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

Abstract

BackgroundPhlebotomine 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.ResultsDNA 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.ConclusionsMolecular 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.

Revision of the species composition and distribution of Turkish sand flies using DNA barcodes

“…Herein, cox 1 barcodes of two specimens collected from the Mediterranean region were molecularly confirmed as P. anatolicus and P. killicki. The first record of P. killicki in Cyprus was also verified based on cox 1 sequence correlation [20].…”

“…Analysis of concatenated mtDNA cytb - cox 1 sequences of S. minuta from across Europe and North Africa determined four distinct lineages, irrespective of their geographical origins [76]. Conversely, S. minuta specimens from Crete, Cyprus and mainland Greece clustered in three distinct lineages by locality [20]. Distributed across all three continents of the Old World, S. minuta exhibits morphologically diverse populations.…”

“…DNA sequence data from several mitochondrial and nuclear loci have been introduced in several studies to mitigate the need for accurate, reliable sand fly identification methods for both taxonomic and epidemiological studies (see [15] for a review). Among these, the DNA barcoding region of the mitochondrial cytochrome c oxidase I ( cox 1) gene, previously proposed by Hebert et al [16], has proven highly useful in the differentiation of sand fly species [17–20]. The increasing availability of DNA barcode reference sequences has allowed retrospective identification of infected sand fly specimens, permitting accurate vector incrimination in high-throughput sand fly biosurveillance studies for the first time [21, 22].…”

Identification of Blood Meal of Sand Flies in a Cutaneous Leishmaniasis Endemic Area, Volta Region-Ghana

“…It is worth noting that sand flies are known to have a low and weak flight pattern [8,9] and as such will not travel too far to get their source of blood meal. Thus, one of the most important steps towards predicting and controlling this disease is by monitoring the sand flies in a region [10]. The identification of the source of blood meal of blood sucking insects were until recently conducted using mainly serological techniques such as the enzyme-linked immune-sorbent assays, latex agglutination test and the precipitin test [3].…”

The Prevalence of Antibodies against Sandfly Fever Viruses and West Nile Virus in Cyprus

“…However, when the CNS is affected, high fever, headache, nausea, vomiting, Kernig signs, neck rigidity, myalgia and sometimes unconsciousness, tremors, paresis and nystagmus as well as encephalitis, severe meningoencephalitis and long-lasting sequelae have been reported (4, 5). The Mediterranean basin is endemic for sandfly-borne phleboviruses infections (4, 6–10) and particularly Cyprus where phlebotomines (11, 12) and outbreaks have been reported (13–16).…”