Alebion carchariae (Copepod: Caligidae) host plasticity and distribution: a new host and locality record from Ascension Island

Abstract

The first record of the parasite Alebion carchariae in the waters of Ascension Island collected from Galapagos sharks, Carcharhinus galapagensis (Carcharhinidae), is described. No previous record of this parasite exists for Ascension Island, nor have Galapagos sharks previously been listed as a host. Specimens of A. carchariae were identified using morphological techniques and DNA barcoding of the cytochrome c oxidase I subunit (COI) gene. This study provides the first COI barcodes for this species and a brief review of known hosts. We recommend further research to understand the life cycle of this parasite, its plasticity in terms of host/habitat selection, and to determine the implications of its presence on the hosts it inhabits.

Introduction

Copepods are the most abundant metazoan animals in the ocean, with over 14,500 accepted species of parasitic copepods recorded to date (Walter & Boxshall, 2022). Despite their abundance, the distribution of these species is patchily recorded across ocean basins, with their occurrence in remote locations poorly understood. Adding to patchily known species distributions can be useful in identifying stepping-stones of transport and colonisation, which facilitate widespread animal distributions. The order Siphonostomatoida contains 2233 species, of which an estimated 69% feed on the epidermal tissue, mucus or blood of marine fishes, ranging from small bait fish to large elasmobranchs (Austin et al., 2014; Ohtsuka et al., 2020). This order contains ~550 genera and nearly 40 families (Austin et al., 2014), including the genus Alebion which is contained within the family Caligidae. Kabata (1979) placed Alebion in the family Euryphoridae; however, Boxshall & Halsey (2004) recognise Alebion and other former euryphorid genera as members of the Caligidae. The caligids are a diverse group of copepods that predominantly parasitise marine fishes (Dojiri & Ho, 2013). The adults exhibit highly modified body forms. The adult female caligid body is dorso-ventrally flattened to allow the cephalothorax to serve as a suction cup to attach to the host (Dojiri & Ho, 2013 and sources therein). Since the first description of the genus Alebion by Krøyer in 1863, nine species have been described (formerly 14, later redescribed as 9; Cressey, 1972; Dippenaar, 2018).

Alebion carchariae has been recorded on at least 16 shark and one fish species (Table 1), with three records in the South Atlantic Ocean spanning from the eastern USA (listed hosts include smooth hammerhead, Sphyrna zygaena; scalloped hammerhead, Sphyrna lewini, see Rokicki & Bychawska, 1991), São Paulo (unspecified host, reviewed in Luque & Tavares, 2007) and off the coast of Senegal, West Africa (Vaissière, 1959). Here we describe the occurrence of this species in the nearshore waters of Ascension Island, a remote island located in the South Atlantic Ocean. Thereafter we provide the first DNA barcode sequence for this species.

Table 1. The diversity of recorded Alebion carchariae hosts

Data on the host species ecology (water type, depth range, habitat) were extracted from https://fishbase.se.

Materials and methods

Six specimens of Alebion carchariae were collected off the north-west coast of Ascension Island in the South Atlantic Ocean (7.9467°S 14.3559°W). On 27 January 2021, one specimen was removed from the snout region of a juvenile (~90 cm TL) Galapagos shark (Carcharhinus galapagensis) at Comfortless Cove (7.9104°S 14.4030°W). On 17 April 2021, an additional five specimens were removed from the face of a juvenile (<100 cm TL) Galapagos shark (Carcharhinus galapagensis) at English Bay (7.8933°S 14.3849°W). Hosts were caught unintentionally using light or medium-action fishing rods in both incidences and released promptly after manually removing the copepod/s from the host. Copepods were preserved in 70% ethanol and stored temporarily in the Ascension Island Government Conservation Centre on Ascension Island.

The copepod specimens were sent to the College of Fisheries and Ocean Sciences at the University of Alaska, Fairbanks (USA) and examined with a Leica M205C stereomicroscope, and photographed using a Leica DMC4500 camera. Two specimens were dissected to examine the legs and genital complex in further detail. The specimens were identified as Alebion carchariae based on the taxonomic descriptors of Cressey (1972), Boxshall & Halsey (2004), Dojiri & Ho (2013) and Dippenaar (2018). After dissection, specimens were stored in ethanol at −20°C. Subsequent DNA extraction and amplicon sequencing was conducted on two individual specimens.

DNA extraction, PCR amplification and sequencing

Prior to DNA extraction, individually selected and identified female A. carchariae were rinsed in sterile Milli-Q® water to remove all traces of ethanol to enable successful DNA extraction. Genomic DNA was extracted using a Qiagen DNeasy Blood and Tissue Kit with a final elution volume of 200 μl elution buffer. A 658 base-pair (bp) region of the mitochondrial cytochrome oxidase I (COI) gene (i.e. the barcode region) was targeted for species identification (Bucklin et al., 2011). Polymerase Chain Reaction (PCR) was carried out using 12.5 μl Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs Inc.), 1.25 μl of each forward and reverse primer (10 μM), 3–5 μl DNA template, and PCR grade water for a final reaction volume of 25 μl. COI was amplified with the universal primer pair LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-′3) (Folmer et al., 1994). PCR thermal cycle protocol consisted of 35 cycles of 98°C for 10 s, 62°C for 20 s, 72°C for 20 s and a final extension at 72°C for 2 min. PCR products were run on a 1.5% agarose TBE buffer gel stained with GelRed. Amplicons from successful PCR reactions were purified using ExoSAP-IT^TM Express PCR cleanup, combined with either the forward or reverse primer for bi-directional sequencing, and sequenced by Azenta Life Sciences (http://www.azenta.com).

Bi-directional COI sequences were assembled to create contiguous reads and checked for ambiguous base calling using Geneious Prime (Ver. R10). COI sequences for copepods in the family Caligidae with species-level identifications were retrieved from the NCBI GenBank public repository (www.ncbi.nlm.nih.gov/genbank) and analysed with COI data for A. carchariae obtained in this study. COI reads were aligned using the MUSCLE algorithm (Edgar 2004) and trimmed to uniform lengths. Neighbour-joining (NJ) gene trees were analysed after 10,000 coalescence simulations using a bootstrap test of 1000 replicates. The COI sequence for Demoleus heptapus (MH242722) represents the outgroup. Pairwise proportional nucleotide distances (p-distance) within and between Caligidae species were calculated in MEGA X (Kumar et al., 2018).

Results

Morphological identification

The body was dorso-ventrally flattened (Figure 1) with genital complex bearing elongated posterior processes extending slightly beyond the caudal rami. Lateral processes of the first abdominal somite reached the distal margin of the second abdominal somite. The genital complex had a slight bulge in the centre and margin armed with spinules, with legs 1–3 biramous, and leg 1 possessing 2 segmented rami (Figure 2A). Importantly, the specimen's exopodal spines of legs 1–3 were modified into large paddle-like structures (Figure 2B), characteristic of the genus Alebion. Additionally, legs 2 and 3 possessed 3-segmented rami (Figure 2C, D, E), while leg 4 was reduced and uniramous (Figure 2F). Leg 5 was represented by the elongated posterior processes extending from genital segment. Both female specimens were 8.6 mm in total length. The cephalothorax lengths of the individuals were 5.1 and 5.3 mm.

Fig. 1. Alebion carchariae female. (A) Body, dorsal view; (B) abdomen and caudal rami; posterior processes of genital segment, dorsal view; (C) spinules on margin of genital complex.

Fig. 2. Alebion carchariae female. (A) Leg 1; (B) Distal portion of exopod of leg 1; arrow indicates modified paddle-like exopodal spine; (C) Leg 2; (D) Leg 3 endopod; (E) Leg 3 exopod; (F) Leg 4.

Analysis of COI sequences

Two COI sequences of 557 base-pairs (bp) in length were sequenced for A. carchariae (GenBank accession nos ON807358 and ON807359). These data represent the first publicly available COI barcodes for A. carchariae and the first species-level COI data for the genus Alebion. Low pairwise distances (p-distances) were observed within individual A. carchariae (0.36%; N = 2) with high between-species distances observed when compared with Caligus spp. (18.0–28.1%; N = 9; Table 2). The high interspecific genetic variability for the COI barcode region is consistent with metazoan species (10–25%; Hebert et al., 2003). However, phylogenetic trees based on COI variation for species in the family Caligidae (Figure 3) revealed a bifurcating pattern with A. carchariae clustering separately from the other Caligidae species (100% bootstrap support). These data suggest A. carchariae and Caligus spp. may not belong in the same family, but additional COI data for species across multiple families in the order Siphonostomatoida are needed to resolve phylogenetic lineages.

Fig. 3. COI gene tree for copepods in the family Caligidae based on COI reads for Alebion carchariae generated in this study and species-level data available in GenBank (www.ncbi.nlm.nih.gov/genbank). Demoleus heptapus (MH242772) represents the outgroup. Neighbour-joining tree with Tamura & Nei (1993) substitution model; 1000× bootstrap values are indicated at nodes; values >50 are shown; scale bar indicates genetic distance.

Table 2. Pairwise genetic distances (p-distance) within and between eight species in the family Caligidae based on COI sequence variation. GenBank accession numbers for Caligus spp. are in parentheses

Discussion

Alebion carchariae (Krøyer, 1863)

New record. Ascension Island (7.9467°S 14.3559°W)

The recorded diversity of A. carchariae hosts suggests this parasite has plasticity in terms of the hosts it infects and the conditions it can tolerate, given the variety of habitats and depths utilised by these hosts. Documented hosts include sharks and fishes commonly distributed across reef habitats, benthic and pelagic to oceanic realms, with preferred depths ranging between 0–500 m. Current knowledge regarding the life cycle of caligids is based on studies of sea lice genera, including Caligus and Lepeophtheirus, which have been relatively well studied due to their economic impact on aquaculture (Boxaspen, 2006; Hemmingsen et al., 2020). The generalised caligid life cycle consists of two naupliar stages and one infective copepodid stage (Hamre et al., 2013). However, the number of stages between the copepodid and adult stage reported to vary between four chalimus stages (Pseudocaligus and Caligus species; Kim, 1993; Piasecki, 1996; Ohtsuka et al., 2009) to four chalimus plus two preadult stages (Lepeophtheirus species; Lewis, 1963; Voth, 1972; Boxshall, 1974; Johnson & Albright, 1991). The copepodid stage of caligids is infective, and the subsequent chalimus stages bear a frontal filament used to attach to the host. Wilson (1907) reported that Alebion glaber does not possess a frontal filament, but instead attaches to the host with prehensile antennae. It should also be noted that there are no published studies regarding the life cycle of caligid genera formerly included in the Euryphoridae, including Alebion. Experimental studies show the infective stage of a salmon louse (Lepeophtheirus salmonis) will aggregate in the upper water column to increase the probability of encountering potential hosts (Heuch et al., 1995). The lifespan of the infective stage of sea lice has been reported as lasting between 1–9 days (Johnson & Albright, 1991), and only upon attachment to a host can the lice fully develop and sexually reproduce. Life cycle estimates have ranged from less than two weeks (in controlled trials at 20 and 30°C; Khoa et al., 2019) to <210 days under laboratory conditions (Mustafa et al., 2001). The potential longevity of these species may enable them to hitchhike onto migratory species and facilitate their distribution (Boxaspen, 2006). For example, previous records of Alebion carchariae closest to Ascension Island include South America and West Africa, and longevity could facilitate its widespread distribution via hitchhiking on hosts traversing ocean basins or transport via ocean currents (enabling subsequent host infection). The described position of attachment of A. carchariae includes shark pectoral fins (C. obscurus, Vaissière, 1959) and the underside of the head (Sphyrna lewini, Vaissière, 1959), and fish gills (Coryphaena equisetis, Vaissière, 1959). Alebion has also been reported to wander the body surface of hosts (Cressey, 1972). Dorso-ventral flattening likely facilitates its successful adhesion by reducing drag when situated on the face (as observed in Nerocila, Nagler & Haug, 2016), and the versatility of its attachment location may increase its chance of success.

Further sampling of the parasitic assemblage around Ascension Island through opportunistic sampling, e.g. during fisheries surveys to collect mature individuals, and water sampling for pre-adult stages, could provide insight into the ecology and life cycle of this poorly studied genus and identify susceptible hosts. For example, data are required to determine the mechanisms that support its widespread distribution and its potential tolerance to a range of conditions (i.e. depths) that may facilitate its association with diverse hosts. Additionally, sampling this parasite may offer a minimally invasive technique to explore parasite-host interactions and/or the genetic structure of host populations such as C. galapagensis sharks around Ascension Island (following Meekan et al., 2017). While only seven A. carchariae were collected from two sharks it was noted that one individual hosted numerous other A. carchariae that were not collected. The implications of the presence of this parasitic copepod on host health and fitness remain unknown and warrants further research.