Phlebotomine sand flies are insects that are highly relevant in medicine, particularly as the sole proven vectors of leishmaniasis. Accurate identification of sand fly species is an essential prerequisite for eco-epidemiological studies aiming to better understand the disease. Traditional morphological identification is painstaking and time-consuming, and molecular methods for extensive screening remain expensive. Recent studies have shown that matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for rapid and cost-effective identification of arthropod vectors, including sand flies. The aim of this study was to validate the use of MALDI-TOF MS for the identification of Northern Amazonian sand flies. We constituted a MALDI-TOF MS reference database comprising 29 species of sand flies that were field-collected in French Guiana, which are expected to cover many of the more common species of the Northern Amazonian region, including known vectors of leishmaniasis. Carrying out a blind test, all the sand flies tested (n = 157) with a log (score) threshold greater than 1. 7 were correctly identified at the species level. We confirmed that MALDI-TOF MS protein profiling is a useful tool for the study of sand flies, including neotropical species, known for their great diversity. An application that includes the spectra generated here will be available to the scientific community in the near future via an online platform.
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are insects of great medical relevance because they are the most frequent vectors of leishmaniasis [1,2] and also transmit various other human pathogens including bacteria and viruses [3]. Leishmaniases are a range of diseases caused by flagellated protozoans of the genus Leishmania (Kinetoplastida: Trypanosomatidae), transmitted through the bite of an infected female sand fly [2]. The epidemiology of leishmaniasis is complex, due to the wide diversity of Leishmania and the sand fly species involved. In the Americas, 56 sand fly species are known to be potential vectors of 15 Leishmania species [2]. In French Guiana, Leishmania (Viannia) guyanensis is the most prevalent Leishmania species in humans and is mainly responsible for localized cutaneous leishmaniasis [4]. Other Leishmania species such as L. braziliensis can be more clinically debilitating, since they can cause mucocutaneous (nose, mouth, and throat commitment) or diffuse cutaneous leishmaniasis, requiring specific medical management [4,5]. On the Guiana Shield, the main vector of L. guyanensis is Nyssomyia umbratilis [6,7]. However, sand fly species transmitting medically important Leishmania species are partially identified or not yet identified, such as those related to the L. braziliensis local transmission cycle [2,7]. Additionally, vector ecology can evolve with environmental changes such as deforestation and urbanization [1,2, 7]. Human activities in deforested areas may result in epidemics, as seen by the reported outbreaks of cutaneous leishmaniasis in French Guiana [5] and Argentina [8], for instance. Therefore, the identification of sand fly species associated with transmission hotspots together with a better description of sand fly communities are essential to improving the understanding of leishmaniasis epidemiology [1,2, 8]. To date, sand fly ecology studies have been limited due to the complexity of species identification, which requires the meticulous and time-consuming labor of entomological taxonomy experts [9]. Molecular identification has been proposed as an alternative method and molecular reference databases have been made available [10,11]. However, cost analysis for huge series of samples shows that extensive screening remains expensive. The recent advent of protein profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is about to revolutionize medical entomology [12]. Several studies have shown that MALDI-TOF MS is suitable for sand fly identification, and in-house databases have been constructed [13–17]. Only four species of sand flies from South America have been tested to date and implemented in an in-house MALDI-TOF MS database [13]. A disadvantage of in-house databases is the restricted access to local utilization. As a solution, the implementation of a centralized online platform has been suggested for sand fly identification [12,13]. The aim of the study was to create a MALDI-TOF MS reference database of French Guiana sand flies. Sand flies were captured from different geographic locations in French Guiana, an Amazonian territory along the Northern South American Atlantic coast. A mass spectra library (MSL) was implemented, based on molecular identification of field-collected sand flies. The reliability of the MSL was then evaluated using a blind test. This MSL will be included in an online platform dedicated to phlebotomine sand fly identification that is currently being developed at Sorbonne University.
To constitute and validate a MSL, collected sand flies were identified by DNA sequencing and divided into two different panels, a construction panel and a validation panel (Table 1 and Fig 1). The separation of the sampling into construction and validation panels was sequential. The first 206 sand flies analysed were attributed to the construction panel because it gave a sufficient number of species (n = 20) and individuals by species (from one to ten) to build a MSL. The remaining 199 specimens, different from the samples of the construction panel, which had not been analysed before, were attributed to the validation panel. The construction panel comprised 260 sand flies collected from four study sites located in a pristine forest (at the Nouragues reserve), in secondary forest (Saint-Georges and Regina) and in logging forest (Counami). The validation panel comprised 199 sand flies, including 93 individuals that were collected at the same study sites and periods as those of the construction panel and 106 that were collected at four additional study sites and study periods. Additional sites were three peri-urban sites of secondary and edge forest (around Cayenne) and one pristine forest site (along the Approuague River in Saut Grand Machicou). Between February 2017 and July 2017, male and female sand flies were collected from different types of forested habitats in French Guiana (Table 1, Fig 2). Sand flies were captured using Centers for Disease Control (CDC) Miniature light traps with Incandescent light ([John W. Hock Company, Gainesville, FL, USA, CDC]), set between 6 pm and 6 am. Back at the laboratory, the sand flies were rapidly killed by freezing at −20°C and dissected immediately thereafter into four parts (head, thorax with legs and wings, abdomen and genitalia), at room temperature. After dissection, the thoraxes with legs and wings were stored dry-frozen at −80°C, and the other body parts were stored in 70% ethanol at −80°C. Thoraxes with legs and wings were put aside for MALDI-TOF MS analysis and abdomens were put aside for molecular identification. Head and genitalia were kept for morphology in a future study (not analysed at the time of this study), in case of discordant or uninterpretable identification results. Before analysis, the period of dry-frozen storage at −80°C varied between 10 days and 7 months, with a mean time of 4 months. Thoraxes with wings and legs were rinsed in ethanol 70% for 10 min in a 1. 5-mL microcentrifuge tube. Tubes were centrifuged at (13,000 rpm, 10 min) and supernatant was discarded. After a second centrifugation (13,000 rpm, 2 min), the remaining ethanol solution was then eliminated using a micropipette and left to evaporate. Proteic extraction consisted in adding 10 μL of 70% formic acid. After a manual homogenization with a micropipette, the homogenate was incubated for 5 min. Then 10 μL of 100% acetonitrile was added and left to incubate for 5 min. The homogenate was centrifuged (13,000 rpm, 2 min) and 1 μL of the supernatant of each sample containing the protein extract was deposited onto a steel target plate (Bruker Daltonics, Wissembourg, France). Once dried, the deposits were covered with a 1-μL alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix prepared in 50% acetonitrile and 2. 5% trifluoroacetic acid. To ensure the reproducibility of the results, a total of ten replicates were spotted for each isolate to be included in the MSL and a total of four replicates for each isolate of the panel to be tested. Mass spectra were acquired with a Microflex LT (Bruker France SAS) using the default acquisition parameters. The spectra were acquired in linear mode in the ion-positive mode at a laser frequency of 60 Hz and mass range of 2–20 kDa. The data was automatically acquired using AutoXecute in FlexControl v3. 4 software (Bruker France SAS), and exported into Maldi Biotyper v4. 1 software for data processing with the default parameters and spectra analysis. All specimens from the validation panel, with a valid MALDI-TOF MS-based identification and mass spectrum quality, were secondarily implemented in the reference database. The species of the blind test that were not previously represented in the MSL or that were represented at a number of specimens less than 10, were spotted in ten replicates until reaching ten references per species, with the same method as applied for the MSL construction.
The intra-clade genetic distance calculated on the 208 reference sequences, before and after the addition of our sequences to the analysis, was less than 3% for all the species. As expected, because of insufficient resolution, the 16S rDNA sequencing identification failed to discriminate morphologically closely related species Trichopygomyia trichopyga / Trichopygomyia depaquiti as well as three species of the genus Nyssomyia: N. umbratilis, N. yuilli and N. antunesi (S2 Table). Ten individuals did not cluster with any species in the available reference sequences. MSL was composed of 20 species of field-collected sand flies. Between one and ten specimens of each MSL species were included, corresponding to 89 specimens. The validation panel was composed of 199 specimens, including from one to 45 specimens of 24 different sand fly species. The details of the sand fly species in each panel are found in Table 2. The spectra had good resolution and intensity, with a large mass/charge interval, ranging from about 2 to 10 kDa (Fig 3A, 3B and 3C). The spectra were highly homogenous and reproducible when obtained from different protein extract deposits of a single specimen (Fig 3A). Variability of mass spectraprotein profiles was observed between different specimens of a single species, including when comparing spectra obtained from specimens of the same sex (Fig 3B). When comparing spectra within a complex of species or between different species, the heterogeneity of mass spectraprotein profiles was observed (Fig 3C). In the heat map grid of the CCI matrix values (Fig 4), the coloured squares of the central diagonal reflected the degree of reproducibility of each specimen’s mass spectra when compared to itself. Hot colours reflected high reproducibility of each specimen’s mass spectra. Around the central diagonal, spectra from various specimens of the same species were compared; hot colours showed a high level of intraspecific reproducibility of mass spectra (diagonally), distributed in a cluster of species (square). A mosaic of hot colours inside a cluster of identical species was indicative of heterogeneity of mass spectraprotein profiles between specimens and reflected intraspecific diversity. Outside of the diagonal, when the spectra of different species were compared, colder colours revealed lower CCI values with very little between-species MSP correlation, confirming the high intra-species specificity of the mass spectra. The matrix of CCI values of sand flies MSL is available in the supplementary data (S3 Table). Cluster analysis of the dendrogram (Fig 5) showed that each specimen belonging to the same species, either males and females, collected from various sites in French Guiana, clustered on the same branch. This result attests to the intra-species specificity of MALDI-TOF MS sand fly protein profiles and of the consistency with molecular identification. In concordance with molecular results, T. trichopyga and T. depaquiti were grouped together in a unique cluster of mass spectra. For the Nyssomyia genus, specimens were separated on two different branches of the dendrogram, whereas three were clustered in a monophyletic group of the maximum likelihood tree by molecular analysis of the 16S rDNA (S1 Fig). According to the distribution of LS values, the interpretable identification result was defined as the best match of four spots with a LS value ≥1. 7 (Fig 6). Of all the sand flies tested by MALDI-TOF MS during the blind test, 79% (157/199) gave interpretable MALDI-TOF MS-based identification results. A total of 37 samples corresponded to species molecularly identified that were missing in the MSL. When a corresponding reference spectrum was available in the MSL, 97% (157/162) of the MALDI-TOF MS-based identification results were interpretable. For specimens that did not have a corresponding reference spectrum in the MSL, 100% (37/37) of the identifications were not interpretable, because the best match of the four spots did not reach the threshold of 1. 7 (mean LS value = 1. 36±0. 095). With the threshold value ≥1. 7, no misidentification was observed. Of the 157 sand flies with interpretable identification results, 100% were correctly classified at the species level with the best match LS value ranging from 1. 7 to 2. 6 (mean LS value = 2. 23±0. 19). Overall sensitivity was 79% when considering all the sand flies tested and 97% when considering only species with a corresponding reference in the MSL. Specificity was 100%. Five sand flies tested with a corresponding reference in the MSL had a LS value <1. 7 and could not be identified. Three of them, a P. hirsutus (LS value = 1. 5), a T. trichopyga/T. depaquiti (LS value = 1. 6) and a T. ininii (LS value = 1. 65) had a MALDI-TOF MS identification result concordant with molecular identification. Two of them, one P. amazonensis (LS value = 1. 63) and one E. infraspinosa (LS value = 1. 2) had a MALDI-TOF MS identification discordant with the molecular identification (compared with P. claustrei and Nyssomyia sp. , respectively). This imply that a threshold lowered below 1. 7 would have decreased specificity and increased the risk in giving wrong identification result. Complete dataset of sand flies identification results obtained by DNA sequencing and by MALDI-TOF MS is available in supplementary data (S2 Table). The MSL was implemented with the mass spectra of nine additional sand fly species. Mass spectra of additional specimens of species already present in the MSL were also implemented to increase the diversity of mass spectra. The resulting reference database was made up of 282 specimens and 29 sand fly species (Table 2).
We confirm that MALDI-TOF MS protein profiling is well adapted to the identification of sand fly species, including in neotropical areas, known for its great diversity of sand fly species. MALDI-TOF MS can be a useful tool for rapid, inexpensive and accurate identification of sand flies but, like molecular methods, better accessibility to reference libraries for the scientific community would extend its utility. In the near future, this Northern Amazonian sand fly spectral database will be included in an online platform dedicated to phlebotomine sand fly identification, as already applied with success for identification of fungi and Leishmania of medical interest [26,37]. Recent studies have shown that MALDI-TOF MS was also accurate for the detection of Rickettsia spp. [41] and Borrelia crocidurae [42] in ticks, Plasmodium spp. in Anopheles mosquitoes [43] and Bartonella spp. in fleas [44], by generating distinct mass spectra protein profiles between infected and uninfected arthropods. The possibility of identifying sand flies to the species level as well as the infection status by Leishmania parasites using MALDI-TOF MS would offer a significant opportunity for sand fly eco-epidemiology studies.
Phlebotomine sand flies are small insects, mostly known for their role in the transmission of Leishmania parasites to humans and other mammals. In French Guiana, the main clinical form of the disease manifests as cutaneous lesions also called American cutaneous leishmaniasis. The transmission of Leishmania from wild mammals to humans depends on the species of sand fly involved in the transmission. To better understand the mechanism of disease transmission, it is essential to accurately identify sand flies, including both vector and non-vector species. Until now, sand flies have mainly been identified using morphological and molecular methods. Recent studies have shown that a new tool based on protein profiling compiled in a library of spectra may be useful for the identification of arthropod vectors. This tool has the advantage of being less time-consuming, less expensive and does not require technical skills. The aim of this study was to assess the usefulness and accuracy of this new tool in identifying Northern Amazonian sand flies.
medicine and health sciences
ecology and environmental sciences
tropical diseases
sand flies
parasitic diseases
parasitic protozoans
organisms
protozoans
leishmania
neglected tropical diseases
insect vectors
research and analysis methods
sequence analysis
infectious diseases
zoonoses
mass spectra
bioinformatics
ecosystems
biological databases
chemistry
mass spectrometry
protozoan infections
disease vectors
matrix-assisted laser desorption ionization time-of-flight mass spectrometry
analytical chemistry
dna sequence analysis
sequence databases
eukaryota
ecology
forests
database and informatics methods
leishmaniasis
biology and life sciences
species interactions
physical sciences
spectrum analysis techniques
terrestrial environments
Identification of French Guiana sand flies using MALDI-TOF mass spectrometry with a new mass spectra library