Heavy Strand Research Articles

Whole mitochondrial genome sequencing and phylogenetic analysis of Gangetic mystus (Mystus cavasius)

Mystus cavasius, known as Gangetic mystus, is a freshwater indigenous catfish. The present study represents the first-ever complete mitochondrial genome sequencing of M. cavasius. The whole mitochondrial genome size is 16,554 bp (GenBank accession number OR018997). The mitogenome organization consists of total 37 genes, with 13 protein-coding genes, two rRNA, 22 tRNAs, and a D-loop regulatory region. Among these, 28 genes are coded on the heavy strand, while eight tRNA genes and ND6 are encoded separately. Phylogenetic analysis reveals that M. cavasius clusters with other Mystus species and bagrid catfishes. This work contributes valuable insights into structural characterization and phylogenetic relationships.

Maternal mitochondrial DNA copy number and methylation as possible predictors of pregnancy outcomes in a Michigan pregnancy cohort

ABSTRACT Background: Little is understood about the role of mitochondria in pregnancy-related adaptations. Therefore, we evaluated associations of maternal early-to-mid pregnancy mitochondrial DNA copy number (mtDNAcn) and mtDNA methylation with birth size and gestational length. Methods: Michigan women (n=396) provided venous bloodspots at median 11 weeks gestation to quantify mtDNAcn marker NADH-ubiquinone oxidoreductase chain 1 (ND1) using real-time quantitative PCR and mtDNA methylation at several regions within four mitochondria-specific genes using pyrosequencing: MTTF (mitochondrially encoded tRNA phenylalanine), DLOOP (D-loop promoter region, heavy strand), CYTB (cytochrome b), and LDLR (D-loop promoter region, light strand). We abstracted gestational length and birthweight from birth certificates and calculated birthweight z-scores using published references. We used multivariable linear regression to evaluate associations of mtDNAcn and mtDNA methylation with birthweight and birthweight z-scores. Cox Proportional Hazards Models (PHMs) and quantile regression characterized associations of mitochondrial measures with gestational length. We also considered differences by fetal sex. Results: Using linear regression and Cox PHMs, mtDNAcn was not associated with birth outcomes, whereas associations of mtDNA methylation with birth outcomes were inconsistent. However, using quantile regression, mtDNAcn was associated with shorter gestation in female newborns at the upper quantiles of gestational length, but with longer gestational length in males at the lower quantiles of gestational length. Maternal LDLR, DLOOP, and MTTF methylation was associated with longer gestational length in females at the upper quantiles and in males at lower gestational length quantiles. Conclusions: Maternal mtDNAcn and mtDNA methylation were associated with gestational length in babies born comparatively early or late, which could reflect adaptations in mitochondrial processes that regulate the length of gestation.

Complete mitochondrial genome sequence and phylogenetic analysis of the hybrid flat fish Platichthys stellatus (♀) × Platichthys bicoloratus (♂)

We report the complete mitochondrial genome of the hybrid flounder Platichthys stellatus (♀) × Platichthys bicoloratus (♂). The mitochondrial genome contained 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 1 control region (D-loop), for a total length of 16,874 bp. The nucleotide composition of the heavy strand was 29.15% C, 26.99% A, 26.14% T, and 17.71% G. A maximum-likelihood phylogenetic analysis showed that the hybrid flat fish was a member of the same clade as P. stellatus (maternal inheritance). Our findings add to the extant data on the subfamily Pleuronecidae and provide insight into their molecular phylogeny and taxonomy.

Characterisation of the complete mitochondrial genome of the imperiled Pearl darter Percina aurora (Perciformes: Percidae)

ABSTRACT The Pearl darter Percina aurora, a member of the species-rich family Percidae, is endemic to the Pearl and Pascagoula river systems in Louisiana and Mississippi, USA. However, this fish is now only found in the Pascagoula River and is listed as threatened under the US Endangered Species Act. This study, for the first time, assembled and characterised in detail the mitochondrial genome of P. aurora. The mitochondrial genome of P. aurora is 16,646 bp in length and comprises 13 protein coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes (rrnS [12s ribosomal RNA] and rrnL [16s ribosomal RNA]). A 33 bp long region was identified as the origin of replication for the light strand (OL), and a putative non-coding control region (CR), 987 bp in length, contains the origin of replication for the heavy strand (OH). The two ribosomal RNA genes, the majority of the PCGs, and the majority of the tRNA genes are encoded on the positive, or heavy, strand. The gene order in P. aurora is identical to that previously reported for confamilial species. All tRNAs exhibit a cloverleaf secondary structure except tRNA-Serine 1 which lacked the D-arm loop. An analysis of selective pressure (Ka/Ks ratios) for all the PCGs showed Ka/Ks values ≤1, suggesting that all these PCGs experience strong purifying selection. This new genomic resource will aid with bioprospecting and biomonitoring of P. aurora using environmental DNA.

The complete mitochondrial genome of the leopard shark Triakis semifasciata (Triakidae)

ABSTRACT The leopard shark Triakis semifasciata (family Triakidae) is threatened by habitat loss and targeted by recreational and commercial fishermen in the Eastern Pacific Ocean, from Oregon, USA, to the Gulf of California, Mexico. Despite environmental issues, there are few genetic and genomic resources available for this species. This study assembled the complete mitochondrial genome of T. semifasciata from a skin metagenomic sample and described it in detail. The phylogenetic position of T. semifasciata amongst closely related species was also examined using mitochondrial protein-coding genes (PCGs). The mitochondrial genome of T. semifasciata is 16,613 bp in length and consists of 13 PCGs, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. A 30 bp long region was identified as the origin of replication for the light strand (OL) between the trnN and trnC genes, and a 974 bp long putative control region (CR) contains the origin of replication for the heavy strand (OH). The gene order in T. semifasciata is identical to that of cofamilial species. An analysis of Ka/Ks ratios for all PCGs yielded values < 1, indicating that all PCGs experience strong purifying selection. All tRNAs exhibit a canonical ‘cloverleaf’ secondary structure except for tRNA-Ser1 which lacks the stem of the dihydrouridine (DHU) arm and in place possesses a simple loop. A phylomitogenomic maximum likelihood (ML) analysis did not support the monophyly of the family Trakidae and placed T. semifasciata in a sister position to Hemitriakis japanica. However, the aforementioned sister position was poorly supported by ML bootstrap values. This study represents a new genomic resource for this commercially and recreationally important species and confirms that mitochondrial genomes can be assembled from skin metagenomic samples as shown before in an unrelated shark species.

The complete mitochondrial genome of Heptathela kimurai (Araneae: Heptathelidae)

Heptathela kimurai (Kishida, 1920) is a spider that belongs to the family Heptathelidae which is a basial lineage of spiders. The molecular information of ancestral species belonging to families like Heptathelidae is comparatively limited when compared to spider species from derived families. Here we present the complete mitochondrial genome sequence (mtDNA) of H. kimurai. The sequence was obtained using massively parallel sequencing technology. The circular genome was 14,224 bp in length, and the AT content was 69.53%. The H. kimurai mitochondrial genome contains 13 protein-coding genes (PCGs), 21 tRNA genes, and 2 rRNA genes. The majority of PCGs were found in the heavy strand.

Mitochondrial DNA transcription and mitochondrial genome-encoded long noncoding RNA in diabetic retinopathy

In diabetic retinopathy, mitochondrial DNA (mtDNA) is damaged and mtDNA-encoded genes and long noncoding RNA cytochrome B (LncCytB) are downregulated. LncRNAs lack an open reading frame, but they can regulate gene expression by associating with DNA/RNA/protein. Double stranded mtDNA has promoters on both heavy (HSP) and light (LSP) strands with binding sites for mitochondrial transcription factor A (TFAM) between them. The aim was to investigate the role of LncCytB in mtDNA transcription in diabetic retinopathy. Using human retinal endothelial cells incubated in high glucose, the effect of regulation of LncCytB on TFAM binding at mtDNA promoters was investigated by Chromatin immunoprecipitation, and binding of LncCytB at TFAM by RNA immunoprecipitation and RNA fluorescence in situ hybridization. High glucose decreased TFAM binding at both HSP and LSP, and binding of LncCytB at TFAM. While LncCytB overexpression ameliorated decrease in TFAM binding and transcription of genes encoded by both H- and L- strands, LncCytB-siRNA further downregulated them. Maintenance of mitochondrial homeostasis by overexpressing mitochondrial superoxide dismutase or Sirtuin-1 protected diabetes-induced decrease in TFAM binding at mtDNA and LncCytB binding at TFAM, and mtDNA transcription. Similar results were obtained from mouse retinal microvessels from streptozotocin-induced diabetic mice. Thus, LncCytB facilitates recruitment of TFAM at HSP and LSP, and its downregulation in diabetes compromises the binding, resulting in the downregulation of polypeptides encoded by mtDNA. Regulation of LncCytB, in addition to protecting mitochondrial genomic stability, should also help in maintaining the transcription of mtDNA encoded genes and electron transport chain integrity in diabetic retinopathy.

Sequence-specific dynamic DNA bending explains mitochondrial TFAM’s dual role in DNA packaging and transcription initiation

Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent—LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

Low depth sequencing reveals the critically endangered Batagur kachuga (Red-crowned roofed turtle) mitochondrial genome and its evolutionary implications

The Batagur kachuga (B. kachuga), commonly known as the Red-crowned roofed turtle, is a critically endangered species native to India and its neighboring countries like Bangladesh, and Nepal. The present study is the first report of the complete mitochondrial genome of B. kachuga (16,517 bp) construed via the next-generation sequencing (NGS) approach from eggshell DNA. There are 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), 13 protein-coding genes (PCGs), and one putative control region (CR/D-loop) in the mitogenome. The CR region from the current study reveals conserved TAS, CD, and CSB domains and two AT-rich tandem repeat regions. Most genes are encoded in the heavy strand except the NADH dehydrogenase subunit 6 (ND6) gene and seven tRNA genes. Most PCGs start with the initiation codon ATG, except the COI (Cytochrome Oxidase Subunit-I) gene, which starts with the GTG codon. The present investigation also predicts the distinctive cloverleaf structures of tRNAs except for tRNA-Ser1 and tRNA-Ser-2, which lack a DHU arm. The comparative analysis of Ka/Ks with other 33 species from Order Testudines, in relation to B. kachuga, revealed negative selection in most PCGs, indicating a process of preservation and purification that aids in eliminating undesirable or detrimental substitutes. Phylogenetic analysis of this species has been analysed using the complete mitogenome of 33 turtle species. The maximum likelihood phylogenetic tree strongly supports each family in different clades and also reveals a close relationship between the Pangashura and Batagur genera. Our study suggests the generation of genome-wide molecular data, in terms of mitogenomes, SNPs, and SSRs, is needed to improve the understanding of this species and their phylogenetics and evolutionary relationships, which will help to improve the conservation efforts of this species.

The complete mitochondrial genome of the ice krill Euphausia crystallorophias Holt & Tattersall, 1906 (Euphausiacea, Euphausiidae), from the Ross Sea, Antarctica

The mitogenome of Euphausia crystallorophias collected from the Ross Sea Region Marine Protected Area (RSR MPA) is described for the first time. The assembled mitogenome was 17,291 bp in length and consisted of two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), and noncoding regions, all of which were identical to those of other euphausiid species. The most common start codon for the 13 PCGs was ATG, and the most common termination codon was TAA. The overall G + C content was 33.2% in the heavy strand. Euphausia crystallorophias was sister to E. superba in the phylogenetic analysis. The mitogenome of E. crystallorophias provided significant DNA molecular data for further identification and phylogenetic analysis within the euphausiids.

Characterization of the Complete Mitochondrial Genome of Schizothorax kozlovi (Cypriniformes, Cyprinidae, Schizothorax) and Insights into the Phylogenetic Relationships of Schizothorax.

Schizothorax kozlovi is an endemic and vulnerable fish species found in the upper Yangtze River in China. Over the past few years, the population resources of S. kozlovi have been nearly completely depleted owing to multiple contributing threats. While the complete mitochondrial genomes serve as important molecular markers for phylogenetic and genetic studies, the mitochondrial genome of S. kozlovi has still received little attention. In this study, we analyzed the characterization of the mitochondrial genome of S. kozlovi and investigated the phylogenetic relationships of Schizothorax. The complete mitochondrial genome of S. kozlovi was 16,585 bp in length, which contained thirty-seven genes (thirteen protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), twenty-two transfer RNA genes (tRNAs)) and two non-coding regions for the origin of light strand (OL) and the control region (CR). There were nine overlapping regions and seventeen intergenic spacers regions in the mitochondrial genome. The genome also showed a bias towards A + T content (55.01%) and had a positive AT-skew (0.08) and a negative GC-skew (-0.20). All the PCGs employed the ATG or GTG as the start codon and TAA, TAG, or single T as the stop codon. Additionally, all of the tRNAs displayed a typical cloverleaf secondary structure, except trnS1 which lacked the D arm. The phylogenetic analysis, based on the maximum likelihood (ML) and Bayesian inference (BI) methods, revealed that the topologies of the phylogenetic tree divided the Schizothorax into four clades and did not support the classification of Schizothorax based on morphology. The phylogenetic status of S. kozlovi was closely related to that of S. chongi. The present study provides valuable genomic information for S. kozlovi and new insights in phylogenetic relationships of Schizothorax. These data could also offer fundamental references and guidelines for the management and conservation of S. kozlovi and other species of Schizothorax.

Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout ageing

Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mitochondrial genome in different tissues throughout ageing. We used ultrasensitive duplex sequencing to profile ~2.5 million mitochondrial genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloguing ~1.2 million mitochondrial somatic and ultralow-frequency inherited mutations, of which 81,097 are unique. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the light strand origin of replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared with primates with a surfeit of reactive oxygen species-associated G > T/C > A mutations, and that somatic mutations in protein-coding genes exhibit signatures of negative selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that ‘re-align’ mito-nuclear ancestry within an organism’s lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.

Novel insights into adaptive evolution based on the unusual AT-skew in Acheilognathus gracilis mitogenome and phylogenetic relationships of bitterling

Acheilognathus gracilis, a bitterling species, distribute in lower reaches of Yangtze River. They are identified as the top-priority bitterling species for conservation as having high evolutionary distinctiveness and are at risk of extinction. In present study, we first sequenced the complete mitogenome of A. gracilis and analyzed its phylogenetic position using 13 PCGs. The A. gracilis mitogenome is 16,774 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, a control region and the origin of the light strand replication. The overall base composition of A. gracilis in descending order is T 27.9 %, A 27.7 %, C 26.1 % and G 18.3 %, shows a unusual AT-skew with slightly negative. Further investigation revealed A. gracilis uses excess T over A in NADH dehydrogenase 5 (nd5), whereas the most of other bitterlings are biased toward to use A not T, implying there is likely to be unique strategy of adaptive evolution in A. gracilis. We also compared 13 PCGs of 30 bitterling mitogenomes and the results exhibit highly conservative. Phylogenetic trees constructed by 13 PCGs strongly support the monophyly of Acheilognathus and the paraphyly of Rhodeus and Tanakia. Current results will provide valuable information for follow-up research on conservation of species facing with serious population decline and can provide novel insights into the phylogenetic analysis and evolutionary biology research.

The complete mitochondrial genome of Siganus virgatus Valenciennes (Siganidae: Acanthuriformes)

Siganus virgatus Valenciennes 1835 is an essential species for examining reef ecosystems; however, its mitochondrial genome has not been studied. In this research, the mitogenome of S. virgatus was sequenced and characterized. The results revealed a circular genome of 16,505 bp that was composed of A (28.1%), C (31.3%), G (14%), and T nucleotides (26.6%). The genome contained 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. Most genes of the mitogenome were transcribed on the heavy strand (H-strand), whereas ND6 and eight tRNA genes (including tRNA-Ala, -Asn, -Cys, -Gln, -Glu, -Ser (1), -Pro, and -Tyr) were transcribed on the light strand (L-strand). Comparative analysis revealed a high degree of conservation of gene content and order among the Siganus mitogenomes. Phylogenetic analysis inferred from whole mitogenomes exhibited a close relationship between S. virgatus and S. guttatus. The newly completed mitogenome of S. virgatus provides essential genomic data for further studies on population genetics and the evolution of the Siganus genus and the Siganidae family.

Diterpenoid honatisine overcomes temozolomide resistance in glioblastoma by inducing mitonuclear protein imbalance through disruption of TFAM-mediated mtDNA transcription

BackgroundGlioblastoma (GBM) represents as the most formidable intracranial malignancy. The systematic exploration of natural compounds for their potential applications in GBM therapy has emerged as a pivotal and fruitful avenue of research. PurposeIn the present study, a panel of 96 diterpenoids was systematically evaluated as a repository of potential antitumour agents. The primary objective was to discern their potency in overcoming resistance to temozolomide (TMZ). Through an extensive screening process, honatisine, a heptacyclic diterpenoid alkaloid, emerged as the most robust candidate. Notably, honatisine exhibited remarkable efficacy in patient-derived primary and recurrent GBM strains. Subsequently, we subjected this compound to comprehensive scrutiny, encompassing GBM cultured spheres, GBM organoids (GBOs), TMZ-resistant GBM cell lines, and orthotopic xenograft mouse models of GBM cells. ResultsOur investigative efforts delved into the mechanistic underpinnings of honatisine's impact. It was discerned that honatisine prompted mitonuclear protein imbalance and elicited the mitochondrial unfolded protein response (UPRmt). This effect was mediated through the selective depletion of mitochondrial DNA (mtDNA)-encoded subunits, with a particular emphasis on the diminution of mitochondrial transcription factor A (TFAM). The ultimate outcome was the instigation of deleterious mitochondrial dysfunction, culminating in apoptosis. Molecular docking and surface plasmon resonance (SPR) experiments validated honatisine's binding affinity to TFAM within its HMG-box B domain. This binding may promote phosphorylation of TFAM and obstruct the interaction of TFAM bound to heavy strand promoter 1 (HSP1), thereby enhancing Lon-mediated TFAM degradation. Finally, in vivo experiments confirmed honatisine's antiglioma properties. Our comprehensive toxicological assessments underscored its mild toxicity profile, emphasizing the necessity for a thorough evaluation of honatisine as a novel antiglioma agent. ConclusionIn summary, our data provide new insights into the therapeutic mechanisms underlying honatisine's selective inducetion of apoptosis and its ability to overcome chemotherapy resistance in GBM. These actions are mediated through the disruption of mitochondrial proteostasis and function, achieved by the inhibition of TFAM-mediated mtDNA transcription. This study highlights honatisine's potential as a promising agent for glioblastoma therapy, underscoring the need for further exploration and investigation.

Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspotsand association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Complete mitochondrial genome of Metathelazia capsulata (Pneumospiruridae) and comparison with other Spiruromorpha species.

Metathelazia capsulata (family Pneumospiruridae) is a lungworm parasitizing the bronchi and bronchioles, described in four species of wild carnivores. Very little molecular data are available on this nematode and none on other species of the Pneumospiruridae family. In this work, we describe for the first time the complete mitogenome (mitochondrial genome) of M. capsulata, being the first described of the family Pneumospiruridae. The mitogenome of M. capsulata has 13,659bp in length, an A + T content of 79.2%. The mitogenome included 12 protein-coding genes (PCGs) (lacking the atp8 gene), 22 tRNA genes, 2 rRNA genes (all the genes are coded by the heavy strand), and an AT-rich region. The PCGs varied in size (232bp-1645bp). Only the tRNA-Trp has the standard cloverleaf secondary structure, while the other 21 do not. The AT-rich region, with a 90.5% A + T content and a length of 389bp, is located between the cox3 and tRNA-Ala genes. Comparison with the mitogenomes of 29 species of Spiruromorpha infraorder, belonging to different families, demonstrates that M. capsulata mitogenome shared the common characteristics of most of them. The phylogeny constructions yielded phylogenies that were in agreement with the obtained previously by using sequences and gene order data of mitogenomes.

Phylogenetic relationships and introduction history inferred from complete mitochondrial genomes of four smelts (Osmeridae) of the modern San Francisco Estuary

We announce complete mitogenomes of four smelts (Osmeridae) of the San Francisco Estuary (SFE): endemic Hypomesus transpacificus, native H. pretiosus and Spirinchus thaleichthys, and non-native H. nipponensis. The genomes each contained 13 protein coding, 2 ribosomal RNA and 22 transfer RNA genes, a control region (D-loop), and a putative origin of replication of the light strand (OL); gene order followed other osmerids. Total genomes were 16,767 base-pairs (bp) for H. transpacificus; 16,672bp for H. pretiosus; 16,811bp for S. thaleichthys; and 16,779bp for H. nipponensis. GC content was from 47.8% (S. thaleichthys) to 49.4% (H. pretiosus). Phylogenetic analyses of mitogenomes placed H. transpacificus sequences within the H. pretiosus [Oregon + SFE] clade and most closely related to H. pretiosus [SFE], whereas affinities of derivative cytochrome b sequences supported a Lake Suwa origin for SFE H. nipponensis.

In silico prospecting of the mtDNA of Macrobrachium amazonicum from transcriptome data

BackgroundMacrobrachium amazonicum is a freshwater prawn widely distributed in South America that is undergoing speciation, so the denomination “M. amazonicum complex” is used for it. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used to elucidate this speciation, but heteroplasmies and pseudogenes have been recorded, making separation difficult. Obtaining genes from cDNA (RNA) rather than genomic DNA is an effective tool to mitigate those two types of occurrences. The aim of this study was to assemble in silico the mitochondrial DNA (mtDNA) of the Amazonian coastal population of M. amazonicum inhabiting the state of Pará.ResultsSequences were obtained from the prawn’s transcriptome using the de novo approach. Six libraries of cDNA from the androgen gland, hepatopancreas, and muscle tissue were used. The mtDNA of M. amazonicum was 14,960 bp in length. It contained 13 protein-coding genes, 21 complete transfer RNAs, and the 12S and 16S subunits of ribosomal RNA. All regions were found on the light strand except tRNAGln, which was on the heavy strand. The control region (D-loop) was not recovered, making for a gap of 793 bp. The cladogram showed the formation of the well-defined Macrobrachium clade, with high support value in the established branches (91–100). The three-dimensional spatial conformation of the mtDNA-encoded proteins showed that most of them were mainly composed of major α-helices that typically shows in those proteins inserted in the membrane (mitochondrial).ConclusionsIt was possible to assemble a large part of the mitochondrial genome of M. amazonicum in silico using data from other genomes deposited in GenBank and to validate it through the similarities between its COI and 16S genes and those from animals of the same region deposited in GenBank. Depositing the M. amazonicum mtDNA sequences in GenBank may help solve the taxonomic problems recorded for the species, in addition to providing complete sequences of candidate coding genes for use as biomarkers in ecological studies.

Complete mitochondrial genome of Parasa sinica: New insights into the phylogeny of Limacodidae

In this study, the whole mitochondrial genome (mitogenome) of Parasa sinica was sequenced. It contains 15,306 base pairs (bp), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one non-coding regulatory area (CR), all of which are shared by other lepidopterans. It follows the same gene order as ordinary lepidopterans. Further, out of these 37 genes, 23 are present on the heavy strand whereas the remaining 14 are located on the light strand. The A + T composition of the mitogenome is relatively high. Although P. sinica has a negative AT-skew and GC-skew, the GC-skew value is significantly lower than the AT-skew value. All PCGs, with the exception of CO1, carry the same start codon (ATN). All tRNAs exhibit the usual cloverleaf secondary structure. We identified the conserved motif “ATAGA + poly-T″ found in other lepidopteran insects at the beginning of the CR. We collected the concatenated PCGs sequences in the mitochondrial genome of 15 species of Zygaenoidea, with the sequences of Geometridae as outgroups, including P. sinica, and constructed phylogenetic trees using Bayesian inference (BI) and maximum likelihood (ML) methods. The monolineage of each superfamily is usually well supported. Based on phylogenetic analysis, P. sinica is a member of family Limacodidae, strongly supporting the monophyly of the Zygaenoidea groups.