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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.

SUBMITTER: Dokianakis E

PROVIDER: S-EPMC5816364 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.

Project description:BackgroundPhlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.Methodology/principal findingsSand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M'sila where P. (Phlebotomus) papatasi was the only sand fly species detected.ConclusionThe present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.

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

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