General features of P. fimetorum mitogenomes
The circular mitochondrial genome of P. fimetorum (GenBank accession OK572962) is 14,619 base pairs in size, and contains 37 genes, which include 13 protein-coding genes (nad1-nad6, nad4L, cox1-cox3, cob, atp6, atp8), 2 rRNA genes (rrnS and rrnL), and 22 tRNA genes (Fig. 1; Table 1); like the reported mitochondrial genomes of other arthropods. The base composition of this chain is adenine 35.6%, thymine 34.8%, cytosine 18.2%, guanine 11.4%. The overall AT-content of the mt genome of P. fimetorum is 70.4% was near to the average AT-content of Acari mitochondrial genomes (75.34±4.78%) [24]. Of those, 22 genes, comprising 9 PCG (cox1-3, atp6, atp8, nad2, nad3, nad6, and cob) and 13 of the tRNA genes (trnA, trnD, trnE, trnG, trnI, trnK, trnM, trnN, trnR, trnS1, trnS2, trnT and trnW) were coded on the majority stand (J-stand). The other 15 genes were on the minority strand (N-stand). The mitochondrial genome of P. fimetorum is the second sequenced species of Parasitidae, and it shares the same genome structure and content as P. wangdunqingi; no gene rearrangement occurs. Gene orders of the mitochondrial genome for two species of mites (P. fimetorum and P. wangdunqingi) are identical to those of arthropods hypothetical ancestor, but diverge from those of other Parasitiformes species. While the degree of mitochondrial genome rearrangement in Parasitiformes mites is already quite high in comparison to other metazoans, the degree of mitochondrial gene rearrangement in Acariformes mites is even greater: all 27 Acariformes species mitochondrial genomes measured in previous studies exhibit a high degree of rearrangement. Generally speaking, gene rearrangement occurs seldom in the mitochondrial genomes of the majority of arthropod groupings, and gene content and arrangement are conservative at the lower classification levels (families and genera), even though there are significant changes at the higher classification levels (phylum) [27]. So far, only 23 species of Parasitiformes think that their mitochondrial gene sequence is the same as the arthropods hypothetical ancestor arrangement pattern, of 50 species of mitochondrial genomes that have been measured. These species belong to 6 families: 11 species are located in the Argasidae [25–26], 1 species is seen in the Allothyridae [25], 1 species is placed in the Nuttalliellidae [27], 3 species are known in the Diplogyniidae [28], 1 species are found in the Parasitidae [28], and the remaining 6 species are contained in the Ixodidae [27]. In addition, 27 species from 7 families show varying degrees of rearrangement. There seem to be 16 species of Ixodidae [29], 1 species of Varroidae [30], 3 species of Phytoseiidae [28], with the rate of rearrangement of the mitochondrial genome being the highest studied, 2 species of Laelapidae [28], 3 species of Macrochelidae [28], 1 species of Blattisociidae [28], and 1 species of Ologamasidae [28].
Table 1
List of annotated mitochondrial genome of Parasitus fimetorum
Genes | Strand | Size | Intergenic length | Start codon | Stop codon | Anticodon |
cox1 | J | 1500 | 3 | ATT | TAA | |
cox2 | J | 679 | 0 | ATG | T | |
trnK | J | 68 | 2 | | | CUU |
trnD | J | 62 | 0 | | | GUC |
atp8 | J | 162 | -4 | ATT | TAA | |
atp6 | J | 666 | 3 | ATA | TAA | |
cox3 | J | 778 | 0 | ATG | T | |
trnG | J | 62 | 0 | | | UCC |
nad3 | J | 339 | -2 | ATT | TAG | |
trnA | J | 63 | 0 | | | UGC |
trnR | J | 61 | 3 | | | UCG |
trnN | J | 67 | -1 | | | GUU |
trnS1 | J | 59 | 2 | | | GCU |
trnE | J | 65 | -2 | | | UUC |
trnF | N | 63 | 0 | | | GAA |
nad5 | N | 1681 | 0 | GTG | T | |
trnH | N | 62 | 0 | | | GUG |
nad4 | N | 1302 | 37 | ATA | TAA | |
nad4L | N | 276 | 17 | ATG | TAA | |
trnT | J | 62 | -1 | | | UGU |
trnP | N | 64 | 1 | | | UGG |
nad6 | J | 435 | -1 | ATT | TAA | |
cob | J | 1114 | 0 | ATG | T | |
trnS2 | J | 62 | 22 | | | UGA |
nad1 | N | 909 | 0 | GTG | TAA | |
trnL2 | N | 62 | 1 | | | UAA |
trnL1 | N | 64 | 0 | | | UAG |
rrnL | N | 1194 | 0 | | | |
trnV | N | 63 | 0 | | | UAC |
rrnS | N | 707 | 0 | | | |
NCR#2 | | 406 | 0 | | | |
trnI | J | 64 | -3 | | | GAU |
trnQ | N | 65 | 20 | | | UUG |
trnM | J | 64 | 0 | | | CAU |
nad2 | J | 963 | -2 | ATT | TAA | |
trnW | J | 63 | -8 | | | UCA |
trnC | N | 62 | 0 | | | GCA |
trnY | N | 63 | 31 | | | GUA |
Table 2
The range of intergenic and overlapping regions of 51 species
Order | Family | Species | Accession number | Interval range | Overlap range | Control region |
Holothyrida | Allothyridae | Allothyrus sp. | KC769586 | 1-20 | 1-8 | 348 |
Ixodida | Argasidae | Antricola mexicanus | KC769591 | 1-15 | 1-8 | 335 |
| | Argas africolumbae | JQ665720 | 1-8 | 1-64 | 343 |
| | Argas lagenoplastis | KC769587 | 1-20 | 1-29 | 343 |
| | Argas miniatus | KC769590 | 1-18 | 1-9 | 346 |
| | Argas sp. | KC769588 | 1-18 | 1-8 | 344 |
| | Carios capensis | AB075953 | 1-7 | 1-57 | 342 |
| | Ornithodoros brasiliensis | KC769593 | 1-22 | 1-9 | 343 |
| | Ornithodoros moubata | AB073679 | 1-8 | 1-47 | 342 |
| | Ornithodoros porcinus | AB105451 | 1-8 | 1-100 | 338 |
| | Ornithodoros rostratus | KC769592 | 1-15 | 1-8 | 340 |
| | Otobius megnini | KC769589 | 1-15 | 1-8 | 340 |
| Ixodidae | Amblyomma cajennense | JX573118 | 1-16 | 1-24 | 304 304 |
| | Robertsicus elaphensi | JN863729 | 1-14 | 1-15 | 308 286 |
| | Aponomma fimbriatum | JN863730 | 1-11 | 1-66 | 307 269 |
| | Archaeocroton sphenodonti | JN863731 | 1-13 | 1-48 | 317 311 |
| | Amblyomma triguttatum | AB113317 | 1-14 | 1-67 | 307 307 |
| | Bothriocroton concolor | JN863727 | 1-16 | 1-23 | 303 298 |
| | Bothriocroton undatum | JN863728 | 1-9 | 1-31 | 302 302 |
| | Dermacentor nitens | KC503258 | 1-324 | 1-11 | 311 306 |
| | Haemaphysalis flava | AB075954 | 1-12 | 1-14 | 310 310 |
| | Haemaphysalis formosensis | JX573135 | 1-11 | 1-20 | 316 310 |
| | Haemaphysalis inermis | JX573136 | 1-144 | 1-8 | 320 318 |
| | Ixodes hexagonus | AF081828 | 1-16 | 1-59 | 359 |
| | Ixodes holocyclus | AB075955 | 1-15 | 1-10 | 450 |
| | Ixodes pavlovskyi | KJ000060 | 1-14 | 1-57 | 354 |
| | Ixodes persulcatus | AB073725 | 1-12 | 1-59 | 352 |
| | Ixodes ricinus | JN248424 | 1-354 | 1-19 | 354 |
| | Ixodes uriae | AB087746 | 1-12 | 1-7 | 476 388 |
| | Rhipicephalus australis | KC503255 | 1-27 | 1-11 | 310 305 |
| | Rhipicephalus geigyi | KC503263 | 1-117 | 1-13 | 306 302 |
| | Rhipicephalus microplus | KC503260 | 1-11 | 1-13 | 310 307 |
| | Rhipicephalus sanguineus | AF081829 | 1-24 | 1-13 | 305 303 |
| | Rhipicephalus simus | KJ739594 | 1-60 | 1-13 | 309 |
| Nuttalliellidae | Nuttalliella namaqua | NC019663 | 1-5 | 1-11 | 339 |
Mesostigmata | Varroidae | Varroa destructor | AJ493124 | 1-360 | 1-62 | 2174 |
| Ologamasidae | Stylochyrus rarior | GQ927176 | 1-16 | 1-13 | 471 399 |
| Parasitidae | Parasitus wandunqingi | MK270528 | 1-31 | 1-8 | 413 |
| | Parasitus fimetorum | OK572962 | 1-37 | 1-8 | 406 |
| Blattisociidae | Blattisocius tarsalis | MK270529 | 1-11 | 1-4 | 425 513 |
| Laelapidae | Coleolaelaps c.f. liui | MK270524 | 1-56 | 1-32 | 312 326 |
| | Hypoaspis linteyini | MK270530 | 1-30 | 1-11 | 398 319 |
| Phytoseiidae | Phytoseiulus persimilis | GQ222414 | 1-39 | 1-11 | 555 578 112 568 |
| | Metaseiulus occidentalis | EF221760 | 1-129 | 1-57 | 311 310 311 311 |
| | Euseius nicholsi | KM999989 | 1-335 | 1-11 | 386 279 |
| Diplogyniidae | Quadristernoseta c.f. intermedia | MK270521 | 1-31 | 1-9 | 437 |
| | Quadristernoseta c.f. longigynium | MK270522 | 1-31 | 1-8 | 479 |
| | Microdiplogynium sp. | MK270523 | 1-31 | 1-8 | 432 |
| Machochelidae | Macrocheles glaber | MK270525 | 1-40 | 1-7 | 400 |
| | Macrocheles muscaedomesticae | MK270526 | 1-32 | 1-2 | 368 |
| | Macrocheles nataliae | MK270527 | 1-38 | 1-131 | 496 425 |
Mitochondrial gene order changes not only between families (Varroidae, Phytoseiidae, Laelapidae, Macrochelidae, Blattisociidae, and Ologamasidae), but also between different genera within the same family (Coleolaelaps c.f. liui (Laelapidae: Coleolaelaps) and Hypoaspis linteyini (Laelapidae: Hypoaspis); Metaseiulus occidentalis (Phytoseiidae: Typhlodrominae) and Phytoseiulus persimilis (Phytoseiidae: Amblyseiinae)). Furthermore, the mite mitochondrial gene rearrangements of 27 species in 7 families display the following characteristics: the rearrangement rate is high, the degree of rearrangement varies among families, and the rearrangement is primarily tRNA rearrangement in nature. Each sequenced mite had at least two tRNA translocations, and the cox2-trnD, nad4L-trnT and control areas, as well as the nad2 gene junction, were all frequent translocations of tRNA rearrangements in the mite mitochondrial genomes.
Gene spacer and gene overlap
Gene overlap region is a section of the mitochondrial genome where the same nucleic acid sequence is involved in the coding of multiple genes. Intergenic region is a portion of the mitochondrial genome that contains non-coding sequences between the coding genes, and its size varies substantially. Gene overlaps were revealed at the 9 gene junctions of P. fimetorum, involving a total of 24 bp. The overlap between trnW and trnC is the longest (7 bp). In addition to the large non-coding regions, the mitochondrial genome of P. fimetorum encloses several small non-coding intergenic regions, most of which are distributed at 12 gene junctions and range in diameter from 1 to 37 bp. The nad4 and nad4L genes contain the intergenic region with the longest size. The gene interval and gene overlap range of 51 species sequenced from the Parasitiformes were summarized in this paper (Table 2). The longest spacer (360bp) is found in Varroa destructor (Varroidae), and the overlap area is found in Macrocheles nataliae (Machochelidae), which also has the highest overlap area (131bp). There is gene spacer and overlap in each mitochondrial genome of the Parasitiformes, and the positions of the spacer and overlap vary substantially between families and between genera. In most circumstances, the development of gene spacers promotes biological evolution because spacer genes allow for the storage of additional genetic information as well as an increase in the likelihood of mutation.
Analysis of protein coding genes and use of codons in mitochondrial genome
The occurrence of the start and stop codons, as well as the sequence similarity with other complete Parasitiformes mtDNA sequences, confirm the existence of a protein coding gene (PCG). The total length of the 13 PCGs is 10,804bp (just the sum of the lengths of 13 protein-coding genes was computed, the gene spacer was not included), accounting for 73.90% of the entire length of P. fimetorum mitochondrial genome. PCGs have an overall A + T content of 69.4%, with a range of 64.3% (cox3) to 75.9% (atp8) of the total. The traditional ATN codons were used as start codons in most protein-coding genes, and the use of these start codons appeared to follow the patterns that are conserved in the mitochondrial genomes like most Acari species and even some arthropods [16]: five (cox1, atp8, nad2, nad3 and nad6) with ATT, four (cox2, cox3, cob, and nad4L) with ATG, two (atp6 and nad4) with ATA, whereas has two (nad1 and nad5) appear to start with GTG (Table 1), which is a rare start codon for animal mitochondrial. Eight PCGs terminate with the conventional stop codons TAA (cox1, nad1, nad2, nad4, nad6, nad4L, atp6 and atp8), one PCG terminate with the conventional stop codons TAG (nad3), whereas the remaining four genes (cox2, cox3, cob and nad5) ended at a single T residue. A typical occurrence in metazoan mitochondrial genomes is the occurrence of an incomplete stop codon, and these incomplete stop codons are thought to be performed through post-transcriptional polyadenylation [31–33].
In genetic information is available biological function, codons are critical as they serve as a link between genes and proteins. Protein coding genes contain a total of 64 triplet codons in their coding sequences. Synonym codons are used to various codons that encode the same amino acid. The amino acids encoded by these 64 triplet codons are divided into 20 separate categories [34–35]. Using MEGA-X and codon W tools, the amino acid usage and codon usage frequency of 13 protein coding genes sequences from two species of Parasitidae were assessed and compared in this study. The mitochondrial PCGs of two parasitic mites was subjected to a relative synonymous codon usage (RSCU) analysis, which suggested that 62 amino-acid encoding codons identified in the mitochondrial PCGs of two parasitic mites are also discovered in other invertebrates [36]. Among the amino acids found in the mitochondrial genomic PCGs of P. fimetorum and P. wangdunqingi, the amino acids leucine (12.880~14.361%), serine (9.444~9.530%), and isoleucine (8.260~9.083%) are the most frequently encountered, while cysteine (0.840~0.972%) is the least frequently encountered. The atp8 gene, for illustration, was appearance in P. fimetorum, which has the fewest types of amino acids and does not contain any of the essential amino acids glycine、arginine and aspartic acid, whereas the nad4L gene was discovered in P. wangdunqingi, which also has the fewest types of amino acids, does not contain any of the essential amino acids proline、glutamine、threonine and tryptophan. The amino acid profile of the proteins encoded by P. fimetorum and P. wangdunqingi are biased by the fact that the two parasite mites have different bases in the two chains. Table 3 depicts the codon use of the 13 protein-coding genes sequences. P. fimetorum has a higher relative codon usage than 1 for 32 codons. Codons with higher relative codon usage than 1 are referred to as preferred codons of mtDNA-encoded protein genes. The preference codon of protein genes encoded by mtDNA in P. fimetorum is higher than that of P. wangdunqingi. Only 29 codons of P. wangdunqingi had a relative usage rate higher than 1. Two mites (P. fimetorum and P. wangdunqingi) prefers to use U-base-terminated codons (P. fimetorum exception of AGU、GAU and CGU, P. wangdunqingi exception of CGU, the other are preferred codons), which are followed by A-base-terminated codons (especially P. fimetorum AGA and P. wangdunqingi UUA, whose relative codon usage is 2.06 and 2.45, respectively). To some extent, P. fimetorum should avoid using G-base-terminated codons (other than AGG, AUG and UGG, and every other relative codon usage is less than 1), and P. wangdunqingi should avoid using C-base-terminated codons as much as possible. From an evolutionary perspective, the codon usage of the closely related species seems to be more similar than that of the unrelated species. It is estimated that the proportion of U3s in the P. fimetorum and P. wangdunqingi mtDNA coding protein gene is higher than the proportion of A3s, C3s, and G3s (Table 4). Kimura (1981) genuinely think that codon usage preference is related to GC content, and that codon usage preference related to GC content is affected by mutation pressure, whereas codon usage preference unrelated to GC content is affected by natural selection pressure [37–39]. By comparing the GC and third GC codon content (GC3s) with P. fimetorum and P. wangdunqingi protein coding genes, the GC and GC3s contents of P. fimetorum were observed to be greater than the P. wangdunqingi. Mutation pressure may cause the difference in codon usage preference between P. fimetorum and P. wangdunqingi.
Table 3
RSCU of preferable codons of mtDNA protein coding sequences in P. fimetorum and P. wangdunqingi
Amino acid Codon | P.fimetorum | P. wangdunqingi | Amino acid Codon | P. fimetorum | P. wangdunqingi |
Number | RSCU | Number | RSCU | Number | RSCU | Number | RSCU |
Phenylalanine | UUU | 177 | 1.48 | 271 | 1.63 | Alanine | GCU | 41 | 1.69 | 43 | 2.05 |
UUC | 63 | 0.52 | 61 | 0.37 | GCC | 32 | 1.32 | 11 | 0.52 |
Leucine | UUA | 121 | 2.01 | 210 | 2.45 | GCA | 21 | 0.87 | 26 | 1.24 |
UUG | 49 | 0.81 | 69 | 0.80 | GCG | 3 | 0.12 | 4 | 0.19 |
CUU | 75 | 1.25 | 104 | 1.21 | Histidine | CAU | 58 | 1.26 | 49 | 1.18 |
CUC | 28 | 0.47 | 31 | 0.36 | CAC | 34 | 0.74 | 34 | 0.82 |
CUA | 66 | 1.10 | 69 | 0.80 | Glutamine | CAA | 74 | 1.68 | 40 | 1.21 |
CUG | 22 | 0.37 | 32 | 0.37 | CAG | 14 | 0.32 | 26 | 0.79 |
Isoleucine | AUU | 161 | 1.36 | 207 | 1.54 | L-Asparagine | AAU | 166 | 1.26 | 113 | 1.48 |
AUC | 44 | 0.37 | 48 | 0.36 | AAC | 98 | 0.74 | 40 | 0.52 |
AUA | 150 | 1.27 | 149 | 1.11 | lysine | AAA | 182 | 1.58 | 74 | 1.24 |
Methionine | AUG | 35 | 1.00 | 49 | 1.00 | AAG | 49 | 0.42 | 45 | 0.76 |
Valine | GUU | 37 | 1.45 | 63 | 1.45 | Aspartic acid | GAU | 17 | 0.74 | 48 | 1.39 |
GUC | 21 | 0.82 | 11 | 0.25 | GAC | 29 | 1.26 | 21 | 0.61 |
GUA | 35 | 1.37 | 64 | 1.47 | Glutamic acid | GAA | 37 | 1.51 | 53 | 1.29 |
GUG | 9 | 0.35 | 36 | 0.83 | GAG | 12 | 0.49 | 29 | 0.71 |
Serine | UCU | 100 | 1.82 | 82 | 1.88 | Cysteine | UGU | 50 | 1.28 | 66 | 1.52 |
UCC | 46 | 0.84 | 34 | 0.78 | UGC | 28 | 0.72 | 21 | 0.48 |
UCA | 65 | 1.19 | 47 | 1.08 | Tryptophan | UGG | 27 | 1.00 | 30 | 1.00 |
UCG | 7 | 0.13 | 10 | 0.23 | UGA | 51 | 0.55 | 59 | 0.57 |
AGU | 47 | 0.86 | 59 | 1.35 | Arginine | CGU | 15 | 0.58 | 20 | 0.83 |
AGC | 64 | 1.17 | 30 | 0.69 | CGC | 13 | 0.51 | 8 | 0.33 |
Proline | CCU | 52 | 1.17 | 43 | 1.74 | CGA | 26 | 1.01 | 20 | 0.83 |
CCC | 65 | 1.46 | 28 | 1.13 | CGG | 12 | 0.47 | 10 | 0.42 |
CCA | 51 | 1.15 | 23 | 0.93 | AGA | 53 | 2.06 | 41 | 1.71 |
CCG | 10 | 0.22 | 5 | 0.20 | AGG | 35 | 1.36 | 45 | 1.88 |
Threonine | ACU | 92 | 1.63 | 65 | 1.70 | Glycine | GGU | 27 | 1.23 | 52 | 1.78 |
ACC | 54 | 0.96 | 28 | 0.73 | GGC | 18 | 0.82 | 7 | 0.24 |
ACA | 59 | 1.04 | 50 | 1.31 | GGA | 26 | 1.18 | 37 | 1.26 |
ACG | 21 | 0.37 | 10 | 0.26 | GGG | 17 | 0.77 | 21 | 0.72 |
Tyrosine | UAU | 202 | 1.46 | 209 | 1.53 | Stop codon | UAA | 176 | 1.91 | 155 | 1.49 |
UAC | 75 | 0.54 | 65 | 0.47 | UAG | 49 | 0.53 | 99 | 0.95 |
Table 4
Codon A3s, U3s, G3s, C3s, GC3s, GC content of protein gene encoded by two mitochondrial genomes of Parasitidae. A3s, U3s, G3s and C3s show the codon contents ending with four bases A, U, G and C, respectively; GC3s represents GC content of third synonymous codon
Species | Accession number | A3s(%) | U3s(%) | G3s(%) | C3s(%) | GC3s(%) | GC(%) |
Parasitus fimetorum | OK572962 | 42.78 | 45.62 | 13.66 | 24.66 | 29.90 | 32.20 |
Parasitus wangdunqingi | MK270528 | 40.69 | 50.64 | 18.84 | 16.20 | 25.50 | 29.80 |
Transfer RNA genes
Using tRNAscan-SE [19] and ARWEN [20] programs, we were able to identify 20 tRNA genes in the mt genomes of P. fimetorum. Other than that, the other two mt tRNA genes of P. fimetorum (trnP and trnS1) could only be found manually through sequence alignment and secondary structure comparison with genes previously identified in other species of Parasitiformes and Acariformes mite. It is common for animals to have a clover secondary structure, which comprises of four arms: the AA-arm, the D-arm, the AC arm, and the T-arm [26]. It has been calculated that the total length of 22 tRNA genes in the mitochondrial genome of P. fimetorum is 1390 bp (only the sum of 22 tRNA genes is calculated, excluding gene overlap), that the A + T content is 72.6 %. All of the tRNAs in the secondary structure of P. fimetorum, with the exception of the serine tRNA (anticodon GCU), can combine to form a typical secondary structure of clover. It is thought that serine tRNA was deficient in its D-arm in practically all animals and was unable to create a stable secondary structure of clover, which is considered to be a characteristic feature of all chelicerate mitochondrial genomes, but this structure is not found in other tRNAs [10]. For the first time, the absence of the T-arm was discovered in the genes of nematode tRNAs, in which 20 out of 22 tRNAs lack the T-arm and the two tRNAs for serine (anticodons GCT and TGA) lack the D-arm [40]. Following this discovery, other tRNAs with similar structure have been found in the chelicerates, including the Acariformes [41], Araneae [42–44], Scorpiones [45–46], Thelyphonida [46], Pseudoscorpiones [47], Acanthocephala [48], Insecta [49], and Protura [50]. The secondary structures of 50 published mitochondrial genomes of Parasitiformes were observed, except for the loss of D-arm of serine tRNA, 44 species had atypical tRNA structure, and the phenomenon of broken arm of tRNA was fairly prevalent. Holothyrida has 1 species, Ixodida contains 30 species, and Mesostigmata includes 13 species with 22 tRNA secondary structures lacking D-arm or T-arm. No tRNAs of these species are deficient in both the D-and T-arms. The tRNA breaking arm phenomena of Parasitiformes is less prevalent than Acariformes. For particular, in Acariformes, 19 out of the 22 mitochondrial tRNAs of the Tetranychidae were found to be lacking either the D- or T-arm; in fact, in some species, the tRNAs for phenylalanine and glutamine were found to be deficient in both the D-and T-arm [24]. Demodicidae has 15 tRNAs that lack either the D-arm or the T-arm, as well as 5 tRNAs that lack both the D-arm and the T-arm [51]. Pyroglyphidae has 20 tRNAs with either D-arm or T-arm lacking [52], Trombiculidae have 18 tRNA without D-arm or T-arm [41], Acaridae and Psoroptidae have 21 tRNA lacking D-arm or T-arm [53], and Unionicolidae have 15 tRNA with missing arm [31–32].13 (trnD, trnG, trnA, trnR, trnF, trnH, trnT, trnS2, trnL2, trnV, trnW, trnC, trnY) of the 22 tRNA genes found in P. fimetorum are within the average range (62.0±1.3 bp) of tRNA genes found in Parasitiformes [24]. The length of 8 tRNA genes (trnK, trnN, trnE, trnP, trnL1, trnI, trnQ, trnM) is greater than the average size of Parasitiformes tRNA gene, with a range between 64~68bp. Only one tRNA gene (trnS1) was found to be shorter than the average length of the tRNA gene, and the loss of the D-arm can explain for only 59bp of the total length of the gene. In addition to the standard Waston-Crick pairings of A-U and G-C, there are a number of mispairings, including G-U, U-U, and A-G, among others. P. fimetorum secondary structure for 22 tRNA genes consisting of 29 base mismatches and irregular pairings, of which 21 mismatches were G-U mismatches (Fig. 2). Only 15 tRNA genes (trnA, trnD, trnG, trnI, trnK, trnL2, trnM, trnN, trnP, trnQ, trnR, trnS1, trnT, trnV, trnW) have complete amino acid receptor arms (7bp), while the remaining 7 tRNA genes have base mismatches (1~2bp varies) in their amino acid receptor. Aside from that, 15 tRNA genes (trnC, trnD, trnE, trnG, trnH, trnI, trnL1, trnN, trnP, trnR, trnS2, trnT, trnV, trnW, trnY) were able to generate a totally paired anticodon arm of 5bp, but the anticodon arms of the other 7 tRNA genes had a 1-2 base mismatch. Missmatch which is may be critical for preserving the integrity of the tRNAs secondary structure.
Ribosomal RNA genes and Non-coding region
There have been successful identifications of rrnS gene (707bp) and rrnL gene (1194bp), which are analogous to other mites, in the whole mitochondrial genome sequence of P. fimetorum. Genetic information about the position and structure of rRNA genes is relatively conservative, and the pace of evolution is also conservative. As with other arthropods, two rRNA genes are found on the N-strand between trnL1 and the biggest non-coding region [54]. It was found that A+T content of rrnS gene and rrnL gene was respectively 69.3% and 75.5%. While rrnS gene is situated in the region between trnL1 and trnV, rrnL gene is located in the region between trnV and non-coding region. There is a consistent pattern in the mitochondrial genome of arthropods, in which gene clusters such as rrnS-trnV-rrnL are organized.
The control region is the longest non-coding section in the mitochondrial genome, and it is responsible for the beginning of mitochondrial DNA replication and transcription. In most cases, this area contains nucleotides with an adenine (A) and thymine (T) content greater than 85 %; therefore, the control region is sometimes referred to as the A+T-rich region [55]. P. fimetorum A+T content of control region were 77.6%, and the similarity with the nucleotide sequence of P. wandunqingi control region was 68.1%. It can be inferred that the two parasite mites evolved independently rather than co-evolved. According to Staffan and Anna (2000) [56], only independent evolution can result in a variation in the nucleotide sequence of a non-coding region, which ultimately results in the degeneration or deletion of a specific non-coding region. Non-coding region sections of the mitochondrial genome of the Parasitiformes differ in terms of their location, quantity, and length, and this is one of the primary reasons for the modest difference in overall length between mite genomes. With four control regions, Phytoseiulus persimilis (Physeiidae) is the species with the greatest number of control regions. Although it is the longest control area in the mites, the control area of Varroa destructor (Varroidae) is the longest (up to 2174bp), and the control area of Rhipicephalus simus (Ixodidae) is the shortest (only 309bp) (Table 2).
Phylogenetic analysis based on cox1 gene sequences
In recent years, a large number of researchers have used mixed gene segments for phylogenetic analysis, with the cox1 gene being one of the most frequently used gene fragment. In terms of nucleotide and amino acid evolution, cox1 is the slowest-evolving protein, making it a valuable marker for examining phylogenetic relationships at a higher level of classification [24]. In this study, the phylogenetic tree of cox1 genes from 51 species of Parasitiformes was constructed using the ML method (maximum likelihood method) in MEGA-X software, Carcinoscorpius rotundicauda and Limulus polyphemus were used as outgroups (Fig. 3). The complete order of Parasitiforms phylogenetic relationship is (Ixodida + Holothyrida + Mesostigmata). The cladistic relationship of the phylogenetic tree showed that 51 species of Parasitiformes were divided into four clades, the first clade was composed of Ixodidae and Nuttalliellidae and formed a sister group with a node support rate of 76; the second clade was composed of Argasidae with the first evolutionary clade formed a sister group and a node support rate of 59; the third clade was composed of Allothyridar and the fourth clade was composed of 8 families of Mesostigmata. The overall node support rate of the phylogenetic tree in this study is strong, but the individual node support rate is poor, which may be owing to the limited sample size. Some families only collect samples from a single species, whereas other families have collected no samples at all. At the base node support rate of the constructed phylogenetic tree is really high. In general, the closer a node is to the tree root, the lower the node support rate will be for that node. This is due to the fact that this study only conducts a phylogenetic analysis on the cox1 gene of a few parasitic mites belonging to the Parasitiformes. Our results suggested that nine families are monophyletic, which is in accordance with the result of Xue (2019) et al. [28].