Phlebotomus (Adlerius) kabulensis (Diptera: Psychodidae) a new record sand fly species from Iran: Morphological and molecular aspects

Alireza Zahraei-Ramazani, Abedin Saghafipour, Yavar Rassi

1Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

2Department of Public Health, Faculty of Health, Qom University of Medical Sciences, Qom, Iran

1. Introduction

Phlebotomine sand fly’s species recorded in Iran belong to the genera,PhlebotomusRondani and Berté 1840, andSergentomyiaFranca and Parrot 1920[1]. The genusPhlebotomushas 12 subgenera[2]. Among them, the species of subgenusAdleriusNitzulescu, in 1931 were reported to be the vectors of leishmaniasis and since then, a few studies have been carried out on this subgenus in Iran. Recently, eight species belonging to this subgenus: (i)Phlebotomus(Adlerius)balcanicusTheodor, 1958;(ii)P.(Adl.)brevisTheodor and Mesghali, 1964; (iii)P.(Adl.)comatusArtemiev 1978; (iv)P.(Adl.)halepensisTheodor, 1958;(v)P.(Adl.)kabulensisArtemiev, 1978; (vi)P.(Adl.)longiductusParrot, 1928; (vii)P.(Adl.)turanicusArtemiev, 1974 and (viii)P.(Adl.) salangensisArtemiev, 1978 have been reported in Iran[3-5].Phlebotomus chinensisNewstead, 1916 is the type-species of this subgenus[6] (Artemiev, 1980), the vector ofLeishmania infantum(L.infantum) in China[7]. The Phlebotomine sand flies of the subgenusAdleriusinvolved have seldom been identified, because despite the taxonomic efforts of Artemiev (1980) and others, the morphological characters have not clearly been distinguished between the females of about 20Adleriusspecies[8]. In addition, identification of maleAdleriusby morphological means replace was difficult by remains a difficult task. They differed slightly because some morphological characters were closely related, for instance the location and number of hairs on coxite are the most important morphological characteristic features in identifying and distinguishingAdleriusspecies from others. At times, it is important to identify the number and location of hairs on the body of different species of sand flies to check how similar or different they are. According to morphological keys, total number of coxite hairs inP. kabulensisare between 27-50 and inP. longiductusthe hairs on coxite are 50-85; therefore, when a specimen collected has 50 hairs on its coxite, it is difficult to say which of the two species it belongs to. Also, the antennae of sand fly is the first morphological character; in preparing morphological slides careful handling and care are needed during collection and mounting because of their minute and delicate body parts. The antennae usually break and disappear. Without the antennae, species identification becomes difficult or impossible. Also, the females of the subgenusAdleriusdo not have morphological keys for species identification and the inclusion of its species can be justified on the basis of characters of the males’ morphology[9,10], Because morphological characters do not distinguish between species of theAdleriussubgenus, it is important to thorough revise the identification key based on the morphological characters. Additionally, access to modern molecular techniques and genetic information regarding these vectors and detailed classification of their systematic positions,it seems far too ambitious to state that genetic information will help to elucidate the biological, morphological, ecological and effective leishmaniasis control methods. In this study, we compared the morphological and morphometric characteristics ofP. kabulensismale specimens and also extracted the sequence of cytochrome b (Cyt b) [Mitochondrial DNA (mtDNA)] gene ofP. kabulensissand fly in order to identify and distinguish females of this species in subsequent studies. Also, we compared the 68 sequences with that of species in the GenBank. This gene is normally 69 inherited maternally and had been used to relate geographical populations of many species of 70Larroussiusin the Mediterranean subregion[11,12]. The aim of this study was to detectP. kabulensisfrom other Adlerus species regarding its morphometric characters and molecular sequencing.

2. Materials and methods

2.1. Study area

Sticky paper traps were used for collectingP. kabulensisspecimens in mountainous areas with vegetation coverage (e.g., gardens,mountainous caves, reservoir hosts, trees, river side and stones) in outdoor places of Ilam province of Iran between 5 p.m. and 7 a.m.(33°38′14″ N, 46°25′21″ E and altitude: 1 381 m above sea level),Lorestan (33.4871° N, 48.3538° E and altitude: 4 050 m above sea level) and Esfahan (32°39′25″ N, 51°40′39″ E and altitude: 1 571 m above sea level) provinces. In Esfahan province, sand flies were collected in August 2016. In Lorestan and Ilam provinces, July 2016 was the time of collection.

2.2. Monitoring sand flies

Sand fly specimens were preserved in glass containers with 96%ethanol. Sand flies were washed twice with distilled water. The head as well as the 4thand the 5thends alongside the genitalia of the male sand fly were mounted separately on every slide and two drops of puri’s medium were added and the cover slip was gently put on the specimen[10]. These slides of male sand flies were prepared for morphological identification and the morphometric measurements using standard keys[10,13-15]. Eight characters were measured on the 13P. kabulensisspecimens in order to know if there was any difference between these specimens in the three provinces. The morphometric measurements were done using ocular micrometer and Olympus Microscope (ch-2).

2.3. Statistical analysis

Begin sentence with difference of morphological measurements were carried out with ANOVA and Kruskal-Wallis test with a priori level of significance set atP＜0.05. Given that statistical tests were significantly different, post Hoc-Bonferroni test was used[16]. For the molecular description and identification ofP. kabulensisfemales by pair-wise alignment comparison in subsequent studies, the authors used DNA sequence of the males for thecytb-mtDNA gene. The remaining sections of the abdomen, wings and legs of the sand fly were preserved in 96% ethanol in 1.5 mL sterile micro tubes for DNA extraction. The sequences were compared with sequences available in GenBank using BLAST available on http://blast.ncbi.nlm.nih.gov.

3. Results

In total of 46 specimens ofP. kabulensiswere collected, of which 14 specimens were from Esfahan, 15 from Lorestan and 17 were from Ilam provinces. The main morphological characters of subgenusAdleriuswere: the coxite has no lobe, the style bears five spines, and the paramere was not truncated and carries no ventral process. The most aedeagus form has a sub-terminal minute finlike barb. The female’s spermatheca was incompletely segmented.In addition, the morphological description of maleP. kabulensiswas; presence of one ascoid on antennal segments 9-15, antenna 8 with two ascoids, aedeagus with normal obtuse-angled sub-terminal notch, a group of 27-50 hairs on coxite with dark body,sub terminal tooth in aedeagus is 12 μm, coxite is moderately wide, distal border of the hairy spot is about 0.50% of the coxite and genital filament/genital pump was seven (Figures 1 and 2).Morphometric measurements among the specimens ofP. kabulensisin the three provinces showed there was no significant difference between (i) the length of A3, (ii) the length of epipharynx, the length of surstyles, (iv) the length of styles, (v) the width of styles, (vi) the length of aedeagus, and (vii) the number of hairs on coxite (Table 1). Therefore, this indicates theP. kabulensisspecimens in the three provinces were within the same population or there was adequate gene flow to keep the groups similar. But there was a significant difference between the lengths of the coxite of the specimens from the three provinces (P= 0.045). Post Hoc-Bonferroni test shows that the length of coxite ofP. kabulensisspecimens in Lorestan province was significantly more than that from Ilam province (Table 2). This showed that they were not morphologically similar judging from the length of the coxite. In the conduct of the molecular experiment,DNA was extracted from 12 specimens ofP. kabulensissand fly from which five were used for mtDNA-PCR. PCR product of a male specimen was sent to the Department of Genetics, Faculty of Health Tehran University of Medical Sciences (TUMS) for sequencing. In this study, PCR experiments forCyt bgene, primers CB3-PDR and N1N-PDR were used. These primers amplified a fragment of 550 bp of the mitochondrial genome of Males specimens which were captured from mountainous areas with vegetation within Poldokhtar Township in Lorestan province. This was the first time the sequenced result ofP. kabulensishas been submitted to the GenBank from Iran(ACCESSION NO: JX885994). The comparison alignment of the 528 nucleotides with the specimens in the GenBank using NCBIBLAST software showed that was 87% similarity with P. (Adl.)chinensiswith the accession number: HM747243.1 and E-value 2e-174 (Figure 3). TheP. kabulensisspecimens have been deposited in the corresponding author’s medical entomology laboratory in Tehran University of Medical Sciences, Iran.

Table 2Post Hoc-Bonferroni test results showing comparison of the differences of the length of coxite of P. kabulensis specimens.

Figure 1. Male P. (Adl.) kabulensis Artemiev, 1978.

Figure 2. Drawing of coxite and dense group of hairs in P. (Adl.) kabulensis Artemiev, 1978.

Table 1Samples used in morphometric study of P. (Adl.) kabulensis specimens in Iran.

Figure 3. Sequence comparison of cytochrome b (Cyt b)-mitochondrial DNA between P. (Adl.) kabulensis specimen (Query, with a molecular number of LPM5) and Phlebotomus chinensis haplotype CYTB-10 (Sbjct, accession no.HM747243.1; partial cds; mitochondrial) in the GenBank.

4. Discussion

ThePhlebotomusgenus entails all known medically important species in the Old World, including incriminated vectors of mammalianLeishmaniaspecies and of sand fly fever serogroup of arboviruses[17,18]. This genus included 12 subgenera andAdleriusNitzulescu (1931) is one of the important subgenus in the Old World as it contains the species of leishmaniasis vectors[7]. It is a Central Asian species and it is possible that a number of Central Asiatic species have entered Afghanistan and Iran from central Asia. Artemiev (1978) revised the subgenusAdleriusspecimens of Afghanistan. He capturedP. (Adl.) kabulensisArtemiev (1978) which is found in dwellings and is rather thermophilic and hydrophilic.Since this species belongs to the subgenusAdleriusgroups; he said, perhaps it can transfer visceral leishmaniasis agents like other species of this subgenus. But so far this has not been reported.Generally, there are five factors that all must be satisfied for vector incrimination: finding parasite in wild specimens, transmission from host to vector (in the laboratory), transmission from vector to vertebrate (Lab.), correlation of vector presence with disease outbreak, and laboratory confirmation that life cycle of parasite can be maintained in the purported vector. Although finding the parasite in collected specimens from the wild does not necessarily indicate that the species is a vector. Before the present study, known sand fly species of the subgenusAdleriusfauna from Iran were four species and this has been confirmed by senior sand fly specialists[1,10].Currently, there are eight species of the subgenusAdleriusin Iran.So, it can be mentioned that through further field studies, one can discover a greater number of new records or even newer species due to the large area of the country. During the conduct of this study across three provinces (Esfahan, Lorestan and Ilam) in Iran,P. kabulensiswas collected in outdoor places situated far from human dwellings.These three provinces are located on the visceral leishmaniasis areas of the foothills to high altitude of the Zagros mountain range in the country and this geographical climate is similar to the dwellings of this species in Afghanistan. However, previous field surveys have given evidence of the subgenus’s anthropophilic nature which is usual in human houses, cattle sheds and gardens[14], but the researchers collected all of them outside human dwellings. The collections were made by sticky paper traps but testing by other methods like CDC light traps may be more efficiency for this species. TheP. kabulensishas not been naturally and/or experimentally proven as vector of leishmaniasis; however, it is important to note that its occurrence alongside known and proven vectors inL. infantumendemic regions enhances the risk of transmission. Nevertheless, theP. kabulensisof the subgeneraAdlerius, which includes the potential vectors of Mediterranean kala-azar, suggests that the role of this species should be given serious attention in Iran. In this country,P. kabulensisis very uncommon and even the few that are present show little considerable variation in morphological characters. In this study,P. kabulensisspecies of sand flies were collected at low densities and in small numbers together with other local vectors; therefore,presently, there is little information available regarding its biology and ecology. Ecologically, the successful completion of the life cycle ofL. infantumdepends entirely on the number of efficient vector specimens. Based on the observed densities in nature,P. kabulensiscannot restrictL. infantumlife cycle to itself. Notwithstanding, we cannot rule out the possibility thatP. kabulensisplays a role in the transmission ofL. infantum, as in cases of canine leishmaniasis.Sand fly taxonomy in most cases is based on morphometric means and is either measurable or countable characters. In this present study, length of the 3rdsegment of antennae, length of style, length of epipharynx, length of coxite, width of style, length of substyle,length of aedeagus and number of hairs on coxite, eight taxonomic characters in all were analyzed. Aside these characteristic features,other measurable characters were illustrated in order to describe the morphology of theP. kabulensis. Some taxonomic characters ofP. kabulensisfrom Iran have shown considerable morphological similarities such as the sub-terminal tooth in aedeagus, Filament/Pump and the hairs on coxite, having been compared with the published data of this species from Afghanistan[14]. The mean and standard deviation of eight morphometric characters of the species aided the researchers in the field of ecological study and control of the vectors. DNA sequences alone can be used for species identification. Previous studies have been showed that the PCR technique is an appropriate tool for detecting sand flies species from others[19-21].P. kabulensiswas studied and identified by morphological, morphometric and molecular characterization. The present study suggests a sand fly survey in Iran. Initial DNA analysis has shown how distinct this species is. More entomological intensive studies are needed in order to discover its distribution and abundance as well as further molecular comparisons to effectively control this species in Iran. This is because of its similarity with the female specimen ofAdleriusgroup sand flies in morphological characters and the morphological identification of the females ofP. kabulensissand fly is impossible too. So, by extracting and sequencing the DNA of the males ofP. kabulensissand fly, we can identify the female of this species by comparing their sequences in the future studies. It is feasible that the populations share vector competence. The natural or experimental infection of theP. kabulensiswithLeishmaniaparasites thus seems attractive for future testing.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgment

Authors express their sincerest appreciation to the Mr. Amrolah Azarm for drawing the tables and set-out figures for publishing.This study was sponsored by the Deputy of Research Affairs, Tehran University of Medical Sciences with project number: 5146-27-01-86.

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