A Novel Mitochondrial Genome Fragmentation Pattern in the Buffalo Louse Haematopinus tuberculatus (Psocodea: Haematopinidae).

Sucking lice are obligate ectoparasites of mammalian hosts, causing serious public health problems and economic losses worldwide. It is well known that sucking lice have fragmented mitochondrial (mt) genomes, but many remain undetermined. To better understand patterns of mt genome fragmentation in the sucking lice, we sequenced the mt genome of the buffalo louse Haematopinus tuberculatus using next-generation sequencing (NGS). The mt genome of H. tuberculatus has ten circular minichromosomes containing a total of 37 genes. Each minichromosome is 2.9-5.0 kb long and carries one to eight genes plus one large non-coding region. The number of mt minichromosomes of H. tuberculatus (ten) is different from those of congeneric species (horse louse H. asini, domestic pig louse H. suis and wild pig louse H. apri) and other sucking lice. Two events (gene translocation and merger of mt minichromosome) are observed in Haematopinus. Compared to other studies, our phylogeny generated from mt genome datasets showed a different topology, suggesting that inclusion of data other than mt genomes would be required to resolve phylogeny of sucking lice. To our knowledge, this is the first report of a ten mt minichromosomes genome in sucking lice, which opens a new outlook into unexplored mt genome fragmentation patterns in sucking lice.

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

Analysis of the Mitogenomes of Two Helotid Species Provides New Insights into the Phylogenetic Relationship of the Basal Cucujoidea (Insecta: Coleoptera).

Helotid beetles are commonly found in places where sap flows from tree trunks and in crevices in bark. The Helotidae family is a rare and primitive group of Cucujoidea. To date, no complete mitochondrial (mt) genome has been sequenced for this family. To better understand the characteristics of the mt genome and the evolution of Cucujoidea, we sequenced and annotated the complete mt genomes of Helota thoracica (Ritsema, 1895) and Helota yehi Lee, 2017 using next-generation sequencing. These are the first record of Helotidae mt genomes. The RNA secondary structures of both species were also predicted in this study. The mt genomes of H. thoracica and H. yehi are circular, with total lengths of 16,112 bp and 16,401 bp, respectively. After comparing the mt genomes of H. thoracica and H. yehi, we observed the gene arrangement, codon usage patterns, base content, and RNA secondary structures of both species to be similar, which has also been noted in other Coleoptera insects. The nucleotide sequence of the coding regions and the control region has small differences. The phylogenetic analysis indicated that Helotidae and Protocucujidae are sister groups and revealed the relationship between seven families; however, the validity of the two series (Erotylid series and Nitidulid series) as larger groups in the superfamily was not supported. The mt phylogenomic relationships have strong statistical support. Therefore, the division of Cucujoidea into series should be re-examined. Our results will provide a better understanding of the mt genome and phylogeny of Helotidae and Cucujoidea and will provide valuable molecular markers for further genetic studies.

Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia.

BACKGROUND: Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. METHODS: In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. RESULTS: Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure, and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts-77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between Lucilia cuprina cuprina, L. c. dorsalis and L. sericata. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata. The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. CONCLUSIONS: Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia.

First Description of the Mitogenome Features of Neofoleyellides Genus (Nematoda: Onchocercidae) Isolated from a Wild Bird (Pyrrhocorax pyrrhocorax).

The Onchocercidae family is composed of more than 30 valid nematode species with notable zoonotic potential. Current limitations in molecular characterization methods and species identification are the main obstacles to a better understanding of the biology of Onchocercidae species, particularly in wildlife. This study describes for the first time the complete mitochondrial (mt) genome sequence of Neofoleyellides sp. isolated from a wild bird (Pyrrhocorax pyrrhocorax) and belonging to the Neofoleyellides genus (Nematoda: Onchocercidae). The mt genome of Neofoleyellides sp. (GenBank accession number: ON641583) was a typical circular DNA molecule of 13,628 bp in size with an AT content of 76.69%. The complete mt genome comprised 36 functional subunits, including 12 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. The most common start codon was ATT/ATG except for nad2 with TTG, and TAA was the termination codon for all protein-coding genes (PCGs). Phylogenetic analysis of the concatenated and aligned amino acid sequences of the 12 PCGs showed that the trees generated using different methods (Bayesian inference and maximum likelihood) with different partition schemes shared similar topologies. The isolated Neofoleyellides sp. was placed in the Onchocercidae family and formed a sister branch with the genera Onchocerca and Dirofilaria. The entire mt genome of Neofoleyellides sp. presented in this study could provide useful data for studying the population genetics and phylogenetic relationships of Onchocercidae species.

The complete mitochondrial genome of an enigmatic predaceous springtail Metisotoma macnamarai from northeast China.

The complete mitogenome of Metisotoma macnamarai (Folsom 1918) (Collembola, Isotomidae), a member of obligatory predaceous genus Metisotoma Maynard, 1951, was sequenced. It has a length of 15,177 bp, comprising 13 protein-coding genes, 22 tRNAs, and 2 rRNAs. The mitogenome has the following base composition: A = 37.1%, T = 33.3%, G = 11.8%, and C = 17.4%. Phylogenetic analysis using maximum likelihood (ML) indicates that M. macnamarai clusters as sister taxon to the genus Isotomurus, with high statistical support.

Variation of mitochondrial minichromosome composition in Hoplopleura lice (Phthiraptera: Hoplopleuridae) from rats.

BACKGROUND: The family Hoplopleuridae contains at least 183 species of blood-sucking lice, which widely parasitize both mice and rats. Fragmented mitochondrial (mt) genomes have been reported in two rat lice (Hoplopleura kitti and H. akanezumi) from this family, but some minichromosomes were unidentified in their mt genomes. METHODS: We sequenced the mt genome of the rat louse Hoplopleura sp. with an Illumina platform and compared its mt genome organization with H. kitti and H. akanezumi. RESULTS: Fragmented mt genome of the rat louse Hoplopleura sp. contains 37 genes which are on 12 circular mt minichromosomes. Each mt minichromosome is 1.8-2.7 kb long and contains 1-5 genes and one large non-coding region. The gene content and arrangement of mt minichromosomes of Hoplopleura sp. (n = 3) and H. kitti (n = 3) are different from those in H. akanezumi (n = 3). Phylogenetic analyses based on the deduced amino acid sequences of the eight protein-coding genes showed that the Hoplopleura sp. was more closely related to H. akanezumi than to H. kitti, and then they formed a monophyletic group. CONCLUSIONS: Comparison among the three rat lice revealed variation in the composition of mt minichromosomes within the genus Hoplopleura. Hoplopleura sp. is the first species from the family Hoplopleuridae for which a complete fragmented mt genome has been sequenced. The new data provide useful genetic markers for studying the population genetics, molecular systematics and phylogenetics of blood-sucking lice.

Tick mitochondrial genomes: structural characteristics and phylogenetic implications.

Ticks are obligate blood-sucking arachnid ectoparasites from the order Acarina, and many are notorious as vectors of a wide variety of zoonotic pathogens. However, the systematics of ticks in several genera is still controversial. The mitochondrial genome (mt-genome) has been widely used in arthropod phylogeny, molecular evolution and population genetics. With the development of sequencing technologies, an increasing number of tick mt-genomes have been sequenced and annotated. To date, 63 complete tick mt-genomes are available in the NCBI database, and these genomes have become an increasingly important genetic resource and source of molecular markers in phylogenetic studies of ticks in recent years. The present review summarizes all available complete mt-genomes of ticks in the NCBI database and analyses their characteristics, including structure, base composition and gene arrangement. Furthermore, a phylogenetic tree was constructed using mitochondrial protein-coding genes (PCGs) and ribosomal RNA (rRNA) genes from ticks. The results will provide important clues for deciphering new tick mt-genomes and establish a foundation for subsequent taxonomic research.

Long-read sequencing reveals a 4.4 kb tandem repeat region in the mitogenome of Echinococcus granulosus (sensu stricto) genotype G1.

BACKGROUND: Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method. METHODS: We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow. RESULTS: We assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441-445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat. CONCLUSIONS: We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such 'new' repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

Mitochondrial DNA Mutations in etiopathogenesis of male infertility.

OBJECTIVE: To understand role of mitochondrial (mt) mutations in genes regulating oxidative phosphorylation (OXPHOS) in pathogenesis of male infertility. Infertility affects approximately 15% of couples trying to conceive. Infertility is frequently attributed to defects of sperm motility and number. Mitochondrion and mitochondrial DNA (mtDNA) play an important role in variety of physiological process. They control the oxidative energy supply and thus are central to growth, development and differentiation. Mitochondrial function is controlled by a fine-tuned crosstalk between mtDNA and nuclear DNA (nDNA). As mitochondria supply energy by OXPHOS, any mutation in mtDNA disrupts adenosine triphosphate (ATP) production and thus result in an impaired spermatogenesis and impaired flagellar movement. As sperm midpiece has few mtDNA copies, thus enhanced number of mutant mtDNA results in early phenotypic defect which manifest as spermatogenic arrest or asthenozoospermia. Oxidative stress and mtDNA mutations are positively correlated and mutations in mitochondrial genome (mt genome) are implicated in the lowered fertilising capacity of the sperm and affects the reproductive potential of an individual. MATERIALS AND METHODS: A thorough review of articles in the last 15 years was cited with reference to the below-mentioned keywords. The articles considered discuss the role of mt genome in the normal functioning of sperm and the factors associated with mt mutations and impact of these mutations on the reproductive potential. RESULTS: Sperm motility is a very important factor for the fertilisation of ova. The energy requirements of sperm are therefore very critical for sperm. Mutations in the mitochondrial genes as COX II, ATPase 6 and 8 play an important role and disrupts ATP production affecting the spermatogenesis and sperm motility. Therefore, the aberrations in mt genome are an important etiopatholgy of male infertility. CONCLUSION: In the context of male infertility, mt mutations, generation of reactive oxygen species and lowered antioxidant capacity are interlinked and constitute a unified pathogenic molecular mechanism. In the era of assisted reproduction technique (ART), it is very important to distinguish between mutations in nuclear and mitochondrial genomes in sperm, as mtDNA mutations are better diagnostic and prognostic markers in infertile men opting for ART.

Complete mitochondrial genome of Dryophytes suweonensis (Anura Hylidae).

Dryophytes suweonensis is an endangered species with fragmented and declining populations from the Korean peninsula. We described 17,448 bp of D. suweonensis mtDNA, which had a shorter D-loop than other closely related species. The variation in nucleotide composition was similar to that of Hyla tsinlingensis but was larger than the one of its sister clade, D. japonicus.

The complete mitochondrial genome of Hyla annectans (Anura: Hylidae).

The complete mitochondrial genome sequence of the Hyla annectans (Anura: Hylidae) was determined in this study. It is a circular molecule of 17,973 bp in length including 37 genes for 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region, showing a typical neobatrachian-type arrangement. The A + T content of H-strand and the control region is 60.7% and 64.6%, respectively. The control region is 2554 bp in length and possesses distinct repeat regions at both 5' and 3' ends.

The complete mitochondrial genome of a stonefly species, Togoperla sp. (Plecoptera: Perlidae).

The complete mitochondrial (mt) genome of a stonefly species, Togoperla sp. (Plecoptera: Perlidae), was sequenced. The 15,723 bp long genome has the standard metazoan complement of 37 genes and an A+T-rich region, which is the same as the insect ancestral genome arrangement.

Complete mitochondrial genome sequences of Atlantic representatives of the invasive Pacific coral species Tubastraea coccinea and T. tagusensis (Scleractinia, Dendrophylliidae): Implications for species identification.

Members of the azooxanthellate coral genus Tubastraea are invasive species with particular concern because they have become established and are fierce competitors in the invaded areas in many parts of the world. Pacific Tubastraea species are spreading fast throughout the Atlantic Ocean, occupying over 95% of the available substrate in some areas and out-competing native endemic species. Approximately half of all known coral species are azooxanthellate but these are seriously under-represented compared to zooxanthellate corals in terms of the availability of mitochondrial (mt) genome data. In the present study, the complete mt DNA sequences of Atlantic individuals of the invasive scleractinian species Tubastraea coccinea and Tubastraea tagusensis were determined and compared to the GenBank reference sequence available for a Pacific "T. coccinea" individual. At 19,094bp (compared to 19,070bp for the GenBank specimen), the mt genomes assembled for the Atlantic T. coccinea and T. tagusensis were among the longest sequence determined to date for "Complex" scleractinians. Comparisons of genomes data showed that the "T. coccinea" sequence deposited on GenBank was more closely related to that from Dendrophyllia arbuscula than to the Atlantic Tubastraea spp., in terms of genome length and base pair similarities. This was confirmed by phylogenetic analysis, suggesting that the former was misidentified and might actually be a member from the genus Dendrophyllia. In addition, although in general the COX1 locus has a slow evolutionary rate in Scleractinia, it was the most variable region of the Tubastraea mt genome and can be used as markers for genus or species identification. Given the limited data available for azooxanthellate corals, the results presented here represent an important contribution to our understanding of phylogenetic relationships and the evolutionary history of the Scleractinia.

The complete mitochondrial genome of the Antarctic stalked jellyfish, Haliclystus antarcticus Pfeffer, 1889 (Staurozoa: Stauromedusae).

In present study, the complete mitogenome sequence of the Antarctic stalked jellyfish, Haliclystus antarcticus Pfeffer (Staurozoa: Stauromedusae) has been sequenced by next-generation sequencing method. The assembled mitogenome comprises of 15,766 bp including 13 protein coding genes, 7 transfer RNAs, and 2 ribosomal RNA genes. The overall base of Antarctic stalked jellyfish constitutes of 26.5% for A, 19.6% for C, 19.8% for G, 34.1% for T and show 90% identity to Sessile Jelly, Haliclystus sanjuanensis, in the northeastern Pacific Ocean. The complete mitogenome of the Antarctic stalked jellyfish, contributes fundamental and significant DNA molecular data for further phylogeography and evolutionary analysis for seahorse phylogeny. The complete sequence was deposited in DBBJ/EMBL/GenBank under accession number KU947038.

The complete mitochondrial genome of a stonefly species, Kamimuria chungnanshana Wu, 1948 (Plecoptera: Perlidae).

This study determined the complete mitochondrial (mt) genome of the stonefly, Kamimuria chungnanshana Wu, 1948. The mt genome is 15, 943 bp in size and contains 37 canonical genes which include 22 transfer RNA genes, 13 protein-coding genes, and two ribosomal RNA genes, the control region is 1062 bp in length. The phylogenetic tree shows that Kamimuria chungnanshana is sister group of Kamimuria wangi.

The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus).

We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or D-loop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids.

Mitochondrial population genomic analyses reveal population structure and demography of Indian Plasmodium falciparum.

Inference on the genetic diversity of Plasmodium falciparum populations could help in better management of malaria. A very recent study with mitochondrial (mt) genomes in global P. falciparum had revealed interesting evolutionary genetic patterns of Indian isolates in comparison to global ones. However, no population genetic study using the whole mt genome sequences of P. falciparum isolates collected in the entire distribution range in India has yet been performed. We herewith have analyzed 85 whole mt genomes (48 already published and 37 entirely new) sampled from eight differentially endemic Indian locations to estimate genetic diversity and infer population structure and historical demography of Indian P. falciparum. We found 19 novel Indian-specific Single Nucleotide Polymorphisms (SNPs) and 22 novel haplotypes segregating in Indian P. falciparum. Accordingly, high haplotype and nucleotide diversities were detected in Indian P. falciparum in comparison to many other global isolates. Indian P. falciparum populations were found to be moderately sub-structured with four different genetic clusters. Interestingly, group of local populations aggregate to form each cluster; while samples from Jharkhand and Odisha formed a single cluster, P. falciparum isolates from Asom formed an independent one. Similarly, Surat, Bilaspur and Betul formed a single cluster and Goa and Mangalore formed another. Interestingly, P. falciparum isolates from the two later populations were significantly genetically differentiated from isolates collected in other six Indian locations. Signature of historical population expansion was evident in five population samples, and the onset of expansion event was found to be very similar to African P. falciparum. In agreement with the previous finding, the estimated Time to Most Recent Common Ancestor (TMRCA) and the effective population size were high in Indian P. falciparum. All these genetic features of Indian P. falciparum with high mt genome diversity are somehow similar to Africa, but quite different from other Asian population samples.

Sequence and comparison of mitochondrial genomes in the genus Nerita (Gastropoda: Neritimorpha: Neritidae) and phylogenetic considerations among gastropods.

In the present study, we determined the mitochondrial DNA (mtDNA) sequence of three Neritas, Nerita versicolor, Nerita tessellata, and Nerita fulgurans. We present an analysis of the features of their gene content and genome organization and compare these within the genus Nerita, and among the main gastropod groups. The new sequences were used in a phylogenetic analysis including all available gastropod mitochondrial genomes. Genomic lengths were quite conserved, being 15,866bp for N. versicolor, 15,741bp for N. tessellata and 15,343bp for N. fulgurans. Intergenic regions were generally short; genes are transcribed from both strands and have a nucleotide composition high in A and T. The high similarity in nucleotide content of the different sequences, gene composition, as well as an identical genomic organization among the Nerita species compared in this study, indicates a high degree of conservation within this diverse genus. Values ​​of Ka/Ks of the 13 protein coding genes (PCGs) of Nerita species ranged from 0 to 0.18, and suggested different selection pressures in gene sequences. Bayesian phylogenetic analyses using concatenated DNA sequences of the 13 PCGs and the two rRNAs, and of amino acid sequences strongly supported Neritimorpha and Vetigastropoda as sister groups.

The complete mitochondrial genome sequence of Sogatella furcifera (Horváth) and a comparative mitogenomic analysis of three predominant rice planthoppers.

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the most destructive pests of rice crops in many Asian countries. Using long-PCR and shotgun library methods, we sequenced the entire mitochondrial genomes (mt-genomes) of two WBPH individuals. Total lengths of the mt-genome of the two WBPH individuals were 16,612 bp and 16,654 bp with an identical AT content of 76.19%. Among the 13 protein coding genes (PCGs), only nad5 used an atypical initiation codon GTG. Most of the tRNA genes had the typical cloverleaf secondary structure except that the dihydrouridine (DHU) arms in two trnS genes and the TΨC arm of trnG gene did not form a stable stem-loop structure. Similar to the brown planthopper (BPH), Nilaparvata lugens (Stål), and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén), some extraordinary features were observed in the WBPH mt-genome. These include similar gene rearrangement pattern, unusually short length of the atp8 gene and variable numbers of tandem repeat (VNTR) structure in control region. Interestingly, the same tandem repeat unit with stable secondary structure appeared in two different planthoppers, WBPH and SBPH, which belong to two different genera of the Delphacidae. This peculiar feature provides a direct evidence for the close relationship between the two planthoppers and updates our understanding of the evolutionary characteristics of mitochondrial control region. Comparison with two other predominant rice planthoppers (BPH and SBPH) revealed that different PCGs of mitochondria exhibit different evolutionary patterns.