, in the 4 different NNN preparations. At the end of the 7th day, the NNN medium using horse blood produced the greatest number of promastigotes for all Leishmania spp. tested, whilst goat blood proved the poorest medium, providing culture results only for L. infantum. This finding may be explained by the fact that Leishmania is a nicotinamide adenine dinucleotide (NAD) auxotroph and horse erythrocytes support NAD-dependent microorganisms.

Different degree of paternal mtDNA leakage between male and female progeny in interspecific Drosophila crosses.

Maternal transmission of mitochondrial DNA (mtDNA) in animals is thought to prevent the spread of selfish deleterious mtDNA mutations in the population. Various mechanisms have been evolved independently to prevent the entry of sperm mitochondria in the embryo. However, the increasing number of instances of paternal mtDNA leakage suggests that these mechanisms are not very effective. The destruction of sperm mitochondria in mammalian embryos is mediated by nuclear factors. Also, the destruction of paternal mitochondria in intraspecific crosses is more effective than in interspecific ones. These observations have led to the hypothesis that leakage of paternal mtDNA (and consequently mtDNA recombination owing to ensuing heteroplasmy) might be more common in inter- than in intraspecific crosses and that it should increase with phylogenetic distance of hybridizing species. We checked paternal leakage in inter- and intraspecific crosses in Drosophila and found little evidence for this hypothesis. In addition, we have observed a higher level of leakage among male than among female progeny from the same cross. This is the first report of sex-specific leakage of paternal mtDNA. It suggests that paternal mtDNA leakage might not be a stochastic result of an error-prone mechanism, but rather, it may be under complex genetic control.

Assessment of the infectivity potential of Leishmania infantum, using flow cytometry.

The protozoan parasite Leishmania infantum causes leishmaniases, a sandfly-borne disease of humans and dogs, in all countries of the Mediterranean basin. The promastigote, infective stage of the parasite, once inoculated to the mammalian host by the vector, is ingested by macrophages. Leishmania lives within the lysosome of the phagocytic immune cells inactivating the enzymes contained. The ability of an isolate to survive within the macrophage and its rate of multiplication in this environment is an important factor determining the infectivity potential of the isolate and the manifestation of the disease. This capacity of the parasite is measured as the percentage of infected cells and the mean value of parasites per cell. The infectivity potential, of clinical isolates of L. infantum infecting THP-1 cells in vitro, was studied by flow cytometry and light microscopy. The percentages of cells in a sample containing a specific number of parasites, as recorded by light microscopy, were used in flow cytometry to manually gate the mean fluorescence intensity which corresponded to the percentage of cells with that number of parasites. The gating obtained, was then used as a "standard reference curve" to evaluate results by flow cytometry compared to those obtained by light microscopy. The results, of the overall percentage of infected cells and the number of parasites per cell in the culture, matched in the two methods. So, flow cytometry can be used as a rapid, cost effective, easy and reproducible method to study the infectivity potential of isolates, either in biological, epidemiological, or clinical tests, particularly for the assessment of drug efficiency trials.

Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

BACKGROUND: Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches. METHODS: Six laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes. RESULTS: Specimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens. CONCLUSIONS: Species identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.

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

The 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.