Molecular Divergence in Flea Ctenocephalides Canis From West and Northwest of Iran

Abstract


Abstract Background
Fleas of the family Pulicidae are the most common ectoparasites infesting domestic livestock worldwide.
The main aim of the present study was to demonstrate the degree of molecular divergence between Ctenocephalides canis eas in the Western and Northwestern of Iran, based on nuclear and mitochondrial genes, including ITS1and ITS 2 and cytochrome c-oxidase 1 (cox1) mtDNA.

Methods
A total of 918 C. canis eas was collected. The obtained morphometric data and DNA sequencing results did not show signi cant differences between C. canis specimens from the different regions or hosts. However, there was a signi cant degree of molecular divergence among the ten populations based on nuclear markers.

Results
The degree of molecular divergence between different isolates of C. canis based on ITS1and ITS 2 genes was 0.15% and 3.36%, respectively. But analysis of the sequencing results shows that there was no molecular divergence between the ten populations based on the Cox1 marker.

Conclusions
Study of internal transcribed spacer ITS1 and ITS2 of rDNA and the partial cox1 mtDNA gene showed that these fragments are useful tools for interspeci c divergence rates, species-level differentiation and con rm the diagnosis of species C. canis.

Background
Fleas are very small insects, laterally compressed and wingless. These insects have a holometabolous development and belong to the Siphonaptera order. So far, 2574 species belonging to 16 families and 238 genera have been identi ed from these insects [16,17,5,14].
All species are parasitic in the adult stage. Some species are vectors of disease. Fleas transmit the disease in different ways including ea bites (Yersiniapestis and some viral pathogens), indirect method or fecal route (Rickettsia typhi and Bartonella henselae), and contaminated salivary glands by which Rickettsia felis is transmitted [3,25,8].
Each ea has a different role in causing health complications and transmitting diseases that will have a different transmission cycle. Some species of eas differ in susceptibility to pathogens and symbiosis, which can carry a high risk of transmitting the pathogen, so accurate identi cation of different species of eas is important. Based on recent studies in Iran and the latest available information, 117 species and subspecies have been identi ed [25, 29 [. The identi cation of different species of eas is based on morphological characteristics. However, there are still signi cant questions and concerns about the phylogenetic relationships of eas and the identi cation of appropriate molecular markers in this important group of insects [28].
Variation in morphological characteristics is observed among different species, making it di cult to identify the species correctly. Researchers have been using molecular markers of various species of eas such as ITS1 [10,2,33], ITS2 [27,33] and parts of the mitochondrial genome such as cox1 [15,24] to address their differences in molecular markers.
Ribosomal DNA (rDNA) has a very stable structure in all insects. There are ve subunits in this structure including 28s, 18s, 5.8s, ITS1 and ITS2 that can be used for separation of species [27,9,2].
In the mitochondrial genome, the cox1 fragment is the largest protein-encoding gene in the mitochondria of metazoan organisms. This part of the genome has been extensively used as a derivative of species and phylogenetic studies and intra-species differences [22,6].
The main objective of this study was to show molecular divergence between C. canis ea isolated from West and Northwest of Iran based on analysis of ITS1, ITS2, and cox1.

Study area
According to meteorological information, Iran has different climatic zones. Our study was done in The West and Northwest of Iran. In the Northwest, the winters are cold with heavy snowfall and subfreezing temperatures. Spring and fall are relatively mild, while summers are dry and hot. The western part is cold and mountainous, so the winters are cold, and the summers are hot. [23] (Fig. 1). The annual rainfall in these areas is high, and these areas are rich in vegetation and have created favorable conditions for domestic livestock breeding. Animals commonly breed in these areas include cows, sheep, goats, and horses.

Flea sampling and identi cation
Adult eas were collected from ve provinces (two cities in each province) in the West and Northwest of Iran, including Kermanshah, Kordestan, Hamedan, Lorestan, and West Azerbaijan. Samples were collected using light traps, human baited traps, direct separation of eas from the host body, and collection of eas from home and animal farms. Totally out of 1937 were isolated, and 918 of them were C. canis.
After collection, samples were stored in 70% ethanol and transferred to the laboratory of the Faculty of Veterinary Medicine, Urmia University, Iran. Fleas were identi ed under a stereomicroscope according to identi cation keys [1].
DNA isolation, PCR ampli cation, and DNA sequencing Total DNA was extracted from the individual ea using a DNA extraction kit (MBST, Tehran, Iran) and following the manufacturer instructions. The extracted DNA samples were stored in sterile microtubes at -20 °C until use. The primers used by Vobis et al. (2004) [27] were applied to identify ea's C. canis. These primers were speci c to identify ITS1 and ITS2 (Table 1). Primers used by Lawrence et al. (2014) [15] were used to amplify the fragment of the mitochondrial gene cox1 of C. canis (table 1).
PCR analysis performed in 50 µL total volume including10 ng of DNA, 10x PCR buffer, 1.25 U Taq Polymerase, 1 µL of each primer (20 µM), 1 µL dNTP, (100 µM), 0.75 mM MgCl2 (50mM) all materials obtained from Sinaclon, Iran. Program to perform PRC reaction for fragments ITS1 and ITS2 including 5 min incubation at 94°C to denature double-stranded DNA, followed by 30 cycles of 1min at 94°C (denaturation), 1 min at 54°C (annealing) and 1min at 72°C (extension) and an additional extension step at 72°C for 5 min these steps. The PCR program was also set up to replicate the cox1 fragment 95 •C for 5 min, followed by 35 cycles of denaturation at 95 •C for 1min; annealing at 55 •C for 45 s and extension at 72 •C for 45 s; with a nal extension step at 72 •C for 10. The PCR products were electrophoresed on 1.5% agarose gel and then stained with Safe stain and visualized under the light. The PCR products were then puri ed for sequencing. The phylogenetic tree was plotted using MEGA 6 software based on the maximum likelihood method with bootstrap 1,000(1,000 replicates).

Results
Out of 1937, ea were collected from humans, sheep, dogs, goats, home, and animal farms (table 2) from which 918 C. canis were isolated (Fig. 2).
The sequencing length for the ITS1 and ITS2 genomes in the study sites were 800bp and 500bp, respectively. When the ITS1 sequence in C. canis was compared in ten different regions with the CLUSTAL O software, the sequences were 99.85% similar, and the degree of molecular divergence was 0.15% (Fig. 5). But after comparing the ITS2 gene sequences in the ten studied regions, the molecular divergence between C. canis based on ITS2 gene was 3.36%. (Fig. 6).
Comparison of cox1 sequencing from the nuclear genome (mtDNA) in ve provinces (accession number: MN173762-771) showed that the sequences were 100% similar and all species belong to C. canis species (Fig. 7).

Discussion
Using molecular methods in the identi cation of ectoparasites has enabled the identi cation and evolution of species with high morphological similarities [18]. The present study provided morphometric, phylogenetic, and molecular divergence comparative data between eas C. canis isolated from Western and Northwestern Iran. The morphological characteristics of family Pulicidae eas in this study were in agreement with previous studies [29,21,25,12,4,7].
The host speci city might affect the level of intraspeci c genetic divergence because generalist parasite species will show a higher level of intraspeci c genetic variation, enabling them to infest a broader host range [26]. Hornok et al. (2018) [11]. studying mitochondrial sequences demonstrated divergence in some synanthropic ea species such as C.felis and Pulex irritans. In our study, morphological data indicated that there was no difference between C. canis specimens from Western and Northwestern of Iran. The result of the morphological study was in line with the results of the molecular study.  [33] showed no signi cant difference between the morphological data of P. irritans in Spain and Argentina. There was a considerable degree of intraspeci c similarity between both populations based on mitochondrial genes. Our results agree the studies of Hornok et al. (2018), [11] who observed no morphological differences between human and wild carnivorous P. irritans specimens in Hungary and Croatia. In contrast, the studies of Krasnov et al. (2015) [13] showed that eas species isolated from different hosts in different geographic regions have morphological differences that can indicate a high level of genetic diversity. We concluded that the cox1 region is a useful marker to approach intraspeci c similarity in C. canis and con rming its diagnosis.
A phylogenetic tree based on the similarity between our sequences with registered sequences in GenBank showed 2 subclades for C. canis: one subclade including C. canis of recent study and C. canis from Turkey and the other with C. canis of Iran, Urmia and, Iran, Makoo. C. felis, C. orientis and P. irritans were in separate clades (Fig. 8).
Studies have shown that ITS1 and ITS2 are one of the best molecular markers for analyzing phylogenetic relationships at the species level in eas [27,19]. In our study, a comparison of nucleotide sequences of the ITS1 gene in ve different provinces showed 99.85% similarity. Comparison of nucleotide sequences also showed single-nucleotide transversion at position 294, which caused the substitution of adenine for thymine in the isolate from Kuhdasht, Urmia, and Kermanshah. This result is in agreement with the results of Ghawami et al. (2018), [10] which compared the nucleotide sequence of the ITS1 gene in P. irritans in two different geographic regions and showed only one nucleotide difference with 99.85% similarity. Our results are similar to Vobis et al. (2004) study, which observed that the nucleotide sequence of the ITS1 gene was relatively constant in different populations of C. felis. Therefore, it can be concluded that ITS1 is a useful marker for the diagnosis of Ctenocephalides genus.
A phylogenetic tree based on ITS1 registered sequences in GenBank showed 2 subclades for C. canis: one subclade including C. canis isolated in Kamiyaran, Sanandaj, Mahabad, Ghilangharb, Hamedan, Bahar and Khorramabad and the other C.canis which was isolated in Kuhdasht, Urmia, and Kermanshah. P. irritans and C. felis were in separate clades (Fig. 9).
The ITS2 nucleotide sequence in different sampled regions showed 96.64% similarity. There are ve isolates found.
The results of the current study agree with the survey by Zurita et al. (2015), [30] which showed intraspeci c variation in four clones of C. canis based on the ITS2 fragment is between 90.1% and 100%. Vobis et al. (2004) also used the ITS2 sequence to illustrate intra-species differences and identify different species of eas.
A phylogenetic tree based on ITS2 in the present study showed 4 subclades for C. canis: one subclade including C. canis isolate in Kuhdasht, Mahabad, Urmia, Khorramabad and Kermanshah, next subclade including isolate in Kamiyaran, Sanandaj, and Bahar. The isolates of Ghilangharb and Hamedan were in separate subclades. P. irritans were in a separate clade, and C. felis were in separate subclades (Fig. 10).

Conclusion
In conclusion, we have found that the morphological characteristics in C. canis are consistent with the molecular ndings. The analysis of the cox 1 partial gene is a useful tool to assess intraspeci c similarity and con rm the diagnosis of species C. canis. ITS1 and ITS2 sequences could be used as useful markers for species-level differentiation and interspeci c divergence rates. As a result, the Cox1 gene is more useful than the ITS1 and ITS2 genes in detecting genus and species and showing intra-species similarity