The complete mitochondrial genome of the stomatopod crustacean Squilla mantis
© Cook; licensee BioMed Central Ltd. 2005
Received: 10 October 2004
Accepted: 09 August 2005
Published: 09 August 2005
Animal mitochondrial genomes are physically separate from the much larger nuclear genomes and have proven useful both for phylogenetic studies and for understanding genome evolution. Within the phylum Arthropoda the subphylum Crustacea includes over 50,000 named species with immense variation in body plans and habitats, yet only 23 complete mitochondrial genomes are available from this subphylum.
I describe here the complete mitochondrial genome of the crustacean Squilla mantis (Crustacea: Malacostraca: Stomatopoda). This 15994-nucleotide genome, the first described from a hoplocarid, contains the standard complement of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding AT-rich region that is found in most other metazoans. The gene order is identical to that considered ancestral for hexapods and crustaceans. The 70% AT base composition is within the range described for other arthropods. A single unusual feature of the genome is a 230 nucleotide non-coding region between a serine transfer RNA and the nad1 gene, which has no apparent function.
I also compare gene order, nucleotide composition, and codon usage of the S. mantis genome and eight other malacostracan crustaceans. A translocation of the histidine transfer RNA gene is shared by three taxa in the order Decapoda, infraorder Brachyura; Callinectes sapidus, Portunus trituberculatus and Pseudocarcinus gigas. This translocation may be diagnostic for the Brachyura. For all nine taxa nucleotide composition is biased towards AT-richness, as expected for arthropods, and is within the range reported for other arthropods. Codon usage is biased, and much of this bias is probably due to the skew in nucleotide composition towards AT-richness.
The mitochondrial genome of Squilla mantis contains one unusual feature, a 230 base pair non-coding region has so far not been described in any other malacostracan. Comparisons with other Malacostraca show that all nine genomes, like most other mitochondrial genomes, share a bias toward AT-richness and a related bias in codon usage. The nine malacostracans included in this analysis are not representative of the diversity of the class Malacostraca, and additional malacostracan sequences would surely reveal other unusual genomic features that could be useful in understanding mitochondrial evolution in this taxon.
The mitochondria are extranuclear organelles present in all metazoans. They contain a circular genome, usually around 16 kilobases in length, with 37 genes (13 protein-coding, two ribosomal RNA genes, and 22 transfer RNA genes). This gene content is widely conserved, but gene order and the DNA sequences of the genes themselves are variable. Because of their small size many more mitochondrial genomes than nuclear genomes have been sequenced, and comparisons among them may serve as models for the evolution of the much larger nuclear genomes . In addition, gene order rearrangements and mitochondrial gene sequences have been widely used for phylogenetic inference [2–7].
At present there are about 650 complete mitochondrial genomes available in public databases. Of these, about 75 percent are of vertebrates. By contrast only 129 complete mitochondrial genomes are available from arthropods, which are the most diverse and speciose phylum of animals. In addition, there is considerable taxonomic bias among the available arthropod sequences; 86, (67 percent) are hexapods. The subphylum Crustacea includes over 50,000 named species and is ecologically and morphologically the most diverse of the arthropod groups, and therefore of all the animals. Crustaceans occupy marine, terrestrial, and fresh water habitats from the deep sea to high mountains; range in adult size from less than one millimeter to more than four meters (leg span); and exhibit extensive variability in body plans when compared to other arthropod groups . At present there are 23 complete crustacean mitochondrial genomes available. Within the Crustacea members of the class Malacostraca, which include crabs, lobsters, and shrimp, are perhaps the most well known to non-scientists. Due to their economic importance this group is often the focus of scientific enquiry. At present there are nine complete malacostracan mitochondrial genomes available, including that of the stomatopod shrimp Squilla mantis. In this paper I describe this genome and compare it to eight other mitochondrial genomes that are available from other Malacostraca.
Mantid shrimps, or stomatopods, are benthic predators distributed in the shallow waters of tropical and subtropical seas. They are best known for their raptorial appendages – pointed or clubbed – which they use to make lightning-fast attacks that disable prey animals by spearing or blunt trauma. Large individuals with the club-type appendages have been known to shatter the sides of aquaria . Squilla mantis (Linnaeus, 1758) (Crustacea: Malacostraca: Stomatopoda), with a maximum length of around 20 cm, is distributed in shallow waters throughout the Mediterranean Sea and Eastern Atlantic . S. mantis is widely consumed by humans throughout its range; UN Food and Agriculture Administration statistics indicate that total catches in the Mediterranean are currently in excess of 6500 tonnes per annum so this species is of some commercial importance .
Results and discussion
Mitochondrial genome composition
Complete mitochondrial genomes available for the Malacostraca. Taxononomic classification from the NCBI taxonomy browser  and from reference .
Nucleotide composition of Squilla mantis mitochondrial genome features. Major and minor strand genes show no significant differences in nucleotide composition.
Number of nucleotides
Proportion of nucleotides
All nucleotides (major strand)
rRNA genes (minor strand)
AT-rich region 1 (major strand)
AT-rich region 12(major strand)
tRNA genes (major strand)
tRNA genes (minor strand)
tRNA genes (both strands)
Unassigned nucleotides (major strand)
Protein coding genes
first codon positions
second codon positions
third codon positions
Majority strand protein coding genes
first codon positions
second codon positions
third codon positions
Minority strand protein coding genes
first codon positions
second codon positions
third codon positions
The large and small subunit ribosomal RNA (rRNA) genes (rnl, rns) have an AT content of 67%, within the range reported for other arthropod ribosomal RNA genes. Alignments of these genes with other arthropod homologues (not shown) as expected show both conserved and unconserved regions that correspond with the putative stems and loops within these genes. There are thus no unusual features to report for the two rRNA genes.
All of the 13 protein-coding genes, except cox1, have putative ATR methionine or ATT isoleucine start codons. The putative first codon of the cox1 gene is ACG threonine. The lack of a standard initiation codon in cox1 genes is common in arthropod mitochondria, so S. mantis is not unusual [17, 18]. Two of the protein-coding genes, cox1 and nad6, lack a full TAA or TAG stop codon. These genes appear to terminate with a single T from which a stop codon is created by polyadenylation of the mRNA during processing. Again, this phenomenon has been observed in other arthropod mitochondrial genomes and is not unusual [17, 19, 20].
Arthropod mitochondrial genomes typically have a long region that has an AT content higher than that of mitochondrial coding regions. This AT-rich region, ranging from 263 to 4601 base pairs in length and usually located between the rns and trnI genes, is often termed the control region because it contains a number of regulatory elements including the origin of replication for the heavy strand of the mitochondrial genome [21, 22]. In some arthropods the AT-rich region is reported to have any or all of these four different motifs: tandemly repeated sequences, a long sequence of T's, a subregion of even higher AT richness, and stem-loop structures [23, 24].
Annotation of the mitochondrial genome of Squilla mantis. Position numbers refer to positions on the major strand, with the first nucleotide of trnI assigned as number 1. Parentheses indicate genes encoded on the minor strand. Intergenic region numbers refer to the number of non-coding bases between features (positive integers) or the number of overlapping bases (negative integers). Asterisks (*) refer to incomplete termination codons that may be extended by post-transcriptional adenylation.
AT-rich region 1
AT-rich region 2
I therefore examined the shorter AT-rich region of 230 nucleotides between the trnS2 and nad1 genes for possible functional motifs. Most arthropod mitochondrial genomes have a few short non-coding regions between some genes, usually from a few bases to 20 bases long, but longer non-coding regions, such as AT-rich region 1 in S. mantis, are rare. It therefore seemed possible that this region might have taken over some of the functions putatively assigned to the longer AT-rich region. However, AT-rich region 1, like region 2, contains none of the motifs listed above. Furthermore, the AT content of this region, at 87%, is similar to that calculated collectively for other unassigned nucleotides in the S. mantis genome (Table 2) and is consistent with the hypothesis that this region has no function.
Unusual genomic features, such as this non-coding region or gene order rearrangements, can be useful as characters for reconstructing evolutionary relationships [13, 25]. A second AT-rich region is notably absent even in Harpiosquilla harpax, which is also a member of the family Squillidae. A survey of other members of the genus Squilla for the presence of a similar region would perhaps enhance our understanding of the history of this unusual genomic feature.
Comparison with other malacostracan crustaceans
A number of features of mitochondrial genomes can be used to infer relationships among taxa. These include phylogenetic analysis using DNA and protein sequences, relative rates of sequence evolution, gene order, and the effective number of codons (Nc). I present a phylogenetic analysis and a discussion of rates of sequence evolution in arthropods (including S. mantis) elsewhere , and discuss gene order and Nc below.
Codon usage in nine malacostracan mitochondrial genomes. Columns indicate GC content and effective number of codons (Nc) for majority strand genes, minority strand genes, and all genes. The least squares linear regression equation and the coefficient of determination are also shown for each data set.
Majority strand genes
Minority strand genes
Least sqaures equation
y = 84.83x+14.08
y = 116.49x+4.47
y = 145.8x-4.044
Coefficient of determination (R2)
R2 = 0.5015
R2 = 0.4493
R2 = 0.9139
This is the first formal description of the mitochondrial genome of a stomatopod crustacean. This genome maintains the same genes and gene order that are inferred as ancestral in the Pancrustacea, but does contain one unusual feature: a 230 base pair AT-rich region between the trnS2 and nad1 genes. This feature has no discernable function, but it may prove useful as a character in understanding the evolutionary history of the genus Squilla. Three other arthropod mitochondrial genomes have two large non-coding regions; the ostracod crustacean Vargula hilgendorfii, a millipede Thyropygus sp., and the tick Boophilus microplus [15, 33, 34]. Of these the latter two are clearly duplications of the orginal control region. Only V. hilgendorfii has, like S. mantis, two apparently unrelated non-contiguous AT-rich regions.
A comparison of nine malacostracan genomes, including that of S. mantis, shows that all nine exhibit the nucleotide composition bias favoring A and T nucleotides that is commonly observed for arthropod genomes, and that this bias is responsible at least in part for the observed codon usage bias in these genomes. However, there are no observable patterns of nucleotide composition bias or codon usage bias that unite particular taxa into common groups. These nine taxa represent only a small fraction of the diversity of the Malacostraca, and additional sequencing from across the diversity of this taxon would provide additional data for understanding the evolution of mitochondrial genomes of the class.
Samples and DNA extraction
A single freshly caught specimen of S. mantis was purchased from the fish market at Heraklion, Crete, Greece. Six grams of abdominal muscle were dissected from the specimen and immediately frozen at -70°C. Approximately one half gram of the frozen tissue was shaved from the specimen using a sterile razor blade and genomic DNA extracted using a QIAGEN genomic-tip 20 and the associated buffer set according to the manufacturer's protocol.
PCR, sequencing, and annotation
Short fragments (300–1000 nucleotides) of the mitochondrial genome were amplified at low stringency (50–55 degree annealing temperatures) using primers designed to work on all arthropods (Table 4). Amplification products were cloned into the T-Easy vector (Promega) and at least three clones from each PCR product were sequenced with vector-specific primers using ABI Big-Dye chemistry. Squilla-specific primers were designed and used to amplify longer fragments of 1000–4000 nucleotides that spanned the gaps between the short fragments. Longer fragments were also ligated into the T-Easy vector and at least three clones from each ligation were isolated. Each clone was sequenced using a primer-walking strategy initiated with vector-specific primers. Sequences were assembled using Sequencher v. 3.1 (GeneCodes Corp.). Protein-coding genes were identified using BLAST searches  and by comparison with other arthropod mitochondrial genome sequences. Transfer RNA genes were identified using tRNAscan-SE . Transfer RNA sequences were folded by eye, but made use of the tRNAscan-SE server output when that was available. The effective number of codons, Nc was calculated using the software package CodonW .
Primers used to amplify short fragments of the Squilla mantis genome. Majority/minority strand are with reference to the ancestral pancrustacean sequence. These primers will work for almost all arthropods and for many other metazoans as well. Reference "JB" is J. Boore pers. comm. Reference "CEC" refers to primers designed for this project.
This work was funded by the UK Biotechnology and Biology Research Council. CEC is currently funded by the UK Natural Environment Research Council. Many thanks to M. Averof for purchasing an individual S. mantis in Crete and to J. Boore for primer sequences and helpful discussions regarding methodology, annotation, and data presentation.
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