Eleonora's falcon (Falco eleonorae Géné, 1839) is a well-known long-distance migrant of the Afro-Palaearctic flyway, a summer breeder of the Mediterranean region and North-west Africa and a winter resident of Madagascar and surrounding areas, thus characterized as a double endemic. Within the context of a long-term monitoring and conservation programme on Antikythira Island, Greece, which accommodates one of the largest concentrations of breeding pairs of Eleonora's falcons globally, birds were subjected to regular inspections for the presence of ticks from 2017 to 2023. In total, 104 adults and 149 nymphs (all belonging to Haemaphysalis genus) were collected. All ticks, apart from 2 nymphs, exhibited broadly salient palpi and did not possess the pronounced palpal segment 2 spurs or spur-like angles that are characteristic of adults, nymphs and most larvae of Rhipistoma, thus placed them in the Ornithophysalis subgenus. Following comprehensive morphological assessment and genetic analysis of the mitochondrial genome by means of next-generation sequencing of both adult and nymphal stages of the ticks, our empirical findings substantiate the delineation of a previously unclassified species. This taxonomic assignment situates the newly described species within the Ornithophysalis subgenus and the Haemaphysalis doenitzi group, marking its presence for the first time within the Western Palaearctic region.

We first sequenced and characterised the complete mitochondrial genome of Toxocara apodeme, then studied the evolutionary relationship of the species within Toxocaridae. The complete mitochondrial genome was amplified using PCR with 14 specific primers. The mitogenome length was 14303 bp in size, including 12 PCGs (encoding 3,423 amino acids), 22 tRNAs, 2 rRNAs, and 2 NCRs, with 68.38% A+T contents. The mt genomes of T. apodemi had relatively compact structures with 11 intergenic spacers and 5 overlaps. Comparative analyses of the nucleotide sequences of complete mt genomes showed that T. apodemi had higher identities with T. canis than other congeners. A sliding window analysis of 12 PCGs among 5 Toxocara species indicated that nad4 had the highest sequence divergence, and cox1 was the least variable gene. Relative synonymous codon usage showed that UUG, ACU, CCU, CGU, and UCU most frequently occurred in the complete genomes of T. apodemi. The Ka/Ks ratio showed that all Toxocara mt genes were subject to purification selection. The largest genetic distance between T. apodemi and the other 4 congeneric species was found in nad2, and the smallest was found in cox2. Phylogenetic analyses based on the concatenated amino acid sequences of 12 PCGs demonstrated that T. apodemi formed a distinct branch and was always a sister taxon to other congeneric species. The present study determined the complete mt genome sequences of T. apodemi, which provide novel genetic markers for further studies of the taxonomy, population genetics, and systematics of the Toxocaridae nematodes.

The phylum Acanthocephala is an important group of parasites with more than 1,300 species parasitizing intestine of all major vertebrate groups. However, our present knowledge of the mitochondrial genomes of Acanthocephala remains very limited. In the present study, we sequenced and annotated the complete mitochondrial genome of Acanthogyrus (Acanthosentis) bilaspurensis (Gyracanthocephala: Quadrigyridae) for the first time based on the specimens recovered from the intestine of common carp Cyprinus carpio Linnaeus (Cyprinidae) in Pakistan. The mitochondrial genome of A. bilaspurensis is 13,360 bp in size and contains 36 genes, representing the smallest mitogenome of acanthocephalans reported so far. The mitogenome of A. bilaspurensis also has the lowest level of overall A+T contents (59.3%) in the mitogenomes of Eoacanthocephala, and the non-coding region 3 (NCR3) lies between trnS2 and trnI, which is different from all of the other acanthocephalan species. Phylogenetic analyses based on concatenating the amino acid sequences of 12 protein-coding genes using maximum likelihood (ML) and Bayesian inference (BI) methods revealed that the family Pseudoacanthocephalidae is a sister to the Arhythmacanthidae rather than the Cavisomatidae, and the families Rhadinorhynchidae and Cavisomatidae showed sister relationships.

Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions (NCR1 and NCR2). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus. Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

In this study, the mitochondrial genome of Eulaelaps silvestris, which parasitizes Apodemus chevrieri, was sequenced and assembled to fill the gap in understanding the molecular evolution of the genus Eulaelaps. The E. silvestris mitochondrial genome is a double-stranded DNA molecule with a length of 14 882 bp, with a distinct AT preference for base composition and a notably higher AT content than GC content. The arrangement between genes is relatively compact, with a total of 10 gene intergenic regions and 12 gene overlap regions. All protein-coding genes had a typical ATN initiation codon, and only 2 protein-coding genes had an incomplete termination codon T. Out of the 13 protein-coding genes, the 5 most frequently used codons ended in A/U, with only 1 codon ending in G/C had an relative synonymous codon usage value >1. Except for trnS1 and trnS2, which lacked the D arm, all other tRNAs were able to form a typical cloverleaf structure; and there were a total of 38 mismatches in the folding process of tRNA genes. Unlike the gene arrangement order of the arthropod hypothetical ancestor, the E. silvestris mitochondrial genome underwent fewer rearrangements, mainly near tRNA genes and control regions. Both the maximum likelihood tree and the Bayesian tree showed that the family Haemogamasidae is most closely related to the family Dermanyssidae. The results not only provide a theoretical basis for studying the phylogenetic relationships of the genus Eulaelaps, but also provide molecular evidence that the family Haemogamasidae does not belong to the subfamily Laelapidae.

Trematodes of the genus Ogmocotyle are intestinal flukes that can infect a variety of definitive hosts, resulting in significant economic losses worldwide. However, there are few studies on molecular data of these trematodes. In this study, the mitochondrial (mt) genome of Ogmocotyle ailuri isolated from red panda (Ailurus fulgens) was determined and compared with those from Pronocephalata to investigate the mt genome content, genetic distance, gene rearrangements and phylogeny. The complete mt genome of O. ailuri is a typical closed circular molecule of 14 642 base pairs, comprising 12 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions. All genes are transcribed in the same direction. In addition, 23 intergenic spacers and 2 locations with gene overlaps were determined. Sequence identities and sliding window analysis indicated that cox1 is the most conserved gene among 12 PCGs in O. ailuri mt genome. The sequenced mt genomes of the 48 Plagiorchiida trematodes showed 5 types of gene arrangement based on all mt genome genes, with the gene arrangement of O. ailuri being type I. Phylogenetic analysis using concatenated amino acid sequences of 12 PCGs revealed that O. ailuri was closer to Ogmocotyle sikae than to Notocotylus intestinalis. These data enhance the Ogmocotyle mt genome database and provide molecular resources for further studies of Pronocephalata taxonomy, population genetics and systematics.

Echinolaelaps echidninus is a gamasid mite that is of medical and veterinary significance as parasites and vectors of disease agents, which can carry pathogens of zoonosis such as Rickettsia tsutsugamushi, Rickettsia Q fever, Rickettsia mooseri, Rickettsia pox pathogens, Corynebacterium pseudotuberculosis and Leptospira. At present, only single mitochondrial genes have been analysed for E. echidninus in the world, and no complete mitochondrial genome has been reported. However, information carried by a single gene is limited. Therefore, the complete mitochondrial genome of E. echidninus was determined for the first time by Illumina Hiseq X-Ten platform in this study. The mitochondrial genome is 15 736 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region of 1561 bp in length. Codon analysis of 13 protein-coding genes revealed that UUU, UUA, AUU, AUA and AAU were the most frequently used, while cox2 had the fastest evolutionary rate and cob the slowest. Comparative analysis of genome structure and breakpoint distances of the mitochondrial genomes of 23 species in 17 genera from 10 families of Gamasida deposited in GenBank revealed a novel gene arrangement type of the E. echidninus mitochondrial genome, and different degrees of rearrangement among different taxa of Gamasida. Phylogenetic analyses of Gamasida were performed using the maximum likelihood and Bayesian inference methods. Echinolaelaps echidninus was clustered with Dermanyssoidea and formed a more supportive sister group with Varroa destructor. This study provides novel insights into rearrangement patterns and evolution of mitochondrial genomes of Gamasida.

In recent years, bat-associated pathogens, such as 2019 novel coronavirus, have been ravaging the world, and ectoparasites of bats have received increasing attention. Penicillidia jenynsii is a member of the family Nycteribiidae which is a group of specialized ectoparasites of bats. In this study, the complete mitochondrial genome of P. jenynsii was sequenced for the first time and a comprehensive phylogenetic analysis of the superfamily Hippoboscoidea was conducted. The complete mitochondrial genome of P. jenynsii is 16 165 base pairs (bp) in size, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region. The phylogenetic analysis based on 13 PCGs of the superfamily Hippoboscoidea known from the NCBI supported the monophyly of the family Nycteribiidae, and the family Nycteribiidae was a sister group with the family Streblidae. This study not only provided molecular data for the identification of P. jenynsii, but also provided a reference for the phylogenetic analysis of the superfamily Hippoboscoidea.

Ticks are a group of blood-sucking ectoparasites that play an important role in human health and livestock production development as vectors of zoonotic diseases. The phylogenetic tree of single genes cannot accurately reflect the true kinship between species. Based on the complete mitochondrial genome analysis one can help to elucidate the phylogenetic relationships among species. In this study, the complete mitochondrial genome of Dermacentor steini (isolate Longyan) was sequenced and compared with the mitochondrial genes of 3 other Chinese isolates (Nanchang, Jinhua and Yingtan). In Dermacentor steini 4 isolates had identical or similar mitochondrial genome lengths and an overall variation of 0.76% between sequences. All nucleotide compositions showed a distinct AT preference. The most common initiation and stop codons were ATG and TAA, respectively. Fewer base mismatches were found in the tRNA gene of D. steini (isolate Longyan), and the vicinity of the control region and tRNA gene was a hot rearrangement region of the genus Dermacentor. Maximum likelihood trees and Bayesian trees indicate that D. steini is most closely related to Dermacentor auratus. The results enrich the mitochondrial genomic data of species in the genus Dermacentor and provide novel insights for further studies on the phylogeographic classification and molecular evolution of ticks.

The mitochondrial genome provides important information for phylogenetic analysis and an understanding of evolutionary origin. In this study, the mitochondrial genomes of Ilisha elongata and Setipinna tenuifilis were sequenced, which are typical circular vertebrate mitochondrial genomes composed of 16,770 and 16,805 bp, respectively. The mitogenomes of I. elongata and S. tenuifilis include 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA), two ribosomal RNA (rRNA) genes and one control region (CR). Both two species' genome compositions were highly A + T biased and exhibited positive AT-skews and negative GC-skews. The genetic distance and Ka/Ks ratio analyses indicated that 13 PCGs were affected by purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. Results of phylogenetic analysis support close relationships among Chirocentridae, Denticipitidae, Clupeidae, Engraulidae and Pristigasteridae based on the nucleotide and amino acid sequences of 13 PCGs. Within Clupeoidei, I. elongata and S. tenuifilis were most closely related to the family Pristigasteridae and Engraulidae, respectively. These results will help to better understand the evolutionary position of Clupeiformes and provide a reference for further phylogenetic research on Clupeiformes species.

To explore the characteristics of the mitochondrial genome (mitogenome) of the squeaking silkmoths Rhodinia, a genus of wild silkmoths in the family Saturniidae of Lepidoptera, and reveal phylogenetic relationships, the mitogenome of Rhodinia fugax Butler was determined. This wild silkmoth spins a green cocoon that has potential significance in sericulture, and exhibits a unique feature that its larvae can squeak loudly when touched. The mitogenome of R. fugax is a circular molecule of 15,334 bp long and comprises 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and an A + T-rich region, consistent with previous observations of Saturniidae species. The 370-bp A + T-rich region of R. fugax contains no tandem repeat elements and harbors several features common to the Bombycidea insects, but microsatellite AT repeat sequence preceded by the ATTTA motif is not present. Mitogenome-based phylogenetic analysis shows that R. fugax belongs to Attacini, instead of Saturniini. This study presents the first mitogenome for Rhodinia genus.

Our present genetic data of Acanthocephala, especially the mitochondrial (mt) genomes, remains very limited. In the present study, the nearly complete mt genome sequences of Sphaerirostris lanceoides (Petrochenko, 1949) was sequenced and determined for the first time based on specimens collected from the Indian pond heron Ardeola grayii (Sykes) (Ciconiiformes: Ardeidae) in Pakistan. The mt genome of S. lanceoides is 13 478 bp in size and contains 36 genes, including 12 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). Moreover, in order to clarify the phylogenetic relationship of the genera Centrorhynchus and Sphaerirostris, and to test the systematic position of S. lanceoides in the Centrorhynchidae, the phylogenetic analyses were performed using Bayesian inference and maximum likelihood methods, based on concatenated nucleotide sequences of 12 PCGs, rRNAs and tRNAs. The phylogenetic results further confirmed the monophyly of the order Polymorphida and the paraphyly of the order Echinorhynchida in the class Palaeacanthocephala. Our results also challenged the validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae) and showed a sister relationship between S. lanceoides and S. picae (Rudolphi, 1819).

The complete mitochondrial sequence of 17,030 bp was obtained from Echinostoma revolutum and characterized with those of previously reported members of the superfamily Echinostomatoidea, i.e. six echinostomatids, one echinochasmid, five fasciolids, one himasthlid, and two cyclocoelids. Relationship within suborders and between superfamilies, such as Echinostomata, Pronocephalata, Troglotremata, Opisthorchiata, and Xiphiditata, are also considered. It contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes and a tandem repetitive consisting non-coding region (NCR). The gene order, one way-positive transcription, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes were similar as in common trematodes. The NCR located between tRNAGlu (trnE) and cox3 contained 11 long (LRUs) and short repeat units (SRUs) (seven LRUs of 317 bp, four SRUs of 207 bp each), and an internal spacer sequence between LRU7 and SRU4 specifying high-level polymorphism. Special DHU-arm missing tRNAs for Serine were found for both tRNAS1(AGN) and tRNAS2(UCN). Echinostoma revolutum indicated the lowest divergence rate to E. miyagawai and the highest to Tracheophilus cymbius and Echinochasmus japonicus. The usage of ATG/GTG start and TAG/TAA stop codons, the AT composition bias, the negative AT-skewness, and the most for Phe/Leu/Val and the least for Arg/Asn/Asp codons were noted. Topology indicated the monophyletic position of E. revolutum to E. miyagawai. Monophyly of Echinostomatidae and Fasciolidae was clearly solved with respect to Echinochasmidae, Himasthlidae, and Cyclocoelidae which were rendered paraphyletic in the suborder Echinostomata.

Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

The taxonomy of Echinococcus has long been controversial. Based mainly on differences in morphology and host-parasite specificity characteristics, 16 species and 13 subspecies were originally described. Subsequently, most of these taxa were regarded as synonyms for Echinococcus granulosus and only 4 valid species were recognised: E. granulosus; E. multilocularis; E. oligarthrus and E. vogeli. But, over the past 50 years, laboratory and field observations have revealed considerable phenotypic variability between isolates of Echinococcus, particularly those of E. granulosus, which include differences in: morphology in both larval and adult stages, development in vitro and in vivo, host infectivity and specificity, chemical composition, metabolism, proteins and enzymes, pathogenicity and antigenicity. The application of molecular tools has revealed differences in nucleic acid sequences that reflect this phenotypic variation and the genetic and phenotypic characteristics complement the previous observations made by the descriptive parasitologists many years ago. The fact that some of these variants or strains are poorly or not infective to humans has resulted in a reappraisal of the public health significance of Echinococcus in areas where such variants occur. A revised taxonomy for species in the Echinococcus genus has been proposed that is generally accepted, and is based on the new molecular data and the biological and epidemiological characteristics of host-adapted species and strains.

The mitochondrial genomes of the genus Echinococcus have already been sequenced for most species and genotypes to reconstruct their phylogeny. However, two important taxa, E. felidis and E. canadensis G10 genotype (Fennoscandian cervid strain), were lacking in the published phylogeny. In this study, the phylogeny based on mitochondrial genome sequences was completed with these taxa. The present phylogeny highly supports the previous one, with an additional topology showing sister relationships between E. felidis and E. granulosus sensu stricto and between E. canadensis G10 and E. canadensis G6/G7 (closely related genotypes referred to as camel and pig strains, respectively). The latter relationship has a crucial implication for the species status of E. canadensis. The cervid strain is composed of two genotypes (G8 and G10), but the present phylogeny clearly suggests that they are paraphyletic. The paraphyly was also demonstrated by analysing the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) of E. canadensis genotypes from various localities. A haplotype network analysis using the short cox1 sequences from worldwide isolates clearly showed a close relatedness of G10 to G6/G7. Domestic and sylvatic life cycles based on the host specificity of E. canadensis strains have been important for epidemiological considerations. However, the taxonomic treatment of the strains as separate species or subspecies is invalid from a molecular cladistic viewpoint.

The nucleotide sequences of mitochondrial DNA fragments containing genes for cytochrome c oxidase subunits I and II, ATPase subunits 6 and 8, large and small subunits of rRNA, and 9 tRNAs were determined using 2 genetically distinct representative specimens of the hydrothermal vent-endemic genus, Alviniconcha, and for the hydrothermal vent-endemic species, Ifremeria nautilei (Provannidae: Caenogastropoda: Mollusca). All 3 specimens showed tRNATrp gene translocation. To determine the time point at which this translocation occurred, we determined the nucleotide sequences of fragments containing 7 tRNA genes for another 3 Alviniconcha and 2 Ifremeria specimens, as well as for 7 related species. Our findings indicated that the translocation occurred in a common ancestor of the Alviniconcha and Ifremeria genera. Relatively long non-coding regions were observed on both sides of the tRNATrp gene and at the original position of this gene in all the Alviniconcha and Ifremeria gastropod specimens. Overall, the mitochondrial non-coding regions appear to provide much more information about genetic structure than the mitochondrial COI gene does and could therefore be a valuable marker for future studies.

In this study, the complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined, and the phylogenetic relationships between the Tibetan Mastiff and other species of Canidae were analyzed using the coyote (Canis latrans) as an outgroup. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16 710 bp, and included 22 tRNA genes, 2S rRNA gene, 13 protein-coding genes and one non-coding region (D-loop region), which is similar to other mammalian mitochondrial genomes. The characteristics of the protein-coding genes, non-coding region, tRNA and rRNA genes among Canidae were analyzed in detail. Neighbor-joining and maximum-parsimony trees of Canids constructed using 12 mitochondrial protein-coding genes showed that as the coyotes and Tibetan wolves clustered together, so too did the gray wolves and domestic dogs, suggesting that the Tibetan Mastiff originated from the gray wolf as did other domestic dogs. Domestic dogs clustered into four clades, implying at least four maternal origins (A to D). The Tibetan Mastiff, which belongs to clade A, appears to be closely related to the Saint Bernard and the Old English Sheepdog.