An integrative approach to identify sand fly vectors of leishmaniasis in Ethiopia by morphological and molecular techniques

Abstract

Background Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. Methods In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. Results Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. Conclusions Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

Phlebotomine Sand Fly Survey in the Republic of Moldova: Species Composition, Distribution and Host Preferences

“…Close relationship between P. balcanicus sequences from Eastern Europe (Romania and Serbia) and recently identi ed P. creticus needs further evaluation as well as the divergence of two lineages within the widely distributed P. simici which has been recently recorded in Austria, suggesting possible northward spread in Europe (Kniha et al, 2021). An integrative approach deploying several molecular methods which study both DNA and proteins to complement traditional morphological "golden standard" was recently successfully applied when describing Phlebotomus creticus, a novel species from eastern Mediterranean (Dvorak et al, 2020a), as well as studying sand y fauna in East Africa (Pareyn et al, 2020). Our ndings further advocate its use as a valuable tool that provides better understanding of taxonomy and biogeography of this morphologically challenging yet medically important sand y subgenus.…”

Morphology and mini‐barcodes: The inclusion of larval sampling and NGS‐based barcoding improves robustness of ecological analyses of mosquito communities

“…In fact, we found two species of this Barbirostris complex here in Singapore and there is no information on their role as potential malaria vectors locally. Detection of cryptic species complexes is also important in other vector systems such as sand flies (Pareyn et al., 2020) and biting midges (Mignotte et al., 2020), where effective control measures rely on accurate insect identification.…”

Entomological study in an anthroponotic cutaneous leishmaniasis focus in Morocco: Fauna survey, Leishmania infection screening, molecular characterization and MALDI‐TOF MS protein profiling of relevant Phlebotomus species

“…This technique of mass spectrometry, originally exploited mainly for the identification of unicellular pathogens, was recently successfully applied also on various multicellular organisms including medically important arthropods as a rapid, simple, reproducible and also cost‐effective alternative (Yssouf et al., 2016). After the initial proof‐of‐concept assay on sand flies (Dvorak et al, 2014), the method was successfully applied in several field studies including identification of sand flies in leishmaniasis‐endemic regions of Algeria (Lafri et al, 2016), a recent inventory of sand fly fauna in Crete with a description of Phlebotomus creticus , a new species from Eastern Mediterranean (Dvorák et al, 2020) and identification of sand flies in Ethiopia including some important vectors of human leishmaniases (Pareyn et al, 2020). These studies demonstrate that if the field specimens are collected and stored with respect to the subsequent MALDI‐TOF MS protein profiling as described by Halada et al., (2018), they can then be easily and with low costs transported to a mass spectrometry facility that performs sand fly species determination, overcoming through such collaboration the shortcoming of prohibitive costs of the necessary machinery.…”