Molecular identification and phylogenetic relationship of Demodex mites based on mitochondrial 16S rDNA.

BACKGROUND AND
OBJECTIVE: Demodex mites are tiny parasites that live around hair follicles of mammals. The two main species of Demodex i.e. Demodex folliculorum and Demodex brevis present in humans are found near the hair follicles of eyes. The present study was to understand the presence of Demodex mites in people suffering from blepharitis in Amritsar, Punjab.
MATERIAL AND METHODS: Demodex mites samples present in blepharitis patients were isolated from the eyelashes. DNA was isolated from three mites and used for PCR amplification of mitochondrial (mt) 16S rDNA. The amplified PCR product were purified and used for molecular identification.
RESULTS: The amplified mt16s rDNA product was sequenced and subjected to BLAST search in the NCBI database for molecular identification. The identified mite belongs to Demodex folliculorum species. The phylogenetic tree constructed by using mt16s rDNA sequence suggests that D. folliculorum is closer to D. canis than to D. brevis.
CONCLUSION: All the three isolates belong to D. folliculorum and the mitochondrial DNA 16S rDNA partial sequence is applicable for phylogenetic relationship analysis. Copyright:

INTRODUCTION

Demodex mites belong to the class: Arachnida; order: Prostigmata; family: Demodicidae; and genus: Demodex.[1] Simon first described the genus Demodex in 1842. Since then a total of 140 Demodex species or subspecies have been identified. Demodex mites generally infest hair follicles, sebaceous glands, meibomian glands, ceruminous glands, and internal organs of 11 orders of mammals, including dog, sheep, cat, pig, and mouse. It is generally considered that Demodex is a host-specific obligate parasite. Two or more Demodex species might simultaneously parasitize the same host.[2] Cross infection between humans and animals has also been reported.[34]

The traditional classification of the Demodex species mainly depends on the morphological characteristics or the phenotype, but it has certain limitations that induce certain difficulties and indeterminacy.[1] Therefore, it is necessary to study the Demodex mites phylogenetic relationship at the molecular level. A research on the Demodex mites has been limited by the three factors. First, less attention has been paid to Demodex because its pathogenicity is not certain. Second, DNA extraction of Demodex is troublesome as mites are tiny with thick chitin which is difficult to rupture. Third, it is difficult to obtain a large number of standard samples of mites because these mites are species specific to their host, and it cannot be maintained or cultured in vitro.[567]

The increased number of studies have shown a strong association of Demodex infestation with various dermatitis and ocular problems such as rosacea, blepharitis, and acne.[689101112] Therefore, interest in Demodex has been raised, and studies at molecular level are increasing.[1314]

Various molecular techniques have been applied for the classification and phylogenetic studies of the Demodex mites. Zhou and Cheng[515] analyzed the polymorphism of Demodex folliculorum and Demodex brevis by the random amplified polymorphic method (RAPD). Toops et al.[16] isolated genomic DNA from Demodex canis and suggested that the Demodex sequences obtained through oligonucleotide primer amplification can be useful in analyzing the phylogenetic relationship and disease progression. Ravera et al.[17] detected D. canis DNA 166 bp chitin synthase gene using the real-time polymerase chain reaction (PCR). Similarly, Zhao and Wu[14] analyzed D. folliculorum, D. brevis, and D. canis by RAPD sequence characterized amplified region marker.

Mitochondrial rDNA, being maternally inherited, with fast evolution and relatively simple and nontissue-specific gene structure, is useful for studying the phylogeny of organisms.[18] In general, cytochrome c oxidase subunit genes and protein-coding genes (CO1, COII, NDI, and NDS) are useful markers for the phylogenetic analysis of closely related species, sub species, and population from different geographical regions, whereas 12S rDNA and 16S rDNA genes are suitable for the phylogenetic analysis at the genera and species level.

In the present study, 16S rDNA partial sequences of three isolates of D. folliculorum have been amplified, sequenced, and then analyzed with 17 D. folliculorum isolates (2 isolates from Spain, 5 isolates from Iran, and 10 isolates from China), 4 isolates of D. brevis from China, and 6 isolates of D. canis (1 isolate from USA, 2 isolates from China, and 3 isolates from Spain) to discuss the phylogenetic relationship in mites. Genetic distance was computed, and phylogenetic tree was reconstructed using MEGA 6.0 (www.megasoftware.net).[19]

MATERIALS AND METHODS

Mite collection method

Samples of the D. folliculorum were obtained from the eyelashes of three patients diagnosed with Demodex blepharitis. The diagnosis of blepharitis was established on the slit-lamp examination with patients who presented with the symptoms of ocular discomfort and exhibited characteristic cylindrical dandruff like deposits on the base of eyelashes [Figure 1]. Demodex mites were identified using the method given by Coston.[20] Four eyelashes were epilated from each patient and placed on a glass slide and were observed under the light microscope to detect the presence of mites [Figure 2]. The mites were preserved in the Tris–EDTA solution and subjected for molecular analysis.

Figure 1

Representative picture of a patient with blepharitis showing characteristic cylindrical dandruff like deposits on the bases of eyelashes

Figure 2

Representative picture of Demodex mites seen in relation to the base of hair follicles as seen on light microscopy

Genomic DNA isolation of mites

Mites were suspended in 1 ml of Tris-EDTA. Samples were centrifuged for 5 min at 3000 rpm, and the DNA was extracted using the method given by Toops et al.[16] The primers used for 16S rDNA amplification were forward 5'CTGTGCTAAGGYAGCGAAGTC3' and reverse 5'TCAAAWGCCAACAKCGAGGTAA3' (GenBank no. JF783994).[21]

Polymerase chain reaction

The following conditions were used for PCR amplification of 16S rDNA sequences. Taq buffer 2 μl, dNTPs 1.8 μl, primer forward 0.4 μl, primer reverse 0.4 μl, MgCl2 0.8 μl, DNA polymerase (Taq) 0.2 μl, MQ water 10.4 μl, and DNA 4 μl to make a final volume of 20 μl. The amplification condition for 16S rDNA was 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, elongation at 72°C for 30 s, and final extension at 72°C for 10 min and followed by 35 cycles. PCR fragments were detected by the 1.5% agarose gel electrophoresis, 1 μl of EtBr is added in the solution, along with the molecular marker (100 bp) ladder at 100 V for 1 h, and then visualized under ultraviolet light in a Gel documentation system (Protein Simple-Alpha Imager Mini system) [Figure 3]. The amplified products were stored at 4°C. PCR products were purified using the Gel/PCR DNA fragments extraction kit (IBI Scientific) according to the manufacturer's instructions. DNA fragments were sequenced in both directions using the same primers used in amplification steps.

Figure 3

Polymerase chain reaction amplification of 16S rDNA from Demodex species. Lane 1: DNA Ladder 100 bp. Lane 2, 3, 4: polymerase chain reaction products of three Demodex species

Sequence alignments

Sequence alignments for 16S rDNA sequences of the Demodex isolates obtained in the present study were aligned with DNA sequences of Demodex species obtained from the GenBank by using MUSCLE. Maximum composite likelihood estimate of the pattern of nucleotide substitution, nucleotide composition, neighborhood joining tree, maximum likelihood tree, variable sites, and parsim-info sites were analyzed using MEGA.

RESULTS

Phylogenetic analysis

The mitochondrial 16S rDNA PCR products of the three D. folliculorum species from Amritsar, India, were successfully amplified [Figure 3] and sequenced (Accession number KX151160.1, KX151161.1, KX151162.1). These three DNA sequences were aligned with 17 sequences of D. folliculorum which was retrieved from the GenBank [Table 1] to reconstruct the phylogenetic tree by maximum likely hood method, neighborhood, and nucleotide composition in MEGA 6.0 to confirm the reliability of phylogenetic relationship.

Table 1

Information of 16S rDNA sequences of 27 species of Demodex species retrieved from GenBank

Mites	Nation	GenBank number	Size (bp)
D. folliculorum	India	KX151160	297
D. folliculorum	India	KX151161	300
D. folliculorum	India	KX151163	243
D. folliculorum isolate DF-4	China	JF783996	337
D. folliculorum DF-2	China	HQ844221	337
D. folliculorum partial mitochondrial 16S	Spain	FN424246	436
D. folliculorum partial mitochondrial 16S	Spain	FN424245	436
D. folliculorum isolate Df-S10	China	KC917237	337
D. folliculorum isolate Df-S7 16S	China	KC917234	337
D. folliculorum isolate Df-S5 16S	China	KC917232	337
D. folliculorum isolate Df-5 16S	China	JF783997	336
D. folliculorum isolate Df-1 16S	China	JF783994	337
D. folliculorum isolate Df-S6 16S	China	KC917233	337
D. folliculorum isolate Df-S3	China	KC917230	337
D. folliculorum isolate Df-S2 16S	China	KC917229	337
D. folliculorum isolate 2 16S	Iran	KF875588	333
D. folliculorum isolate 4 16S	Iran	KF875590	313
D. folliculorum isolate 1 16S	Iran	KF875587	229
D. folliculorum isolate 3 16S	Iran	KF875589	320
D. folliculorum isolate 4 16S	Iran	KF875590 (2)	313
D. brevis isolate Db-S3	China	KC917240	342
D. brevis isolate Db-S2	China	KC917239	342
D. brevis isolate Db-3 16S	China	JF783999	342
D. brevis isolate Db-1 16S	China	JF783998	342
D. canis 16S	Spain	JX390979	239
D. canis 16S	Spain	JX390978	337
D. canis isolate canis1 16S	USA	JX193756	319
D. canis isolate Dc-3 16S	China	JF784001	340
D. canis isolate Dc-2 16S	China	JF784000	340
D. canis 16S rRNA gene strain Dc 1	Spain	HE817763	436

D. folliculorum: Demodex folliculorum, Demodex brevis: D. brevis, Demodex canis: D. canis

The alignment of the three sequences of 16S rDNA partial sequences obtained in the present study and the 17 sequences of the D. folliculorum retrieved from the GenBank using MEGA resulted in a total of 300 base pairs including gaps. The MEGA (version 7) analysis presented a (96%) 288/299 sequence identity with the 15 D. folliculorum isolates. It presented a (98%) 243/249 sequence identity with one sequence of D. folliculorum. The overall number of the conserved site in the three isolates was 210/212, variable sites 2/212, and no parsim-info. The average content of T, C, A, and G in D. follculorum was 43.5%, 7.3%, 27.7%, and 21.6% respectively, and the nucleotide frequencies were biased toward A+T, averaging 71.2% [Table 2].

Table 2

Nucleotide composition of 16S rDNA gene of three isolates of Demodex folliculorum

Species	A%	C%	T%	G	A+T	G+C	Total
D. folliculorum KX151160	27.3	7.1	44.4	21.2	71.7	29	297
D. folliculorum KX151161	27.1	8.4	43.1	21.4	70.2	30	299
D. folliculorum` KX151163	28.8	6.2	42.8	22.2	71.6	29	243
Average	27.7	7.3	43.5	21.6	71.2	29	279.7

D. folliculorum: Demodex folliculorum

Nucleotide pattern and protein translations

In the present study, many silent mutations were observed, and most of them were at the third codon position. The nucleotide sequence and the proportion (percentage) of a particular nucleotide (A, C, T, and G) in the DNA strand are important in the phylogenetic analysis and describe a specific evolutionary trend. In the present study, as shown in Table 3, the average nucleotide frequencies of A, C, G, and T in D. folliculorum are 30.70% (A), 41.42% (T/U), 7.26% (C), and 20.61% (G). The transition/transversion rate ratios are k1 = 1.938 (purines) and k2 = 25.043 (pyrimidines). The overall transition/transversion bias is R = 3.507. The analysis involved 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 190 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6.0.

Table 3

Maximum composite likelihood estimate of the pattern of nucleotide substitution

	A	T/U	C	G
A	-	2.73	0.48	2.63
T/U	2.02	-	11.98	1.36
C	2.02	68.3	-	1.36
G	3.92	2.73	0.48	-

The two phylogenetic trees (NJ and ML) showed similar topological structures. The three isolates of the three phenotypes with finger-like terminus clustered as one branch with high node support, and they form the sister clade indicating that three phenotypes were of the Demodex species. D. canis was closer to D. folliculorum than to D. brevis. D. folliculorum species isolated from China were gathered with the three D. folliculorum isolates from Amritsar, India, and then clustered with D. canis and with the D. brevis [Figures 4 and 5].

Figure 4

Neighborhood joining tree of the three Demodex species

Figure 5

Maximum likelihood tree of the three Demodex species

DISCUSSION

The current study showed that the three isolates have a conserved mitochondrial 16S rDNA sequence with some variations among the same species. These belong to the D. folliculorum species based on the morphological characteristics and sequencing. In addition, phylogenetic studies revealed that all three isolates clustered with other sequences of D. folliculorum, retrieved from GenBank database, with minor single-nucleotide polymorphisms variations. The three isolates were more in A+T nucleotide frequencies than G+C nucleotide frequencies and the estimated transition/transversions bias (R) is 0.93. These findings are in agreement with other studies that showed molecular analysis using several mitochondrial gene fragments.

De Rojas et al.[22] analyzed the Demodex mitochondrial 16S rDNA and showed that the A+T nucleotide frequencies were higher than the G+C, which is in consistent with Rhinonyssid mites. Herbert et al.,[23] Tsao and Yeh,[24] and Liu et al.[25] stated that the average genetic distance among the Demodex species is larger than the intraspecific distance. Kumar et al.[26] and Zhou et al.[27] observed the transition/transversions among the three Demodex species are smaller, which indicate that the base substitutions have reached the interspecies level. Zhao and Wu[1] found that the differentiation between D. folliculorum and D. canis is smaller than that between D. folliculorum and D. brevis. Hence, the mitochondrial 16S rDNA partial sequence can be used to identify the difference among the three Demodex species. In the present study, it has been observed that D. folliculorum is more closely related to D. canis than to D. brevis.

De Rojas et al.[22] found the synthetically analytic results of the divergence, genetic distance, and transition/transversions of the two geographic isolates Spain and China, it can be found that the mitochondrial 16S rDNA partial sequence cannot discriminate the difference between the two geographic D. folliculorum isolates. Moreover, 16S rDNA fragments of three D. folliculorum (face) from Xi'an, China and six D. folliculorum (eyelids) from Spain are completely identical and belong to one haplotype, which suggest that neither mitochondrial 16S rDNA partial sequence cannot discriminate the Demodex mites from Chinese faces and Spanish eyelids nor it can tell the difference between the D. folliculorum from the distinct body localization from Spain.

From the above studies, two explanations may arise: one reason is that the mitochondrial 16S rDNA partial sequences were highly conserved and cannot be used for the interspecies identification and second, D. folliculorum has only one species, and subspecies differentiation happened due to the changes of parasitic position and geographic condition. During present studies, it has been observed that three isolates of Demodex show 99% query cover with the D. folliculorum isolates from China.

Zhao and Wu[1] studied the phylogenetic trees inside the genus Demodex, high bootstrap value of 100% supports the presence of three sister branches, where two species, D. folliculorum and D. canis, clustered together (bootstrap 96%–100%), but the third one, D. brevis, seem to be more distant (bootstrap 98%–100%). The phylogenetic tree proved that at the molecular level, the D. folliculorum, D. canis, and D. brevis are three distinct species, and D. folliculorum is closer to D. canis than to D. brevis, which is in consistence with their morphological classification. This may due to the reason: both D. folliculorum and D. brevis are a human parasite, but at a different location, former in the hair follicles and later in the sebaceous glands, where the environment is different. D. canis mites are present in the hair follicles of the dog where the environment is similar to that of the human hair follicle. It might be a similar environment that results into the similar phenotype and genotype of D. folliculorum and D. canis. In conclusion, all three isolates belong to D. folliculorum, and the mitochondrial DNA 16S rDNA partial sequence is applicable for phylogenetic relationship analysis at the lower level, i.e., genus and species but not for interspecies identification.

CONCLUSION

Demodex mites present in the patients of blepharitis from Amritsar belongs to D. folliculorum species. The mitochondrial DNA 16S rDNA parital sequence was applicable for phylogenetic relationship analysis at lower level but not for interspecies identification.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.