Research Article |
Corresponding author: Romain Gastineau ( romain.gastineau@usz.edu.pl ) Corresponding author: Marcel Koken ( mhmkoken@gmail.com ) Academic editor: Nina Bogutskaya
© 2023 Romain Gastineau, Christian Otis, Brian Boyle, Claude Lemieux, Monique Turmel, Jérôme St-Cyr, Marcel Koken.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Gastineau R, Otis C, Boyle B, Lemieux C, Turmel M, St-Cyr J, Koken M (2023) The mitochondrial genome of the bioluminescent fish Malacosteus niger Ayres, 1848 (Stomiidae, Actinopterygii) is large and complex, and contains an inverted-repeat structure. ZooKeys 1157: 177-191. https://doi.org/10.3897/zookeys.1157.97921
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We determined the complete mitogenome sequence of the bioluminescent fish Malacosteus niger using long-read sequencing technologies. The 21,263 bp mitogenome features a complex structure with two copies of a 1198-bp inverted-repeat and a region of 2616-bp containing alternating copies of 16 and 26 bp repeat elements. Whole mitogenome phylogenies inferred from both nucleotide and amino-acid datasets place M. niger among Melanostomiinae. The need for additional complete mitogenome sequences from the subfamily Malacosteinae is discussed.
Bioluminescence, Deep Sea Dragonfish, inverted-repeat, long-read sequencing, Malacosteinae, mitogenome, Stomiidae
Sunlight is filtered by seawater and only blue/green light with wavelengths between 460 and 490 nm penetrates into the deep sea. Many deep-sea animals with well-adapted eyes can see this very weak light down to about 1500 m (
The peculiar mesopelagic black loose-jaw dragonfish, Malacosteus niger
Malacosteus niger model made by 10TONS (www.10tons.dk). These most peculiar and unique fishes are frequently damaged by fishing gear and this model allows for the first time to visualize a faithful representation. Courtesy of Esben Horn.
Like almost all deep-sea fish, M. niger encode only blue opsin photoreceptor genes in order to detect the down welling as well as the bioluminescent light omnipresent in the environment. However, M. niger adapted its eyes by linking antenna-pigments (a red-absorbing bacterio-chlorophyll c) to its blue opsin proteins (
We initiated this study by analyzing the mitogenome of M. niger with short read sequencing technologies. However, all our efforts to assemble the complete M. niger mitogenome proved unsuccessful. The mitogenome always came out as two distinct contigs of similar coverage and all attempts to join them were fruitless whatever parameters were used for assembly. It quickly appeared that repeated sequences were at the base of this problem and that long-read sequencing technologies were clearly needed to resolve the complex mitochondrial genome. The results presented here show yet another peculiarity of M. niger with the discovery of a large and complex mitochondrial genome harboring inverted-repeat-like structures.
The Malacosteus niger specimen used in this study was caught during the Bear Seamount cruise DE200611 (station 012, 18/6/2006). Pieces of the caudal fin and muscle were sent to the “Plateforme d’Analyses Génomiques” of the “Institut de Biologie Intégrative et des Systèmes” of the Université Laval (Québec, Canada) for DNA library preparation and sequencing. For DNA extraction, 300 mg of muscle were crushed in liquid nitrogen and digested at 65 °C for 30 min in 1.0 ml lysis buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM EDTA, 2.0% SDS and 20 mg/ml proteinase K. An equal volume of CTAB buffer containing 50 mM Tris-HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2.0% CTAB, 1.0% PVP 40,000 was added to the lysate and incubation was pursued for an additional 30 min at 65 °C. This mixture was extracted with phenol: chloroform: isoamylalcohol (25:24:1), and following centrifugation, 5 µl of RNase A (100 mg/ml) was added to the aqueous phase and incubated at room temperature for 20 min. This mixture was then extracted twice with an equal volume of chloroform: isoamylalcohol (24:1) and DNA was precipitated with two volumes of EtOH, dried and dissolved in 100 µl of TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
The library preparation protocol for short reads sequencing was as follows. Genomic DNA (500 ng in 55 ul TE buffer) was mechanically fragmented for 40 s using a Covaris M220 (Covaris, Woburn MA, USA) with default settings. Fragmented DNA was transferred to PCR tubes and library synthesis was performed using a NEB Next Ultra II kit (New England Biolabs) according to the manufacturer’s instructions. To barcode the samples, TruSeq HT adapters (Illumina, SanDiego, CA, USA) were used. The library was sequenced on the Illumina MiSeq platform (300-bp paired-end reads). Of the 15 335 342 raw paired-end reads obtained, 11 121 576 remained after elimination of low-quality reads.
For long-read sequencing, DNA was quantified using a Qubit fluorometer (ThermoFisher) and quality checked on a Femto Pulse System with a genomic DNA 165-kb kit (Agilent, Santa Clara, CA, USA). A DNA aliquot of 8 µg was fragmented with a Covaris g-tube (Covaris Woburn, MA, USA) and small fragments were removed using Short Read Eliminator XS (Circulomics/PacBio, Menlo Park, CA, USA). The library for Oxford Nanopore MinIon sequencing was prepared using 1.69 µg of DNA and the LSK-109 ligation sequencing kit (Oxford Nanopore, Littlemore, UK), following repair and end-polishing of the sheared DNA using the NEBNext Companion Module for Oxford Nanopore Technologies ligation kit (New England Biolabs, Ipswich, MA, USA). Finally, 0.595 µg of library were loaded on a R9.4.1 MinION flow cell and sequencing was performed on a GridIon benchtop platform (Oxford Nanopore).
All bioinformatics analyses were performed on the THOT superdome flex server at “Université Laval”. MiSeq reads were first processed with AfterQC (
Annotation was performed with the help of MITOS (
Protein-coding genes and the corresponding amino-acid sequences were extracted from the mitochondrial genomes of 15 taxa of Stomiiformes, including M. niger. Xiphias gladius Linnaeus, 1758 was used as an outgroup. Nucleotide and inferred amino-acid sequences of all conserved mitochondrial genes (ATP6, ATP8, cox1, cox2, cox3, cytB, ND1, ND2, ND3, ND4, ND4L, ND5, ND6) were first concatenated for each species/dataset and then aligned using MAFFT 7 (
The mitochondrial genome has been submitted to GenBank with accession number OP326280. The raw fasta file, the annotated gbk file and a fastq file containing the longest Oxford Nanopore read supporting the assembly can be found on Zenodo following this link: https://doi.org/10.5281/zenodo.7330521.
The size and sequence coverage of the mitogenome contigs obtained after SPAdes assembly of short sequencing reads are indicated in Table
Sizes, coverage and gene contents of the M. niger mitogenome contigs obtained after assembly of short sequencing reads.
Size of the contig (bp) | 12 311 | 4467 | 1198 | 750 |
---|---|---|---|---|
Coverage | 162.46X | 156.70X | 270.75X | 121.78X |
Gene content | ND2, cox1, cox2, ATP8, ATP6, cox3, ND3, ND4L, ND4, ND5, ND6, cob, 18 tRNA genes | ND1, rrnS, rrnL, 3 tRNA genes | None | None |
Oxford Nanopore sequencing was undertaken to confirm and resolve the contig overlaps that were identified using the short read approach. Table
Basic statistics of the Nanopore reads before and after Filtlong filtering.
Nanopore reads (before filtering) | Nanopore reads (after filtering) | |
---|---|---|
Mean read length (bp) | 3854 | 4093 |
Mean read quality | 13.2 | 14.4 |
Median read length (bp) | 1754 | 3304.0 |
Median read quality | 13.2 | 14.4 |
Number of reads | 212 934 | 153 |
Read length N50 (bp) | 8515 | 6327 |
Total bases (bp) | 820 590 847 | 626 269 |
Top 5 longest reads (bp) | 78 432, 77 491, 63021, 58 837, 58 554 | 20 972, 17 597, 14 526, 14 300, 12 821 |
The 21 263 bp mitogenome of M. niger (GenBank: OP326280) contains 46% G+C and encodes 13 proteins, 22 tRNAs and 2 rRNAs (Fig.
Sequences of the repeat elements identified in the region of the M. niger mitogenome located between the tRNA-Met and tRNA-Phe genes.
Sequence of the repeated motif | Number of occurrences |
---|---|
(5’-CATATATCAATATCGACATATGTCAATATTGACATATATCA-3’) | 16 |
(5’-GTCAATACAAACGCATGTGTTTTTAT-3’) | 26 |
Phylogenetic analyses of the 11 411 nucleotides and 3795 amino-acid datasets were conducted using the GTR+I+G and mtMAM+I+G+F evolutionary models, respectively. Separation between the Stomiidae and Gonostomatidae was weakly supported in trees inferred from both datasets, but several nodes within the Stomiidae clade proved to be more robust, especially in the amino acid inferred phylogeny. Both the nucleotide and amino-acid trees revealed that the Malacosteinae M. niger is sister to the Melanostomiinae Tactostoma macropus Bolin, 1939 (LC377784), forming a clade with the Melanostomiinae Photonectes margarita (Goode & Bean, 1896) (AP018417) and Trigonolampa miriceps Regan & Trewavas, 1930 (AP012961). This group of taxa is sister to a clade containing the Stomiinae Stomias atriventer Garman, 1899 (MG321595) and the Chauliodontinae Chauliodus sloani Bloch & Schneider, 1801 (AP002915) in the amino-acid phylogeny (Fig.
With 21 263 bp, the mitogenome of M. niger is among the largest described so far among the Actinopterygii. Recent studies have unveiled very large mitochondrial genomes among metazoan invertebrates, with a current record of 93 065 bp being held by the parasitic cnidarian Polypodium hydriforme Ussov, 1885 (MN794187) (
Inverted-repeat structures are a common feature among plastomes (
Unveiling unusual features in mitogenomes often faces technical limitations, such as those described in the current study that resulted from the use of short sequencing reads. Discovery of metazoan mitogenomes with anomalous characteristics will certainly become more common with increased use of long-read sequencing. In recent studies, long-read sequencing has been decisive in resolving the complex control regions of mitogenomes from Gastropoda (
Additional mitogenome sequences from the Malacosteinae are clearly needed to resolve the phylogenetic position of these bioluminescent fishes. Malacosteus niger is the only representative of the Malacosteinae that has been sampled so far among the three genera described in this subfamily. It will be particularly important to analyze the mitogenomes from the two remaining genera (Aristostomias Zugmayer, 1913 and Photostomias Collett, 1889) that harbor a total of 14 valid species. These studies are expected to shed light not only on the phylogenetic positions of these bioluminescent fishes but also on the putative presence, origin and evolution of the inverted-repeat structure among the mitogenomes of Malacosteinae.
Dr Tracy Sutton (Oceanic Ecology lab, Nova South-Eastern University, FL, USA) is thanked for determination and provision of several M. niger specimens. We thank Stéphane Larose of the bioinformatics platform from Laval Université for his constant and patient help with IT. We would like to thank Esben Horn (https://www.10tons.dk/) for his marvellous model of Malacosteus niger and for letting us use a photo of it in this publication.
This work was supported by the 2017–2022 research funds granted for implementation of a co-financed international research project from the Polish Ministry of Science and Higher Education. CL and MT are supported by grant RGPIN-2017-04506 from the Natural Sciences and Engineering Research Council of Canada. BB, JSC and CO are supported by the “Programme d’appui aux plateformes technologiques stratégiques” from the Ministère de l’Économie, de l’Innovation et de l’Énergie Québec.