The term environmental DNA (eDNA) refers to DNA extracted from cells that are not collected directly from a target organism, but are obtained from the environments they inhabit such as oceans, river water, soil, and air (Ficetola et al., 2008; Fonseca et al., 2010; Andersen et al., 2012; Sigsgaard et al., 2016). Invertebrate-derived DNA (iDNA) is an offshoot of this approach that involves the extraction of genetic material of animals via the flesh-eating or haematophagous invertebrates that parasitise them (Schnell et al., 2015; Schubert et al., 2015; Lee et al., 2016). To date, most iDNA studies have focused on terrestrial vertebrates and have extracted host DNA from insects, ticks, or leeches. There have been no analogous studies in marine environments, despite the potential usefulness of the approach for sampling large marine megafauna such as cetaceans, sirenians, pinnipeds, marine reptiles (sea turtles), elasmobranchs, and teleosts. For such taxa, iDNA sampling offers both ethical and practical advantages as it is minimally-invasive and thus preferable to the direct collection of blood or tissue, particularly where target species are rare or endangered.

The whale shark (Rhincodon typus), similar to many large marine vertebrates, poses significant challenges for researchers trying to understand their biology and ecology and for managers attempting to develop conservation strategies (Graham and Roberts, 2007; Rowat et al., 2009). Tagging, genetic and modeling studies suggest that individuals can disperse widely (Sequeira et al., 2013), although there is evidence of isolation between populations in the Indo-Pacific and Atlantic oceans (Vignaud et al., 2014). However, the scale at which population structure can be discerned is dependent on sample sizes, with low numbers reducing analytical power to reject the null hypothesis of a panmictic population (Castro et al., 2007). For whale sharks, low sample size tends to be a result of the rarity of the species and the difficulties in obtaining biopsies (usually of skin tissue) for genetic analyses, which requires appropriate permits and ethical approvals for invasive sampling of a species that is categorized as “Endangered” by the International Union for the Conservation of Nature and the use of trained divers and technicians. In this situation there are many benefits to the use of an iDNA approach, provided that external invertebrate parasites or commensals that harbor the intact DNA of the whale shark host can be successfully identified. Although eDNA techniques have recently been used to obtain whale shark DNA (Sigsgaard et al., 2016), iDNA still offers a major advantage because it enables haplotypes to be linked to individual whale sharks for the analysis of the genetic structures of populations.

Copepods (Crustacea, Maxillopoda, Copepoda) are an excellent candidate species for iDNA studies on marine vertebrates. Free-living forms are a dominant element of marine zooplankton and may even exceed insects in terrestrial environments in terms of sheer abundance of individuals. Less recognized is that approximately half of the nearly 30,000 described species live in parasitic or commensal associations with a diverse range of taxa, including fish and mammals (Boxshall, 2005). The order Siphonostomatoida contains largely parasitic copepods that feed on the blood, epidermal tissue, or mucus of many marine teleost fishes, sharks, and rays. Approximately 550 genera, representing nearly 40 families, are placed in the order and include economically important species such as sea lice (Brachiura) that parasitise farmed fish (Gunn and Pitt, 2012). Eleven siphonostomatoid families have been reported as symbionts of a diverse range of elasmobranchs (Dippenaar, 2009) and one family, the Pandaridae, is composed of 23 genera that include species that are ectoparasites or commensals of large sharks (Cressey and Boyle, 1978; Walter and Boxshall, 2017). Within the Pandaridae, the genus Pandarus currently has 17 recognized species of which P. rhincodonicus (Norman et al., 2000) is noteworthy as it is appears to be associated exclusively with the whale shark, where it is predominately found on the leading and trailing edges of fins and on the lips. Although it is thought to be a commensal that feeds off bacteria and other microorganisms on the skin of the shark (Norman et al., 2000), dietary studies of the species are incomplete.

Here, we demonstrate that P. rhincodonicus sampled from sites across the Indian Ocean contain sufficient DNA from their whale shark host to routinely recover mitochondrial sequences that allow analyses of the genetic structure of host populations, and the recovery of the complete whale shark mitogenome and ribosomal nuclear genes. The implications of these findings are discussed in relation to the use of copepods for iDNA studies of marine teleosts and sharks and the ecological status of Pandarus spp. as commensals.

Our study is the first demonstration of the use of iDNA sampling of a marine invertebrate in order to obtain mitochondrial genetic information from an elasmobranch host. We successfully amplified mitochondrial DNA fragments of whale sharks in the size range of ~1,000 bp from a copepod, one of the largest host DNA fragments to be recovered by an iDNA study (Schnell et al., 2012, 2015; Lee et al., 2016; Pérez-Flores et al., 2016; Rodgers et al., 2017). This suggests the presence of largely intact whale shark DNA in or on copepods, although the exact source could not be identified as copepods were analyzed whole. Targeted efforts to amplify DNA fragments of whale sharks from the intestine and epidermal layers of P. rhincodonicus will be useful to resolve this issue and clarify the role of the copepod as a commensal or parasite. We found iDNA sequences from multiple copepods sampled from the same whale shark host to be identical, suggesting copepods have a persistent, long-term association with their host shark, which contrasts with the more generalist and mobile host associations of other invertebrate ectoparasites in terrestrial and aquatic ecosystems, such as leeches, blow flies, and mosquitoes (Calvignac-Spencer et al., 2013). However, these findings should be treated with some degree of caution and a multi-locus approach using microsatellite markers (Olson et al., 2012) might offer a more powerful means to validate these findings. As reported by Vignaud et al. (2014), our estimates of genetic structure across the Indian, Pacific, and Atlantic oceans indicated the presence of two distinct populations, one in the Indo-Pacific and the other in the Atlantic Ocean. Isla Holbox was the only location sampled in the Atlantic Ocean and the minimally-invasive iDNA approach could potentially be used to improve the coverage of sampling in this region.

We also demonstrated that complete mitochondrial genome and nuclear ribosomal RNA sequences of whale sharks could be obtained using iDNA sampling. Although not the first for the species (Alam et al., 2014; Chen et al., 2016), the sequences generated by our study represent the first for the Mozambique population of whale sharks. The successful recovery of the mitogenome and nuclear ribosomal RNA from the shotgun sequencing of P. rhincodonicus was probably due to the high copy number of mitochondrial and nuclear ribosomal genes in somatic cells, coupled with high sequencing depth (~14 million paired-end reads). Efficiency of the recovery of whale shark mitogenomes from copepod tissue samples might be improved by sequencing gDNA extracted from the part of the copepod body that contains higher concentrations of whale shark tissue. In addition, given the intactness of whale shark gDNA (>1,000 bp) that was present in P. rhincodonicus, long range PCR of additional mitogenome regions followed by high throughput amplicon sequencing could also be explored to dramatically increase the coverage of the whale shark mitogenome for population genetic and phylogeographic applications (Deiner et al., 2017; Pavlova et al., 2017). Given that mtDNA only represents a fraction of the evolutionary history of a species due to its strict maternal inheritance and small genome size (16 kb), studies of the population genetics of whale sharks may benefit from the combination of both mtDNA and nuclear data (Godinho et al., 2008; Schmidt et al., 2009; Vignaud et al., 2014). The presence of nuclear ribosomal RNA reads in the copepod shotgun sequencing library suggests that it is possible to amplify both nuclear DNA and mtDNA of whale sharks from the copepod and the recent publication of the first draft genome for the whale shark (Read et al., 2017) will greatly facilitate the design of a targeted next-generation sequencing panel to enhance future studies of whale shark population genetics.

The Pandaridae comprises 23 genera and more than 100 species, many of which are ectoparasites or symbionts of large sharks (Walter and Boxshall, 2017), and there are nearly 30 other families of copepods that include species that are parasites of fishes (Boxshall, 2005). These offer an opportunity to extend iDNA methods to other taxa of large marine vertebrates. Conversely, the presence of gDNA of whale sharks in the extracted DNA of P. rhincodonicus will need to be considered as a possible contaminant in future whole genome sequencing or population genomic projects that target this copepod.

MM and CA: Original concept, sample collection, analyses, interpretation of results, writing, proof-reading. MT: Bioinformatics analysis. N-WW: Control region sequencing. AM: interpretation of results, writing, proof-reading. SP, DR, GS, TD, and AP: sample collection proof-reading. HG: analyses, writing, interpretation of results, proof-reading.