Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

Abstract

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, Lepeophtheirus salmonis

“…The polymerase chain reaction (PCR) primer sets were designed for 12 fragments of the Pacific salmon louse ( Fig. 2 and Supplemental Table 1) based on the EST sequences encoding mitochondrial DNA (mtDNA) genes and the complete mtDNA genomic sequence previously reported by Tjensvoll et al (2005). PCR amplification was performed using 1.0μL of extracted total genomic DNA of L. salmonis with an initial denaturation step of 2min at 95°C and then 30 cycles as follows: 30s of denaturation at 95°C, 30s of annealing at 55°C, and 3min of extension at 72°C.…”

“…In the normalized library, the average contig had 1.33 sequences with the largest contig consisting of ten individual sequence reads. A combined total of 14,994 EST sequences from all of the libraries were assembled into 5,256 unique contigs, of which 1,407 were composed of single sequences, 3,849 composed of two or more sequences, and 1,326 of three or of the complete 14.5-kb mtDNA genome of a previously described Atlantic isolate (GenBank: AY625897; Tjensvoll et al 2005). The EST contigs for Pacific L. salmonis mtDNA genes differed from Atlantic mtDNA by an average of 8%.…”

“…2). Structurally, the Atlantic (Tjensvoll et al 2005) and Pacific mtDNA genomes are identical in gene organization, direction of transcription, and transfer RNA locations (Fig. 2).…”

Sequence variation in four mitochondrial genes of the salmon louse Lepeophtheirus salmonis

“…The complete gene sequences of COI, Cyt b, A6 and 16S rRNA were amplified from 180 Lepeophtheirus salmonis using primers constructed from the complete mitochondrial sequence obtained by Tjensvoll et al (2005) (Table 1). The PCR mixture (50 µl) contained 1 × PCR buffer with 1.5 mM MgCl 2 (Amersham Pharmacia Biotech), 25 mM of each dNTP (Promega), 0.2 µM of each primer (Invitrogen), 1U Taq DNA polymerase (Amersham Pharmacia Biotech) and 300 ng DNA.…”

“…The complete gene sequences of COI, Cyt b, A6 and 16S rRNA were amplified from 180 Lepeophtheirus salmonis using primers constructed from the complete mitochondrial sequence obtained by Tjensvoll et al (2005) Sequencing and sequence analysis. PCR products were purified with QIAquick PCR purification kit (Qiagen) as described by the manufacturer.…”