Advanced molecular methods are having widespread impact and implications in parasitology [36–47]. In the present study, we utilised massively parallel sequencing and semi-automated bioinformatic annotation for the characterisation of the complete mt genomes of D. viviparus (from Bos taurus) and Dictyocaulus sp. cf. eckerti from red deer, and explored the genetic relationships of these two lungworms and selected representatives of the Strongylida. Using sliding window analysis (Figure 3), we also identified regions in these mt genomes which might serve as suitable markers for future molecular explorations of the systematics, population genetics or epidemiology of Dictyocaulus species.
Given the controversy surrounding the taxonomy/systematics of some species of Dictyocaulus[1, 5, 8, 9, 48–54], concatenated mt proteomic sequences might be applied effectively as barcodes to genetically characterise and compare dictyocaulids from various ungulate hosts, including domestic and wild bovids and cervids. This is particularly pertinent, given that Dictyocaulus from red deer is genetically distinct from Dictyocaulus species from roe deer (Capreolus capreolus), fallow deer (Dama dama) and moose (Alces alces) [7, 8, 54]. Current evidence shows a distinct genetic differentiation between Dictyocaulus sp. cf. eckerti and D. viviparus, supported by previous results for the ITS-2 region . A detailed appraisal of previously published findings for D. viviparus[12, 17] revealed a maximum nucleotide sequence variation of 3%, 3.5% and 3%, respectively, in partial cox 1, cox 3 or nad 5 regions (375–396 nt) among 72–252 individual worms. These levels of within-species variation are much lower than estimated levels of difference (9%, 15% and 18%, respectively) between Dictyocaulus sp. cf. eckerti and D. viviparus for the same gene regions (this study). At the amino acid level, variation in COX-1 (131 amino acids) within D. viviparus was ≤1.5%, but there was no amino acid difference between Dictyocaulus sp. cf. eckerti and D. viviparus (because of relative conservation). This contrasts the situation for COX3 (125 amino acids) and NAD5 (132 amino acids), for which maximum variation within D. viviparus was 1.6% and 2.3%, respectively (cf. ), and differences between D. viviparus and Dictyocaulus sp. cf. eckerti were substantially higher, at 10.4% and 13.6%, respectively. Together with previous ITS-2 data , this information indicates that Dictyocaulus sp. cf. eckerti and D. viviparus are separate species.
Whether the parasite from red deer (i.e., Dictyocaulus cf. eckerti) represents a distinct species from (or a population variant of) “D. eckerti” (cf. ) remains to be established. A previous study showed that the magnitude of sequence difference (~6-8%) in ITS-2 rDNA of Dictyocaulus from fallow deer from Germany  is greater than variation (0.4-2.6%) among selected individuals of Dictyocaulus from red deer , although the degree of genetic variation within the operational taxonomic unit from fallow deer is not yet known. D. eckerti was originally described from the reindeer, Rangifer tarandus, from Western Siberia (cited in ), which raises questions as to the specific identity of Dictyocaulus from various cervid species [5, 8, 13, 55]. Although Durette-Desset et al.  proposed that dictyocaulids of European cervids be called Dictyocalus noerneri, molecular and morphological data have shown that roe deer and moose can harbour D. capreolus (e.g., [8, 54]) and that chamois (Rupicapra rupicapra) might harbour a unique species . This controversy and the findings of numerous previous studies emphasize the need for detailed investigations of Dictyocaulus specimens from various species of wild and domestic bovid and cervid hosts from different continents, which could be achieved using a combined morphological and mt proteomic barcoding approach.
There is also significance in using mt genetic markers for studying the genetic composition of populations of Dictyocaulus spp., given that there are few morphological features for the specific differentiation of some developmental stages (i.e., larvae)  and given that cryptic species have been detected within the Strongylida [56, 57]. In nematodes, mt DNA is usually more variable in sequence within a species than ITS-2 and other rDNA regions , indicating that mt gene regions are well suited for studying the population genetics of parasitic nematodes [14, 56, 58, 59]. The sliding window analysis conducted herein displayed distinct patterns of nucleotide diversity between the two mt genomes representing Dictyocaulus (Figure 3). Low variability is useful for the design of oligonucleotide primers that flank mt regions with high variability for population genetic or epidemiological investigations. Some previous studies have shown the utility of some mt gene regions. For instance, Hu et al.  employed primer sets, originally designed to cox 1 of flatworms , for PCR-based mutation scanning and selective sequencing analysis of D. viviparus individuals from 17 different populations (farms). Within-species variation in cox 1 was low (0.3–2.3%) , similar to findings for some parasitic nematodes of plants and insects [61–65] but distinctly different from findings for gastrointestinal trichostrongyloid nematodes of domestic ruminants [66, 67]. In the present study, cox 1 is revealed to be the gene with the lowest nucleotide diversity between the two Dictyocaulus species. In another study, Höglund et al.  showed distinct genetic substructuring in D. viviparus populations from Sweden using four mt regions (in the cox3, nad5, trn C_M_D_G and rrn L genes) covering 11% (1542 bp) of the mt genome of D. viviparus (Figure 1). Although some of the gene regions (e.g., cox 3 and nad 5) used to date provided some resolution of genetic variation (<6% among haplotypes, upon pairwise comparison), the present sliding window analysis indicates other candidate genes for capturing greater diversity. That the cox 1 region (of 393 bp) is the most conserved relative to other regions (see Figure 3; M1) is a feature that facilitates the design of primers for PCR, but might come at the cost of missing signal (of sequence variation) required for analysis. Here, numerous sequence tracts in the mt genome have been identified as potentially suitable for population genetic or taxonomic studies (Figure 3). The comparison among the mt genomes of different species of nematodes characterised to date now provides the prospect for designing generic or specific primers to capture regions of variability most suited to the resolution needed for analysis, and also provides opportunities for a range of DNA amplification-based approaches, including multiplexed diagnostic methods (cf. ).
PCR primers can be designed rationally in conserved regions flanking “variable sequence tracts” within the mt genome considered to be most informative for population genetic studies of Dictyocaulus from different bovid and cervid hosts. Utilizing such primer sets, PCR-based single-strand conformation polymorphism (SSCP) analysis  could be applied to screen large numbers of individual specimens representing different host species and populations for haplotypic variability (at any developmental stage). A previous study has shown the merit of SSCP for exploring the genetic variation in various populations of D. viviparus in Sweden , and could be applied to large-scale studies of Dictyocaulus specimens representing distinct operational taxonomic units (based on ITS-2) and different ungulate host species.
Using a range of variable mt regions, in combination with classical parasitological techniques, it might also be possible to assess the cross transmission of particular species or genetic variants (haplotypes) of Dictyocaulus between/among cattle and cervid hosts, and their pathogenicity in different host species (cf. ). Furthermore, it will also be significant to extend mt genome sequencing to a range of lungworm species of domestic and wild ruminants, and assess their genetic relationships. The present study shows the relevance of the mt genomes of Dictyocaulus species for future systematic investigations and, importantly, stimulates a reassessment of the phylogenetic position of the family Dictyocaulidae in relation to the Metastrongylidae, Trichostrongylidae and other families within the order Strongylida (cf. ).