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Molecular and Morphological Identification of Local Sand Fly Species (Diptera: Psychodidae) in Saudi Arabia.

Byline: Reem A. Al-Ajmi, Tahany H. Ayaad, Mashael Al-Enazi and Ahmed A. Al-Qahtani


The identification of sand fly species is important for prediction of disease risks. A method of typing Sergentomyia and Phlebotomus taxa in three areas in Saudi Arabia was investigated by establishing a polymerase chain reaction (PCR) and direct partial sequence of 18S ribosomal RNA (rRNA) gene using specific designed primers. Morphological investigation of adults sand fly showed that Phlebotomine flies are identified into five species, three of them belong to genus Phlebotomus (P. papatasi, P. bergeroti, P. sergenti), and two belong to genus Sergentomyia (S. antennata and S. clydei). Several direct PCR - amplified sequences of each species analyzed using Maximum Composite Likelihood (MCL), Neighbor-Joining (NJ) and MEGA 5 methods revealed phylogenetic relationships of taxa. The obtained data of 18S subunits of rRNA gene showed moderate degree of interspecific variability between species of the same genus and among species of different genera.

These results provide a powerful tool for further molecular retyping of Phlebotomus and Sergentomyia species in endemic areas of Saudi Arabia which may help planning for appropriate epidemiological surveillance programs that could be used to detect natural infections of sand fly vectors with pathogens, and hence, designing the appropriate control measure that limit spreading of such serious vectors.

Key Words: Sergentomyia, Phlebotomus, PCR, direct sequencing, sand fly, molecular and morphological characteristics, 18S rRNA, Saudi Arabia.


Phlebotomine sand flies (Diptera: Psychodidae) are small-sized blood sucking insects feeding on a wide range of hosts and potentially acting as vectors of pathogens responsible for human and animal diseases worldwide. Out of over 800 sand fly species that have been described to date, approximately 10% are proven or suspected vectors of bacteria (Bartonella bacilliformis), viruses (Phlebovirus, Vesiculvirus) as well as Leishmania spp. protozoa, the causative agent of leishmaniasis (Yahia et al., 2004; Kato et al., 2008; Depaquit et al., 2009; Kato et al., 2010). It is suggested that only a restricted number of specific sand fly species can support the development and transmission of specific species of Leishmania (Munstermann, 2004; Kato et al., 2007). P. papatasi is the most prevalent vector of Leishmania major, the causative agent of zoonotic Cutaneous leishmaniasis in North Africa and Middle East (Lane and Crosskey, 1993).

It has been also implicated in the transmission of Phlebovirus viruses in Europe (Depaquit et al., 2009). The occurrence of sand fly species in a given region may represent a risk for L. major transmission or even the introduction of a non endemic parasite species from overseas into the Saudi Arabian regions provided that the reservoir host for such species is present in the area. Thus, identifications of both sand fly and Leishmania spp. are of great importance for predicting expansion of the disease in endemic areas, and also help in designing new strategic programs that limit spreading of such serious vectors (Kato et al., 2007; Fujita et al., 2012).

Recently both L. major and L. tropica from Phlebotomus papatasi and Phlebotomus sergenti, respectively are identified using the semi-nested PCR method against kDNA and the Internal Transcribed Spacer 1 Polymerase Chain Reaction Restriction Fragment Length Polymorphism (ITS1-PCR-RFLP) in Al-madinah Al-munawarah province of Saudi Arabia (El-Beshbishy et al., 2013).

The most abundant phylogenetic analysis of morphological characters led to the conclusion that the Subfamily Phlebotominae comprises six genera: Phlebotomus, Sergentomyia and Chinius in the Old World and Lutzomyia, Brumptomyia and Warileya in the New World (Lane and Crosskey, 1993). Six subgenera of the genus Phlebotomus (Phlebotomus, Paraphlebotomus, Synphlebotomus, Euphlebotomus, Adlerius, Larroussius) and more than 90 species were identified depending on morphological criteria including terminal genitalia of males and internal structures of females, such as spermatheca and cibarium and also pharynx in the head region (Belkaid et al., 2000 ; Lango, 2005; Singh and Philips-Singh, 2010).

This traditional method of identification requires refined storage conditions for samples, highly skilled techniques, taxonomic expertise, presence of intraspecific variability (Aslamkhan and Aslamkhan, 2000 ; Kuwahara et al., 2009), time consuming (Mukhopadhyay et al., 2000; Barroso et al., 2007) as well as expert analysis of cryptic species (Bauzer et al., 2007). Thus, the development of alternative molecular data sets have been introduced only recently as powerful tools for the identification of sand flies is an important issue. Modern techniques that are based on DNA identification of sand flies are generally not limited to specific developmental stages or to specific sex but also extend to intraspecific variability (Collins and Paskewitz, 1996).

Of these techniques, the ribosomal RNA (rRNA) gene architecture and the highly conserved sequences of certain domains of the gene have been used to reevaluate higher level relationships within the family Phlebotominae and within genera Phlebotomus and Sergentomyia (Depaquit et al., 1988; Aransay et al., 2000). The rRNA gene is a multi-copy gene of tandem repeated transcription units. Each transcription unit includes regions that produce three of the major rRNA subuints in insects (18S, 5.8S and 28S) after processing (Burton, 2008). The transcribed unit, which vary in copy number in different insect species, are highly similar to each other in a particular individual, although not necessarily identical.

The 18S rRNA subunit was used to identify and differentiate between several sand fly species based on RFLP-PCR. Additionally, studying the18S rRNA subunit sequence of sand fly species was investigated by Fujita et al. (2012) to differentiate between 13 sand fly species in Peruvian Andes. Also the sequence of 18S rRNA gene was used to identify different Lutzomyia species captured in different areas in Ecuador (Terayama et al., 2008).

The current study was conducted to assess the appropriate genetic diversity among phlebotomine sand fly species collected from three different regions in Saudi Arabia including Riyadh, Al-Madinah Al-Munawarah and Assir provinces, and comparing their phylogenetic relationships with those available and registered in the GeneBank.


Sand flies collection and identification

Sand fly adults of both female and male (n=250) were collected during the year 2012 from field localities including Riyadh, Al-Madinah Al-Munawarah and Assir (Table I) the endemic areas of Cutaneous leishmaniasis, the most prevalent type of leishmaniasis in Saudi Arabia that is caused by L. major and L. tropica. Flies populations were analyzed and identified morphologically according to Killick-Kendrick et al. (1985) and Al-Dawood et al. (2004).

Immediately after sand fly collection using sticky and light traps, specimens were preserved in 70% ethanol. Each fly was dissected into two parts; the first part consists of the head and terminalia and used for morphological identification according to the morphological taxonomic keys of (Alves et al., 2008; Singh and Philips-Singh, 2010). While the other body part was stored at -80C and used for the molecular identification.

DNA extraction

Individual ethanol-fixed specimens were homogenized and lysed using a DNA extraction kit (DNeasy tissue kit, Qiagen, California, USA) following manufacturer's instructions. Then 5ul portions of the DNA extracts were subjected to the PCR gene amplification.

PCR - gene amplification and sequencing

Several sequences from GeneBank were used as references to design primers binding to highly conserved sequences within the 18S ribosomal RNA gene and flanking a short and highly variable region to discriminate between the five species for which sequence data were undertaken in this study. Primers SandF1: 5'-AGGCTCATTCAGTCGCTTTC-3'and SandR1: 5'-TGCAAGCTTATGACTCACACTT-3' were designed to bind within highly conserved regions. A region of 540 bp was amplified, of which a central 200 bp portion was predicted to be diagnostic. Polymerase chain reactions (PCRs) were performed in a final volume of 50ul which includes 5ul PCR buffer, 7ul MgCl2, 10ul Q solution, 1ul dNTPs, 0.5ul Taq polymerase, 1.25ul forward, 1.25ul reverse primers, 19ul distilled water and 5ul DNA.

PCR conditions (PCR palm cycler R Corbett research, USA) were as follows: 40 cycles: denaturation at 94C for 1min, annealing at 55C for 1min, extension at 72C for 1min and final extension at 72C for 7min. Analysis of PCR products. Five micro liters of PCR products were analyzed using electrophoresis on 2% agarose/1X TAE buffer gels in the presence of 0.5 ug/ml ethidium bromide and visualized under UV transilluminator then submitted directly for sequencing with the same F1 and R1 primers using the sequencing kit, Big Dye terminator V3.1 cycle (cycle sequencing kit, Applera, Foster city, California, USA). Sequences were analyzed using ABI 3700 DNA analyzer (Applied Biosystem, Foster city, California, USA) (Aransay et al., 1999). Direct sequence editing and assembly were performed using ChromasPro. MEGA 5 program and Neighbor-Joining (NJ) methods with bootstrap values of 1000 replicates.

The consensus sequences for the Saudi Arabian species of the genus Phlebotomous (P. papatasi, P. bergeroti and P. sergenti) and genus Sergentomyia (S. antennata and S. clydei) have been deposited in GeneBank, with accession numbers JQ920354, JQ929127, JQ929126, JQ929128 and JQ929125, respectively.

Data analysis

The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (-716.9640) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach (Kato et al., 2008) and then selecting the topology with superior log likelihood value. There were a total of 384 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).


Morphological results

All specimens were identified morphologically according to the keys of Killick-Kendrick et al. (1985) and Al-Dawood et al. (2004) into 5 species belonging to two genera, Phlebotomus and Sergentomyia.

Genus Phlebotomus

Of the collected specimens 178 were identified belonging to genus Phlebotomus, where all individuals were characterized by having hairs with round sockets, unarmed cibarium and male style carrying four to five spines. Phlebotomus (Phlebotomus) papatasi was the most abundant species (52.8%) collected from all the studied areas. Phlebotomus (Phlebotomus) bergeroti constituted 14.4% of the total collected sand flies. Ten specimens were identified as Phlebotomus (Paraphlebotomus) sergenti. Sergentomyia (Sergentomyia) antennata represented about 8% of the total collected sand flies. Sergentomyia (Sintonius) clydei accounted for 20.8% of the total collected sand flies.

The PCR amplified products showed bands of variable molecular weights ranging from 480-550bp for each of the random chosen individuals (Fig. 1).

PCR-amplicons of several direct sequences of 18S subunit of rRNA for each species that are submitted in GeneBank under the accession numbers: JQ929125, JQ929126, JQ929127, JQ929128 and JQ920354 for P. papatasi, P. Bergeroti, P. sergenti, S. antennata and S. clydei, respectively.

Analysis of individual sequences belonging to P. papatasi species showed little differences in the 18S rRNA subunit among the randomly chosen twelve individual sequences and found to match to the GeneBank reference AJ244414.1 only in the region between nucleotides 1 to 20. However, significant variations were observed among individuals belonging to P. sergenti and P. bergeroti. Most of these variations were detected in the region of nucleotides from 1 to 45 compared to the reference sequence AJ244405.1 or those of nucleotides in the range from 304 to 465 as compared to the GeneBank reference P. bergeroti.1 sequence. The calculated inter species variations of the genus Phlebotomus (P. papatasi, P. bergeroti and P. sergenti) ranged from 3.6 to 4.1%. However, these variations were significantly higher when species belonging to genus Sergentomyia (S. clydei and S. antennata) compared to each other, reaching up to 7.6%.

These sequences were used for construction of phylogentic trees using ChromasPro. MEGA 5 program and Neighbor-Joining (NJ) and Maximum Likelihood methods. Phylogenetic trees (Figs. 2, 3) revealed moderate degrees of interspecific variability between species of the same genus and among species of different genera.

Since no reference gene sequence was available in the GenBank database, the partial sequence of 18S rRNA of a randomized individual from the present work was chosen and used as a reference sequence for the other eleven corresponding ones.


The morphological classification requires considerable skill as well as taxonomic expertise. In addition, the presence of intraspecific variation and cryptic species frequently complicates classifications based on morphological features (Bauzer et al., 2007). Sometimes damage caused by improper storage makes the identification more difficult. Molecular characteristic markers therefore, have been explored in the present investigation for the development of simpler and more accurate techniques to confirm and retype the morphologically identified sand flies.

The adult morphological features confirmed that the identified phlebotomine sand flies include P. papatasi, P. bergeroti, P. sergenti, S. sergenti and S. clydei species (Young and Duncan, 1994; Munstermann, 2004).

Genetic variability based on 18S rRNA in the present work was highly significant among individuals belonging to the genus Sergentomyia (7.6%). However, insects belonging to the genus Phlebotoumus showed very little specific variability (3.6-4.1%). Individuals related to P. sergenti species showed more intra-individual differences than other species. Similarly 12S and 18S rRNA genes have been analyzed to study the phylogeny of some Lutzomyia species (Beati et al., 2004). Moreover, the discrepancies in the classification of some Lutzomyia species between generally accepted morphologic groupings and the phylogentic relationships of the sequence of 18S rRNA gene have been investigated by Terayama et al. (2008). It has also been previously proven that 18S rRNA gene of arthropods contains some of the slowest evolving sequences (conserved) in living organisms and therefore, used for examining ancient evolutionary phylogenetic status (Hill and Dixon, 1991; McClintock et al., 1991).

The complete sequence of rRNA genes of Drosophila melanogaster (Tautz et al., 1988) and Phlebotomine sand flies (Aransay et al., 2000) have also been investigated for interpretation of the phylogenetic relationships.

The genotyping method has been shown to be more accurate and easy-to-use for identification of sand fly species, requiring less expertise and less risk of different interpretations than the conventional morphology-based classification. Besides that damage to samples, which affects the morphologic classification in many cases, does not affect the genotyping analysis (Lane and Crosskey, 1993; Barroso et al., 2007; Terayama et al., 2008).

Additionally, the phylongenetic tree of different Phlebotomine species based on Neighbor-Joining (NJ) and Maximum Likelihood methods included in the present investigation (Figs. 2, 3) shows that each species is much related to the same species reported as reference species in the GenBank.


In conclusion, the sequence analysis of the 18S region of rRNA gene is a valuable tool in studying inter-species variability in organisms related to the genus Sergentomyia. However, it is not suggested in studying the intra-species variability among different sand fly species.


This research project was supported by a grant from the "Research Center of the Female Scientific and Medical Colleges", Deanship of Scientific Research, King Saud University, Riyadh. The authors would also like to express their thanks to Dr. Ghada H. Badr, an Assistant Professor and the coordinator for the BioInformatics Research Group (BioInG) at KSU and Mrs. Hessah A. Alraqibah, a Lecturer and member in the same group, for their help in data analysis and construction of phylogenetic trees.


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Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:7SAUD
Date:Dec 31, 2015
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