Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes indicates that the genus Amblyomma is polyphyletic

Our understanding of the phylogenetic relationships among tick lineages has been limited by the lack of resolution provided by the most commonly used phylogenetic markers. Mitochondrial genomes are increasingly used to address controversial phylogenetic relationships. To date, the complete mitochondrial genomes of eleven tick species have been sequenced; however, only three of these species are metastriate ticks, the most speciose lineage of ticks. In this study, we present the nucleotide sequences of the complete mitochondrial genomes of five more species of metastriate ticks: Amblyomma elaphense, Amblyomma fimbriatum, Amblyomma sphenodonti, Bothriocroton concolor and Bothriocroton undatum. We use complete mitochondrial genome sequences to address the phylogenetic placement of two morphologically 'primitive' species -Am. elaphense and Am. sphenodonti - with respect to the genus Amblyomma. Our analysis of these five mitochondrial genomes with the other eleven tick mitochondrial genomes, as well as analysis of nuclear rRNA genes, provides strong evidence that the genus Amblyomma is polyphyletic with the inclusion of Am. sphenodonti and Am. elaphense. A new genus or two new genera may be required to describe Am. sphenodonti and Am. elaphense. It is also possible that these two species are sisters to two established genera, Bothriocroton in the case of Am. sphenodonti, and Haemaphysalis in the case of Am. elaphense. However, other arrangements of these taxa cannot be excluded with the current data. Thus, while Am. sphenodonti and Am. elaphense do not belong in the genus Amblyomma, the phylogenetic placement of these two species cannot be resolved without more data from metastriate ticks, either greater sampling of mitochondrial genomes, or a large data set of nuclear genes.     

Mutations in mitochondrial tRNA genes: non-linkage with syndromes of Wolfram and chronic progressive external ophthalmoplegia.

We have recently identified a point mutation in the mitochondrially encoded tRNA(Leu(UUR)) gene which associates with a combination of type II diabetes mellitus and sensorineural hearing loss in a large pedigree. To extend this finding to other syndromes which exhibit a combination of diabetes mellitus and hearing loss we have sequenced all mitochondrial tRNA genes from two patients with the Wolfram syndrome, a rare congenital disease characterized by diabetes mellitus, deafness, diabetes insipidus and optic atrophy. In each patient, a single different mutation was identified. One is an A to G transition mutation at np 12,308 in tRNA(Leu(CUN)) gene in a region which is highly conserved between species during evolution. This mutation has been described by Lauber et al. (1) as associating with chronic progressive external ophthalmoplegia (CPEO). The other is a C to T transition mutation at np 15,904 in tRNA(Thr) gene. Both mutations are also present in the general population (frequency tRNA(Leu(CUN)) mutation 0.16, tRNA(Thr) mutation 0.015). These findings suggest that evolutionarily conserved regions in mitochondrial tRNA genes can exhibit a significant polymorphism in humans, and that the mutation at np 12,308 in the tRNA(Leu(CUN)) gene is unlikely to be associated with CPEO and Wolfram syndrome.     

The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus

We determined the complete 14,985-nt sequence of the mitochondrial DNA of the horseshoe crab Limulus polyphemus (Arthropoda: Xiphosura). This mtDNA encodes the 13 protein, 2 rRNA, and 22 tRNA genes typical for metazoans. The arrangement of these genes and about half of the sequence was reported previously; however, the sequence contained a large number of errors, which are corrected here. The two strands of Limulus mtDNA have significantly different nucleotide compositions. The strand encoding most mitochondrial proteins has 1. 25 times as many A's as T's and 2.33 times as many C's as G's. This nucleotide bias correlates with the biases in amino acid content and synonymous codon usage in proteins encoded by different strands and with the number of non-Watson-Crick base pairs in the stem regions of encoded tRNAs. The sizes of most mitochondrial protein genes in Limulus are either identical to or slightly smaller than those of their Drosophila counterparts. The usage of the initiation and termination codons in these genes seems to follow patterns that are conserved among most arthropod and some other metazoan mitochondrial genomes. The noncoding region of Limulus mtDNA contains a potential stem-loop structure, and we found a similar structure in the noncoding region of the published mtDNA of the prostriate tick Ixodes hexagonus. A simulation study was designed to evaluate the significance of these secondary structures; it revealed that they are statistically significant. No significant, comparable structure can be identified for the metastriate ticks Rhipicephalus sanguineus and Boophilus microplus. The latter two animals also share a mitochondrial gene rearrangement and an unusual structure of mt-tRNA(C) that is exactly the same association of changes as previously reported for a group of lizards. This suggests that the changes observed are not independent and that the stem-loop structure found in the noncoding regions of Limulus and Ixodes mtDNA may play the same role as that between trnN and trnC in vertebrates, i.e., the role of lagging strand origin of replication.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Complete mitochondrial genomes and novel gene rearrangements in two dicroglossid frogs, Hoplobatrachus tigerinus and Euphlyctis hexadactylus, from Bangladesh

We determined the complete nucleotide sequences of mitochondrial (mt) genomes from two dicroglossid frogs, Hoplobatrachus tigerinus (Indian Bullfrog) and Euphlyctis hexadactylus (Indian Green frog). The genome sizes are 20462 bp in H. tigerinus and 20280 bp in E. hexadactylus. Although both genomes encode the typical 37 mt genes, the following unique features are observed: 1) the ND5 genes are duplicated in H. tigerinus that have completely identical sequences, whereas duplicated ND5 genes in E. hexadactylus possessed dissimilar substitutions; 2) duplicated control region (CR) in H. tigerinus has almost identical sequences whereas single control region (CR) was found in E. hexadactylus; 3) the tRNA-Leu (CUN) gene is translocated from the LTPF tRNA cluster to downstream of ND5-1 in H. tigerinus, and the tRNA-Pro gene is translocated from the LTPF tRNA cluster to downstream of CR in E. hexadactylus; 4) pseudo tRNA-Leu (CUN) and tRNA-Pro genes are observed in E. hexadactylus; and 5) two tRNA-Met genes are encoded in both species, as observed in the previously reported dicroglossid mt genomes. Almost all observed gene rearrangements in H. tigerinus and E. hexadactylus can be explained by the tandem duplication and random loss model, except translocation of tRNA-Pro in E. hexadactylus. The novel mt genomic features found in this study may be useful for future phylogenetic studies in the dicroglossid taxa. However, the mt genome with interesting features found in the present study reveal a high level of variation of gene order and gene content, inspiring more research to understand the mechanisms behind gene and genome evolution in the dicroglossid and as well as in the amphibian taxa in future studies.     

A novel heteroplasmic tRNA(Leu(CUN)) mtDNA point mutation associated with chronic progressive external ophthalmoplegia

We have sequenced all mitochondrial tRNA genes from a patient with chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy, who had no detectable large mtDNA deletions. Direct sequencing failed to detect previously reported mutations and showed a heteroplasmic mutation at nucleotide 12,276 in the tRNA(Leu(CUN)) gene, in the dihydrouridine stem, which is highly conserved through the species during evolution. RFLP analyses confirmed that 18% of muscle mtDNA harbored the mutation, while it was absent from DNA of fibroblasts and lymphocytes of the proband and in 110 patients with other encephalomyopathies. To date, besides large and single nucleotide deletions, several point mutations on mitochondrial tRNA genes have been reported in CPEO patients, but only three were in the gene coding for tRNA(Leu(CUN)).     

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region.

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B. microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.     

The Use of a 4% (w/w) Deltamethrin Collar (Scalibor® ProtectorBand) in the Extended Control of Ticks on Dogs

Deltamethrin (4%, w/w) formulated in a polymer matrix system (Scalibor ProtectorBand, Intervet International BV) was evaluated for the prevention of ticks on dogs. Controlled laboratory trials were conducted, wherein dogs wearing anti-tick collars were compared with control dogs and artificially infested by adult Ixodes ricinus and/or Rhipicephalus sanguineus ticks. The activity of the collars increased from 24 to 48 h post-treatment (pt) wherein tick numbers were reduced to 78.7% for I. ricinus and to 77% for R. sanguineus. The proportion of ticks controlled after the second infestation on D7 pt increased to 99% for I. ricinus and 99.5% for R. sanguineus. For I. ricinus adult ticks, an efficacy of 95.4% was reached over a period of 5.5 months and approximated 90% for R. sanguineus. Hence, the efficacy of the deltamethrin-impregnated dog collar was >90% for 6 months against both tick species. There was no significant difference between the efficacy of the collar against either R. sanguineus or I. ricinus. A subsequent field study wherein 82 dogs were wearing the collar and monitored for tick infestation indicated that the duration of efficacy was between 5 and 6 months.     

The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E. coli tRNA(Leu) than that previously expressed in E. coli without the N-terminal 21 residues.     

Ixodes philipi (Acari: Ixodidae): phylogenetic status inferred from mitochondrial cytochrome oxidase subunit I gene sequence comparison

Ixodes philipi ticks were collected from the nest burrows of streaked shearwaters, Calonectris luecomelas, on 3 different islands of Japan (Awashima: 38 degrees 45'N, 139 degrees 24'E; Mikurajima: 33 degrees 52'N, 139 degrees 36'E; and Omorijima: 36 degrees 8'N, 133 degrees 10'E). The mitochondrial cytochrome oxidase subunit I (COI) gene sequence was determined for each tick. The COI sequences of 9 other ixodid tick species also were determined, and they were used for taxonomic positioning of I. philipi. A metastriata tick, Amblyomma triguttatum, was used as an outgroup reference for the analysis. Phylogenetic examination indicated that the I. philipi ticks are on the branch with Ixodes turdus and Ixodes acutitarsus weakly, and the bootstrap value of this branching was low. Three different analyses, maximum parsimony, genetic distance, and maximum likelihood, support this conclusion. To further refine this analysis, 2761 base pairs (bp) of sequence, which included the genes for tRNA(Met), NADH dehydrogenase subunit 2 (ND2), tRNA(Trp), tRNA(Cys), tRNA(Tyr), and COI, were determined and compared for 6 I. philipi ticks from the 3 different collection sites. Although a base substitution (T to C in the ND2 gene for an Awashima tick) and 2 transitions (G to A in the COI gene for 1 Omorijima tick) have occurred, the overall sequences were highly conserved. Preserved mitochondrial sequences in the ticks from 3 widely separated locations suggest the possibility of gene flow, which was probably accomplished by migratory seabirds.     

Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading

During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.     

The Australian fresh water isopod (Phreatoicidea: Isopoda) allows insights into the early mitogenomic evolution of isopods

The complete mitochondrial (mt) genome sequence of the Australian fresh water isopod Eophreatoicus sp.-14 has been determined. The new species is a member of the taxon Phreatoicidea, a clade of particular interest, as it is often regarded as the sister group to all other Isopoda. Although the overall genome organization of Eophreatoicus sp.-14 conforms to the typical state of Metazoa—it is a circular ring of DNA hosting the usual 37 genes and one major non-coding region—it bears a number of derived characters that fall within the scope of “genome morphology”. Earlier studies have indicated that the isopod mitochondrial gene order is not as conserved as that of other crustaceans. Indeed, the mt genome of Eophreatoicus sp.-14 shows an inversion of seven genes (including cox1), which is as far as we know unique. Even more interesting is the derived arrangement of nad1, trnL(CUN), rrnS, control region, cob, trnT, nad5 and trnF that is shared by nearly all available isopod mt genomes. A striking feature is the close proximity of the rearranged genes to the mt control region. Inferable gene translocation events are, however, more suitable to trace the evolution of mt genomes. Genes like nad1/trnL(CUN) and nad5/trnF, which retained their adjacent position after being rearranged, were most likely translocated together. A very good example for the need to understand the mechanisms of translocations is the remolding of trnL(UUR) to trnL(CUN). Both tRNA genes are adjacent and have a high sequence similarity, probably the result of a gene duplication and subsequent anticodon mutation. Modified secondary structures were found in three tRNAs of Eophreatoicus sp.-14, which are all characterized by the loss of the DHU-arm. This is common to crustaceans for tRNA Serine(AGY), while the arm-loss in tRNA Cysteine within Malacostraca is only shared by other isopods. Modification of the third tRNA, Isoleucine, is not known from any other related species. Nucleotide frequencies of genes have been found to be indirectly correlated to the orientation of the mitochondrial replication process. In Eophreatoicus sp.-14 and in other Isopoda the associated nucleotide bias is inversed to the state of other Malacostraca. This is a strong indication for an inversion of the control region that most likely evolved in the isopod ancestor.     

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA^Leu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA^Leu(CUN) and tRNA^Leu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA^Leu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.     

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

Tick species parasitizing people in an area endemic for tick-borne diseases in north-western Italy

In 1995 and 1996, 318 ticks were recovered from 240 people in Liguria (Province of Savona, Italy). Most of the ticks (284; 89.3%) were sheep ticks, Ixodes ricinus. Rhipicephalus sanguineus (31; 9.8%) and Dermacentor marginatus (3; 0.9%) were also recorded. All three life stages of I. ricinus were found on humans while only nymphs and adults of R. sanguineus and adults of D. marginatus were collected. Human tickbites were most frequent in the municipalities of the Province where roe deer density was highest. The number of tickbites in this area was about 500 per 100,000 residents over the period of observation. Most cases of tickbite were observed in children (11.2%), students (25.6%), workers (22.4%) and retired people (24%). Although ticks were recovered from people throughout the year, the highest frequencies of I. ricinus bites were in May, June and July. In the study area natural hosts were also studied. Six tick species were identified (I. ricinus, I. hexagonus, D. marginatus, R. bursa, R. sanguineus, R. turanicus). I. ricinus and R. sanguineus were the most abundant species. I. ricinus was recorded more frequently from ruminants particularly roe deer, while R. sanguineus was found to be associated with the dog.     

Comparative activity of three repellents against the ticks Rhipicephalus sanguineus and Argas persicus

The residual repellent activity of N,N-diethylphenyl-acetamide (DEPA), N,N-diethyl-3-methyl benzamide (DEET) and dimethylphthalate (DMP) against the hard tick Rhipicephalus sanguineus (Latreille) and the soft tick Argas persicus (Oken) from topical application on rabbits and hens respectively in the laboratory have been studied. It was found that a 25% concentration of DEET gave strong repellency (greater than 90%) against larvae and nymphs of R. sanguineus for 15 and 7 days respectively and against A. persicus larvae and nymphs greater than 90% for 11 and 5 days respectively. Strong repellency (greater than 90%) against adults of the hard and soft ticks was observed for 9 and 7 days respectively with 40% DEET. DEPA at 25% concentration showed strong repellency (greater than 90%) against larvae and nymphs of R. sanguineus and A. persicus for 11 and 5 days respectively. Adults of hard and soft ticks were strongly repelled (greater than 90%) for 9 and 5 days respectively by 40% DEPA. DMP was found to be the least effective against all stages of both species of tick.