Estimation of vector competence of Moroccan Sand y Populations Using a Proteomic Approach

Background Phlebotomine vector born disease are distributed throughout the world, and different pathogens are associated with varying degrees of disease severity. In Morocco, sand y populations are incriminated in the transmission of several pathogens, such Leishmania spp. and phlebovirus, in particular the most relevant sand y species as the case of Phlebotomus papatasi. This species is the main vector of Leishmania major in Morocco, and incriminated in phlebovirus transmission. Not to pass over without investigating its possible role on the transmission of entomopathogenic parasite already reported in literature. Methodology and nding The present study focused on proteomic analysis in Phlebotomus papatasi from four localities in Morocco. Proteomic analysis in female P. papatasi was performed with Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 239 identied proteins for Leishmania spp., 26 identied proteins for phlebovirus and 1132 identied proteins for nematode were detected in the 20 pools of P. papatasi from investigated localities. The microscopic examination of 1752 sandies collected showed, the infection of two specimens of female’s P. sergenti with Tetranematid Didilia spp. and potential Microlariae in Imintanout locality (Z2). Conclusion This study presents the rst report of nematodes in sand ies of Africa. Also, the rst use of proteomic tools for estimation of vector competence of P. papatasi which could be a specic tool to a better understanding of the vectorial capacity of Moroccan sandies population.


Introduction
Phlebotomine sand ies (Diptera: Psychodidae, Phlebotominae) are small, fragile, blood-sucking insect with a wide range of hosts [1], allowing the pathogen transmission to humans and other animals [2].
These are considered as vectors of various diseases notably canine and human leishmaniasis, bartonellosis and several arboviruses [3]. Morocco is one of the countries where leishmaniasis constitutes a major public health concern and sand y species exist with a signi cant geographical spread [4]. Moroccan populations of sand ies, as the other Mediterranean region's populations, are not only vectors of protozoa, but also viruses [5].
Phlebotomus papatasi is widely distributed around the Mediterranean basin [6]. This species is very common and has a signi cant ecological plasticity [7][8][9]. It is incriminated in the transmission of cutaneous leishmaniasis (CL) and of sand y fever that has been established in the Mediterranean regions [10]. Beside this, P. papatasi is the main vector of zoonotic leishmaniasis due to L. major in Morocco, the most abundant form in terms of number of cases [11].
The wide distribution range of P. papatasi extends across North Africa, through Eurasia, and into India, it appears to be highly dependent upon environmental conditions [12]. Phlebotomus papatasi associated with all CL to L. major endemic foci across the country in arid areas of Morocco [8]. P. papatasi is most active in the hot, dry season and it was the most abundant species when ambient temperature is in the 32-36 °C range [13]. P. papatasi was found well adapted to arid climate conditions [14,15].
Technics as liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level [16]. Evidently proteins play an important role in different molecular networks and perform many biochemical functions of organisms. Label-free liquid chromatography-mass spectrometry (LC-MS/MS) routinely quanti es and identi es thousands of proteins across multiple samples in a single run, providing an unprecedented opportunity to examine changes in the proteomics pro le of a biological fraction or organism [17]. In the present study, we analyse four Moroccan populations of P. papatasi females in order to intend and improve the information available about this specie's in Morocco by the estimation of its vector competence in the transmission of several pathogens.
We used specimens trapped by CDC technique because it gives better specimens for the accurate proteomic analysis as sticky traps technique for collection could have affected the specimens and their proteomic analysis accuracy. [19]. The rest of collected sand ies were examined using a binocular microscope for the presence of potential nematodes species. The infected specimens were dissected to remove the nematode from the sand y body and identi ed morphologically according to Moroccan sand y Key [20]. Sand ies collected were conserved in 70% ethanol and transported to the RI-MUHC.

Protein digestion with trypsin
The head and genitalia of the sand es were mounted on slide and species identi cation were made using identi cation key [20]. A standard TCA protein precipitation was rst performed to remove detergents from P. papatasi specimens. Protein extracts were then re-solubilized in 10 µL of a 6M urea buffer. Proteins were reduced by adding 2.5 µL of the reduction buffer (45 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 37ºC, and then alkylated by adding 2.5 µL of the alkylation buffer (100 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min at 24ºC in dark. Prior to trypsin digestion, 20 µL of de-ionized distilled water was added to reduce the urea concentration to 2M. Ten µL of the trypsin solution (5 ng/µL of trypsin sequencing grade from Promega, 50 mM ammonium bicarbonate) was added to each sample. Protein digestion was performed at 37ºC for 18 h and stopped with 5 µL of 5% formic acid. Protein digests were dried down in vacuum centrifuge and stored at -20 ºC until LC-MS/MS analysis.

LC-MS/MS
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed at the RI-MUHC proteomic facility as described by Atayde et al. 2019 [16]. Sample proteins were precipitated with 15% trichloroacetic acid (TCA)/acetone and digested with trypsin at a nal concentration of 2 ng/ml. After an 18-hr incubation at 37C, the reactions were quenched by the addition of for mic acid to a nal concentration of 1% prior to the LC-MS/MS analysis. The LC column was a PicoFrit fused silica capillary column (New Objective) self-packed with C-18 reverse-phase material (Phenomenex). This column was installed on the Easy-nLC II system (Proxeon Biosystems) and coupled to the Q Exactive mass spectrometer (Thermo Fisher Scienti c) equipped with a Proxeon nanoelectrospray Flex ion source. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). Peptides were loaded on column at a ow rate of 600 nl/min and eluted with a two-slope gradient at a ow rate of 250 nl/min. Solvent B rst increased from 2% to 40% in 85 min and then from 40% to 80% in 25 min. LC-MS/MS data were acquired using a data-dependent top 15 method and standard values were used for all the parameters of the mass spectrometer.

Protein identi cation
The peak list les were generated with Proteome Discoverer (version 2.1) using the following parameters: minimum mass set to 500 Da, maximum mass set to 6000 Da, no grouping of MS/MS spectra, precursor charge set to auto, and minimum number of fragment ions set to 5. Protein database searching was performed with Mascot 2.6 (Matrix Science) against the Moroccan Leishmania species (L. major, L. infantum, L. tropica), Phleboviruses, Nematode protein databases. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.1 Da, respectively. Trypsin was used as the enzyme allowing for up to 1 missed cleavage. Cysteine carbamidomethylation was speci ed as a xed modi cation and methionine oxidation as variable modi cations. Data analysis was performed using Scaffold (version 4.10.0). Only proteins with minimum 3 peptides and peptide score higher than 20 were considered.

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The study was conducted using 3 pools of 5 specimens of P. papatasi representing each four studied localities. We performed mass spectrometry (LC-MS/MS) to analyze the content of the puri ed Moroccan sand y P. papatasi proteins. Pools were analyzed for the presence of proteins speci c of Leishmania spp., phlebovirus and nematode proteins. Besides, this analysis allows us to make differentiation of biomarkers peptides in P. papatasi populations between different localities. We compared the list of the identi ed proteins in different localities and found that the majority of the protein hits have been observed for all localities. As mentioned earlier, only proteins with high peptide and spectral counts have been chosen for the comparison (Table 1). Detailed spectrum and peptide report and sample of each locality are available in Additional le (S1). We identi ed different level of abundance for P. papatasi proteins, showing spot number, UniProt identi er, protein name, function annotated from BLAST similarity search (S1). All identi ed proteins of signi cance are involved with housekeeping functions, structural proteins of pathogen phlebotomine borne diseases, and entomophogenic proteins.
Our initial analysis has permitted to determine that nematode proteins detected by LC-MS/MS analysis were the most abundant with 1132 proteins identi ed, followed by Leishmania spp. and phlebovirus with 239 and 26 identi ed proteins respectively (Table 1 & Fig. 1). Identi cation of Speci c Leishmania spp. Biomarker Proteins determined by Proteomics Leishmania spp. peptides showed presence in different locality even in the non-endemic locality (NE) (Fig. 1). Comparing spectral counts for Leishmania spp. peptides from different localities studied, our results show a great signi cant difference for L. tropica proteins between different localities which is not for others Leishmania spp. By peptide group-based spectral count differentiation, the difference is signi cant (p value<0.05) ( gure 1). We describe Leishmania species determination on entomological samples based on partial sequencing of Leishmania spp. proteins such as the heat-shock protein 70 gene (hsp70), Alpha tubulin protein, Ubiquitin (Ubi) (Fig .2, Fig .3, and Fig. 4). Those proteins involved in a variety of cellular functions for Leishmania spp. We observed presence of Leishmania spp. biomarker proteins in P. papatasi in the different study sites even in the non-endemic area (Z4) (Fig .2, Fig .3, and Fig. 4). Some of interesting protein was detected is viscerotropic leishmaniasis antigen protein for L. tropica. This entity responsible for anthroponotic cutaneous leishmaniasis in Morocco [18].

Identi cation of Speci c Phlebovirus Biomarker Proteins determined by Proteomics
In regard to phlebovirus, the number of speci c proteins according to locality is lower; we did not get many hits for them. We recorded some phlebovirus proteins such Sand y fever virus (SFSV), Severe fever with Syndrome Virus (SFTSV) and Uukuniemi phlebovirus (UUKV).

Identi cation of Speci c Nematode Biomarker Proteins determined by Proteomics
Of interest, in all localities investigated during our study, we observed that nematodes and Onchoceridae proteins are the most abundant speci c biomarkers proteins in the sand ies, having high unique peptides identi ed and spectral count ( Table 1) ( Fig.6, Fig. 7). However, direct observations by light microscopy of 1752 sand ies specimens collected from all localities revealed infection of only 2 specimens with entomopathogenic nematodes and potential laria in Imintanout locality central area of Morocco (Fig. 8a  & Fig. 8b). From morphological characteristic of those specimens, they are two females of P. sergenti. According to the body feature (body length was 3400 μm and the body width 200 μm), size and egg diameter (26.4 ± 2.2 μm), the nematodes were identi ed as Tetradonematidae, Didilia species (Fig.9).

Discussion
To estimate and study the vector competence of Phlebotomus papatasi in Morocco, we performed a proteomic analysis for Moroccan entities of Leishmania spp., phleboviruses, as well as to check for the presence of entomopathogenic parasite in the different localities were the study was conducted.
Proteomics methods have rarely been applied to compare vector competence of its natural populations of sand ies. Out of the 22 sand ies species, ve species of the genus Phlebotomus are vectors of the three nosogeographic entities of leishmaniasis in Morocco: Phlebotomus papatasi proven vector of L. major [21], P. ariasi, P. longicuspis and P. perniciosus vectors of L. infantum [22], and P. sergenti proved vector of L. tropica [23]. Proteomics may improve our understanding of parasite biology and pathogenesis. On the other hand proteomic has also paved the way for the screening of pathogenic and entomopathogenic proteins of Phlebotomus papatasi. The analysis of the peptide digests of P. papatasi by LC-MS/MS resulted in a huge amount of information, and identi ed peptides with their corresponding proteins. Because peptide counts are not a reliable quantitative measure for sample comparison, we analyzed our proteomic data using the spectral count to provide a more e cient analysis [24]. Our results revealed that some speci c biomarker proteins such Hsp-70, a-tubulin and Ubiquitin of L. infantum and L. tropica proteins where present in P. papatasi. Those proteins have been investigated to permit the discrimination of medically important Leishmania species worldwide without the need for parasite isolation [25][26][27][28]. Among them, some proteins are highly conserved along the eukaryotic evolutionary tree and available on the genome sequences for several Leishmania spp. and important in genome organization of Leishmania species [29,30]. The presence of all Moroccan Leishmania nosogeographic species proteins in P. papatasi could be explained by the overlapping of three Leishmania spp. in Morocco, which renders Moroccan leishmaniasis epidemiological pro le not accurate. Effectively, L. tropica has been recorded in endemic L. major foci [31,32]. Moreover, visceral (VL) leishmaniasis cases have been found in established foci of zoonotic cutaneous leishmaniasis ZCL in Morocco [33]. Presence of viscerotropic L. tropica antigen protein could stem from the presence of zymodeme (L. tropica-279) responsible of canine VL in Morocco [34].
Moroccan populations of sand ies are not only vectors of protozoa, but also viruses. Es-Sette et al. (2015) identi ed the Toscana virus and its distribution in our country [5]. In central Morocco, Antibodies against the Naples virus and Sicilian virus have been observed in human populations living in this part of Morocco. Interestingly, antibodies of these two viruses were detected in areas where P. papatasi is present and abundant [35], raising the question of the potential incrimination of P. papatasi in the transmission of those viruses. Seemingly, our proteomic analysis for phlebovirus detection revealed a low number of identi ed peptides; for instance the polymerase SFSV protein recorded in Zagora (Z3) and Marrakech (NE). Also some emerging tick-borne virus [36][37][38][39] for instance; SFTSV glycoprotein detected in Imintanout (Z2), Zagora (Z3) and Marrakech (NE) and Errachidia (Z1), Zagora (Z3) and Marrakech (NE) for UUKV nucleocapsid protein were identi ed. Lower number of identi ed peptides could be due to RNA degradation while P. papatasi morphological identification was performed prior to proteomics analysis [40].
Unlike to what we found in our phlebovirus proteomic screening, a huge number of identi ed proteins were recorded for nematodes and Onchocercidae with a signi cant importance (Fig.1). Therefore, this nding lead us to microscopically monitor P. papatasi sand ies and other species of the different localities studied. In the past, entomoparasites of phlebotomine sand ies have been reported in the literature. In fact, Tetradonematid parasite nematodes were described to be found in the body cavity of phlebotomine sand ies in Pakistan, Saudi Arabia, Afghanistan and Portugal [41,42]. However, they never been observed in Africa until now. This nematode was reported to infect Luzomyia longipalpis in a laboratory colony maintained at the National institute of Health in Bogota, Colombia [43]. AS well as to be detected in P. sergenti and P. ariasi in Portugal [44,45]. Interestingly, Onchoceridae parasite has never been reported in sand ies. In our study the examination of 1752 sand y specimens for nematodes and Onchocercidae presence from the fourth localities studied showed two observations of P. sergenti specimens infected by nematodes in Imintanout locality, which is known to be an endemic foci of L. tropica-mediated CL in Morocco [34]. From a morphological point of view the rst nematode is a Didilia spp and the second is a potential micro laria. This is the rst observation of Tetranematid Didilia spp. in Africa and the rst report of Micro laria in sand ies.
Our results highlight two ndings, included proteomics results P. papatasi and microscopic examination of sand ies. Firstly, the vector competences of Moroccan population of sand ies are lack estimated, as we see in ours study for P. papatasi main vector of L. major in Morocco [11]. Secondly, we found a high abundance of nematodes proteins in P. papatasi and we observed the rst naturally infection by Didilia spp. and Micro laria in female P. sergenti in Morocco. These observations open questions about the role of sand ies in the transmission cycle of these parasites in Morocco.

Conclusions
In conclusion, the present study identi ed proteins for Moroccan nosogeographic entities of Leishmania, phlebovirus and nematode detected in wild caught of P. papatasi .Micro laria and Tetranematid Didilia spp., were identi ed in the same area where leishmaniasis occur. These results represent the rst use of LC-MS/MS proteomics tool for estimation of vector competence of sand ies and the rst detection of nematodes in African sand y population.
Thus, more investigations on study of vector competence of Moroccan populations of sand ies are needed.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.