Introduction
Elucidating the life cycles of parasites with complex life histories is useful for understanding the pathology of infections (Catalano et al., Reference Catalano, Hutson, Ratcliff and Whittington2011) and to elucidate trophic linkages (Williams et al., Reference Williams, MacKenzie and McCarthy1992), migrations (e.g. Carballo et al., Reference Carballo, Cremonte, Navone and Timi2012) and environmental changes (Palm et al., Reference Palm, Kleinertz and Rückert2011). Although the rate at which new parasites are being identified is accelerating, elucidation of life cycles has waned (Blasco-Costa & Poulin, Reference Blasco-Costa and Poulin2017).
Until recently, jellyfish have been considered trophic dead ends (i.e. rarely eaten; Hays et al., Reference Hays, Doyle and Houghton2018), and so their potential role as vectors for transferring intermediate stages of digenean parasites to vertebrate hosts was seldom considered. New technologies, including DNA metabarcoding, animal-borne cameras and stable isotope analysis, however, reveal that a surprising diversity of vertebrates prey on jellyfish, including flying seabirds, penguins, turtles and fishes (Hays et al., Reference Hays, Doyle and Houghton2018). Indeed, jellyfish are now recognized to be consumed by at least 124 species of fishes (Arai, Reference Arai1988; Purcell & Arai, Reference Purcell and Arai2001; Arai, Reference Arai2005; Pauly et al., Reference Pauly, Graham, Libralato, Morissette and Deng Palomares2009). Digeneans are often more prevalent in jellyfish than in other zooplankton intermediate hosts such as copepods (Marcogliese, Reference Marcogliese1995) and thus may be important for transferring parasites to definitive hosts.
Jellyfish have been reported as intermediate hosts for the metacercariae of at least 17 digenean species and nearly 70 species of jellyfish (medusae, siphonophores and ctenophores) host digeneans (Browne, Reference Browne2015). The metacercariae found in jellyfish, however, are difficult to identify, which has hampered our ability to identify them and their definitive hosts. Life cycles of digeneans infecting jellyfish have thus been primarily elucidated by feeding experiments (e.g. Stunkard, Reference Stunkard1980b). Such studies have shown that some lepocreadiid cercariae emerge from their first intermediate mollusc host, swim in the water column, and directly penetrate jellyfish, which are the second intermediate host, where they form metacercariae (Stunkard, Reference Stunkard1969, Reference Stunkard1972, Reference Stunkard1980a, Reference Stunkardb; Køie, Reference Køie1975). When the jellyfish are eaten by fish, the metacercariae develop into sexual adult digeneans (Stunkard, Reference Stunkard1969, Reference Stunkard1980a, Reference Stunkardb). Lepocreadiid genera that use jellyfish as intermediate hosts include species of Opechona Looss, 1907 (e.g. Køie, Reference Køie1975; Stunkard, Reference Stunkard1980b; Martorelli, Reference Martorelli2001), Cephalolepidapedon Yamaguti, 1970 (e.g. Ohtsuka et al., Reference Ohtsuka, Kondo and Sakai2010), Lepidapedon Stafford, 1904 (Køie, Reference Køie1985), Lepocreadium Stossich, 1904 (e.g. Stunkard, Reference Stunkard1972, Reference Stunkard1980a; Bray & Cribb, Reference Bray and Cribb1996) and Lepotrema Ozaki, 1932 (e.g. Ohtsuka et al., Reference Ohtsuka, Kondo and Sakai2010).
Analysis of ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) enables the identification of digenean species (Nolan & Cribb, Reference Nolan and Cribb2005; Blasco-Costa et al., Reference Blasco-Costa, Cutmore, Miller and Nolan2016). Sequences from unidentified metacercariae can be matched to those of adults and, coupled with morphological data and knowledge of host biology, allow insights into the life cycles of the parasites (Cribb et al., Reference Cribb, Anderson, Adlard and Bray1998). Molecular identification has not been applied to metacercariae found in jellyfish, but this approach may reveal jellyfish to be significant intermediate hosts of digeneans that infect marine vertebrates.
This study utilized the ITS2 of rDNA. This region is highly variable yet relatively conserved within platyhelminth species, and, thus, is useful for identifying larval forms (Adlard et al., Reference Adlard, Barker, Blair and Cribb1993; Tandon et al., Reference Tandon, Prasad, Chatterjee and Bhutia2007; Skov, 2009). The aims of this study were to identify metacercariae from jellyfish by comparing ITS2 sequences of the metacercariae from jellyfish to those of adult digeneans in fishes that predate upon jellyfish, and to thus investigate the role of jellyfish in the life cycles of digeneans.
Materials and methods
Collection of specimens
Jellyfish were sampled opportunistically from within Port Phillip Bay (38°05′17″–38°17′1″S, 144°36′54″–144°43′58″E), Australia, between September 2009 and February 2012. Three species were collected: three Cyanea annaskala Von Lendenfeld, 1882 (Scyphozoa; Semaeostomeae); seven Pseudorhiza haeckeli Haacke, 1884 (Scyphozoa; Rhizostomeae) and six Aequorea eurodina Péron & Lesueur, 1810 (Hydrozoa; Leptothecata; table 1). Cyanea annaskala and P. haeckeli were collected using a dip net from a boat or by zip-lock bag while snorkelling. Aequorea eurodina were collected from the shoreline after having been washed ashore. Although these medusae were dead, the digenean metacercariae in them were alive when collected. Jellyfish were returned to the laboratory (alive if possible) and their diameters were measured. Medusae were examined under a Leica Wild M8 stereomicroscope (Leica Microsystems Pty Ltd, North Ryde, NSW, Australia) and digeneans were removed. The location of digeneans within the jellyfish (bell, tentacles, oral arms (P. haeckeli only), oral pillar/disk and gut) was recorded.
Digenean species, species of hosts dissected, number of hosts (n), prevalence (P) of infection, mean intensity of digeneans and range, and size of host (bell diameter (BD) for jellyfish, total length (TL) for fish).
a Mean intensity only calculated from three fish specimens. SE, standard error.
Three fish species known to feed upon jellyfish were obtained between June 2009 and April 2011 (table 1): Scomber australasicus Cuvier, 1832; Seriolella brama (Günther, 1860); and Seriolella punctata (Forster, 1801). Five S. brama were trawled within Port Phillip Bay (37°58′30″–38°14′34″S, 144°46′01″–144°52′38″E); three S. punctata were trawled near Portland, Victoria (within the vicinity of 38°20′S, 141°36′E) and two S. australasicus individuals were caught in Moreton Bay, Queensland (27°07′54.28″S, 153°21′10.63″E). Although S. australasicus were sampled at a location distant from that of the jellyfish, their distribution overlaps with that of A. eurodina and so was considered worthy of investigation.
Fish were refrigerated or stored on ice (S. punctata and S. brama) or frozen (S. australasicus), and had been dead for 10–48 h before dissection. The total length of each fish was measured, and the digestive tract was removed and separated into stomach, pyloric caeca and intestine. Each section was examined for digeneans using a stereomicroscope, then shaken in a saline solution (one part seawater to three parts tap water) before re-examination (following Cribb & Bray, Reference Cribb and Bray2010).
Digeneans were removed from the jellyfish or fish/gut washes with a pipette and fixed in near-boiling seawater solution (Cribb & Bray, Reference Cribb and Bray2010). They were assigned to a morphotype and preserved in 10% formalin for morphological analysis or 96% absolute alcohol for molecular sequencing. Prevalence and mean intensity of each morphotype were calculated according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Morphological analyses
Prior to DNA extraction, ethanol-preserved samples were examined in fresh water on a concave slide using an Olympus BX50 compound microscope (Olympus, Notting Hill, VIC, Australia). Digeneans were photographed using a QImaging Go-21 CMOS camera (Adept Turnkey, Coburg, VIC, Australia) mounted on the microscope and measured using an ocular micrometre. The morphological characters measured were as follows: body length, body width, length of forebody, oral sucker width, oral sucker length, ventral sucker width and ventral sucker length. The forebody was measured as the distance between the anterior extremity of the body and the anterior margin of the ventral sucker. The prepharynx was measured as the distance from distal end of oral sucker to proximal end of pharynx.
Digeneans preserved in formalin were rinsed in water and over-stained with Mayer's haematoxylin. They were then rinsed in fresh water, destained with 1% hydrochloric acid and neutralized in 1% ammonium hydroxide solution (Miller & Cribb, Reference Miller and Cribb2007). The specimens were dehydrated through a series of ethanol solutions between 70 and 100%, cleared in methyl salicylate and mounted in Canada balsam. The digeneans were viewed using an Olympus BX50 compound microscope and drawn with a camera lucida. Images were digitized using a Wacom tablet and Adobe Illustrator.
Molecular analyses
Genomic DNA was isolated from single specimens using proteinase K and either the phenol/chloroform extraction procedure (Sambrook et al., Reference Sambrook, Fritsch and Maniatis2001) or a QIAamp® DNA Mini Kit (QIAGEN, Hilden, NW, Germany). Due to the small size of the trematodes (some <200 μm), the following modifications were made. Prior to extraction, each trematode was pipetted into a vial with a minimal amount of ethanol. The vial lids were left open until the ethanol had evaporated (removing the risk of losing the digenean when aspirating off solution). TE buffer and proteinase K were added, the solution was centrifuged and vortexed and then placed overnight in a rotating incubator. The ITS2 region was amplified using the forward primer ‘3S’ (5′-GGTACCGGTGGATCACGTGGCTAGTG-3) (3′ end of 5.8S rDNA) (Bowles et al., Reference Bowles, Hope, Tiu, Liu and McManus1993) and the reverse primer ‘ITS2.2’ (5′-CCTGGTTAGTTTCTTTTCCTCCGC-3′) (5′ end of 28S rDNA) (Cribb et al., Reference Cribb, Anderson, Adlard and Bray1998). The polymerase chain reaction (PCR) reactions were carried out in 20 μl volumes, each with 4 μl of Hotstar Q solution (QIAGEN), 2 μl of 10× PCR reaction buffer, 0.8 μl of 10 mM deoxyribonucleotide triphosphate (dNTP), 0.75 μl of each primer (Invitrogen, Carlsbad, CA, USA) (10 μM), 0.25 μl of HotstarTaq (QIAGEN), 6.45 μl of nuclease-free water and 5 μl of template DNA. Amplification included an initial step of 95°C for 15 min followed by 35 cycles of denaturation at 96°C for 45 s, annealing at 48°C for 30 s and extension at 72°C for 45 s followed by a final extension step of 72°C for 4 min and a holding temperature of 15°C. Reamplification was necessary for the metacercariae samples (most likely due to the small size of each specimen). For these reactions, 1 μl of PCR product was used instead of 5 μl of DNA template, and 10.45 μl of nuclease-free water. Positive and negative controls were run for all amplifications. The number of sequences obtained for the digenean species from each host was C. annaskala (n = 3), P. haeckeli (n = 4), A. eurodina (n = 1), Seriolella brama (n = 4), S. punctata (n = 1) and Scomber australasicus (n = 2). The amplified DNA was purified using ExoSAP-IT (USB Corporation, Cleveland, OH, USA) following the manufacturer's recommended protocol. The purified product was sequenced by Macrogen, South Korea.
Forward and reverse sequences were edited to produce a single sequence for each specimen using BioEdit version 7.0.9.0 (Hall, Reference Hall1999). As the sequences obtained included the entire ITS2 region and sections of the adjoining 5.8S and 28S, the ITS2 sequence was isolated using the annotation tool of the ITS2 database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/) using the default parameters (Keller et al., Reference Keller, Schleicher, Schultz, Müller, Dandekar and Wolf2009). Sequences were aligned in MEGA 5.05 (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011) using MUSCLE with the defaults selected, except maximum iterations, which were changed to ten. Alignment was checked by eye in Mesquite (Maddison & Maddison, Reference Maddison and Maddison2011) and the ends were trimmed to match the shortest sequence. Distance matrices were constructed using MEGA 5.05 (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011) to calculate the number of base differences per sequence. Pairwise deletion was selected to remove ambiguous positions. A basic local alignment search tool (BLAST) search was undertaken in GenBank to look for similar sequences. Sequences were lodged with GenBank for each parasite host combination, including all sequence variants.
Results
All adult trematodes from S. punctata and S. brama were identified as Cephalolepidapedon warehou Bray & Cribb, Reference Bray and Cribb2003 (family Lepocreadiidae; table 1) by comparison with the original description. Adult trematodes from S. australasicus (table 1) were in poor condition for morphological study but were consistent with the genus Opechona (elongate body, infundibuliform oral sucker, pseudo-oesophagus present). There are only two species of Opechona known in Australian waters, Opechona austrobacillaris Bray & Cribb, Reference Bray and Cribb1998 and O. kahawai Bray & Cribb, Reference Bray and Cribb2003. Sequences from the present species differ from those of O. austrobacillaris reported by Bray et al. (Reference Bray, Cribb and Cutmore2018) by 20 bases of ITS2 rDNA, but unfortunately no ITS2 sequences are available to corroborate the identification of O. kahawai. Opechona kahawai has previously been reported from a species of Arripis (Arripidae, type host) and from Seriola lalandi Valenciennes, 1833 (Carangidae) by Hutson et al. (Reference Hutson, Ernst, Mooney and Whittington2007). Infection of this species in S. australasicus appears plausible, but the taxonomy of this difficult genus certainly requires further work, so we take the conservative position of identifying it as Opechona cf. kahawai.
Digenean metacercariae from the scyphozoan jellyfish P. haeckeli and C. annaskala were identified as C. warehou, inferred from ITS2 sequences identical to those from adult digeneans (table 2). Morphological features (fig. 1a) were broadly consistent with those of adult C. warehou according to Bray & Cribb (Reference Bray and Cribb2003), although the metacercariae clearly undergo extensive development in the definitive host. Metacercariae from the hydrozoan jellyfish A. eurodina were identified as O. cf. kahawai using ITS2 sequences identical to those of adult O. cf. kahawai (table 2 and fig. 1b); again, the metacercariae clearly develop extensively in the definitive host.
(a) Ventral view of metacercaria of Cephalolepidapedon warehou from Pseudorhiza haeckeli from Port Phillip Bay (point of bifurcation of intestinal caeca anterior to ventral sucker not visible). Drawing a composite of four worms. (b) Ventral view of metacercaria of Opechona cf. kahawai from Aequorea eurodina from Port Phillip Bay. Drawing a composite of two worms. Scale bars: 100 μm.
Digenean species, host, locations, replicate information and GenBank accession numbers for trematode sequences.
Molecular results
Sequences of C. warehou from seven adult digeneans from S. brama and S. punctata were identical to those from seven metacercariae from P. haeckeli and C. annaskala (table 2). All sequences contained complete ITS2 sequences of 290 bases, except two shortened sequences from C. annaskala metacercariae. Apart from the missing ends, all sequences were identical except one for one specimen from S. brama, which differed by only one transition.
Sequences of two adult O. cf. kahawai from S. australasicus matched sequences from two metacercariae from two specimens of A. eurodina (table 2). All sequences contained complete ITS2 sequences of 293 bases. One sequence from S. australasicus was identical to one from A. eurodina and the other two sequences differed from these by one transition and three base pairs transitions (and by one transition to each other).
Morphological description of metacercariae
Cephalolepidapedon warehou metacercariae from jellyfish
Measurements are provided in table 3. Body elongate, rounded posteriorly (fig. 1a). Tegument spinose, with spines in regular rows in forebody, sparse or absent in hindbody. Pigment copious, scattered throughout parenchyma of forebody, to about posterior margin of ventral sucker. Oral sucker funnel-shaped, terminal. Ventral sucker slightly smaller than oral sucker, rounded, pre-equatorial on slight protuberance. Prepharynx long; pharynx distinct, oval; forebody long. Caeca terminate blindly. Testes two, rounded to oval and entire, in mid-hindbody.
Measurements of digeneans found in jellyfish and fish.
Measurements (μm) as range, mean ± standard deviation and number of specimens. Forebody was measured as the distance between the anterior extremity of the body and the anterior margin of the ventral sucker. Sucker width ratio is given with oral sucker as one. Distance to pharynx was measured from distal end of oral sucker to proximal end of pharynx. Body width:oral sucker width ratio is given with body as one.
The morphology of the metacercariae resembles that of the adult digeneans from their fish hosts in the distinct funnel-shaped sucker and the long prepharynx (table 3). However, the forebody is proportionally longer in the metacercariae and the pigmentation is far heavier in the forebody.
Opechona cf. kahawai metacercariae from jellyfish hosts
Measurements are provided in table 3. Body elongate (fig. 1b). Tegumental spines minute, most obvious in forebody. Eye-spot pigment heavy in region from oral sucker to more than halfway to ventral sucker. Oral sucker large, infundibuliform; may be withdrawn into forebody, with wide aperture, terminal or slightly ventrally subterminal. Prepharynx, pharynx, oesophagus and pseudo-oesophagus obscured by pigment in forebody. Intestinal bifurcation in forebody (exact location unable to be seen due to heavy pigmentation). Forebody long. Ventral sucker rounded, smaller than oral sucker, in posterior half of body, slightly protuberant. Excretory pore terminal. No testes, seminal vesicle, ovaries or other reproductive organs visible.
Inability to discern the digestive system in these metacercariae reduced possible points of comparison with sexual adults from fishes. As for C. warehou, the pigmentation was far heavier than is reported in the adults and the forebody was proportionally longer. Adult digeneans from S. australasicus were unable to be measured due to deterioration related to freezing in the host fish.
Prevalence and intensities of infection
Opechona cf. kahawai occurred only within A. eurodina, whereas Cephalolepidapedon warehou occurred in both P. haeckeli and Cyanea annaskala. Prevalence of O. cf. kahawai in the jellyfish was 100%, whereas that of C. warehou ranged from 33 to 100% (table 1). The maximum intensity of C. warehou was higher than that of O. cf. kahawai (table 1). Cephalolepidapedon warehou occurred in different areas of its jellyfish hosts, whereas O. cf. kahawai occurred only in the bell of A. eurodina (table 4). The metacercariae had not obviously damaged the tissues of the jellyfishes, but they were easily dislodged with a pipette suggesting a ‘softening’ of the surrounding tissue.
Number of digeneans within each location of their jellyfish hosts.
Cephalolepidapedon warehou occurred in the intestines of S. brama and S. punctata. All specimens of S. brama were parasitized, unlike S. punctata. Intensity of C. warehou was also higher in S. brama (table 1). Mature and immature specimens of C. warehou were found in both fish species. Opechona cf. kahawai occurred in the intestines of both specimens of Scomber australasicus, with intensities of four and 11 (table 1). Mature and immature O. cf. kahawai were present.
Discussion
Digenean metacercariae recovered from three jellyfish species of Port Phillip Bay were identified as two species of lepocreadiid digeneans (C. warehou and O. cf. kahawai) using DNA sequencing. This is the first time DNA sequencing has been used to identify digenean metacercariae in jellyfish and highlights their role as potentially important intermediate hosts of digenean trematodes. Three new host records for C. warehou were recorded, and one new host was recorded for O. cf. kahawai, thus allowing inference of partial life cycles for two digenean species.
Cephalolepidapedon warehou
Our study has revealed several new aspects of the life cycle of C. warehou. Our observations of C. warehou parasitizing the scyphozoans P. haeckeli and Cyanea annaskala are the first records of C. warehou parasitizing jellyfish and the first time an intermediate host for C. warehou has been identified. Moreover, although C. warehou was described from S. punctata in Tasmania (Bray & Cribb, Reference Bray and Cribb2003), our study identifies S. brama as another definitive host.
The presence of a metacercaria in any intermediate host does not necessarily demonstrate the host is involved in transmission; dead ends are also possible. However, the use of jellyfish as intermediate hosts by C. warehou is consistent with the close association of S. brama and S. punctata with jellyfish. Juveniles of both species of fish aggregate under jellyfish in Tasmania (Last et al., Reference Last, Scott and Talbot1983) and S. brama associate with Catostylus mosaicus, P. haeckeli and A. eurodina in Port Phillip (Browne, pers. obs.). As adults, S. brama and S. punctata feed primarily upon pyrosomes, salps and hyperiid amphipods (Bulman et al., Reference Bulman, Althaus, He, Bax and Williams2001; Horn et al., Reference Horn, Burrell, Connell and Dunn2011). Unidentified jellyfish were also found in the stomach contents of S. punctata (Horn et al., Reference Horn, Burrell, Connell and Dunn2011). The high percentage of ‘unknown’ stomach contents recorded in both species of fish (Bulman et al., Reference Bulman, Althaus, He, Bax and Williams2001; Horn et al., Reference Horn, Burrell, Connell and Dunn2011) may well include jellyfish. Indeed, jellyfish are difficult to identify in gut contents (Arai, Reference Arai1988; Arai et al., Reference Arai, Welch, Dunsmuir, Jacobs and Ladouceur2003), particularly when stomachs are frozen or preserved (Bulman et al., Reference Bulman, Althaus, He, Bax and Williams2001; Horn et al., Reference Horn, Burrell, Connell and Dunn2011). For Atlantic Ocean warehou, Seriolella porosa, ctenophores comprise up to 78% of gut contents during summer (Mianzan et al., Reference Mianzan, Mari, Prenski and Sanchez1996). As no records of digeneans parasitizing salps could be found in the literature, and many other lepocreadiid species use jellyfish and other gelatinous zooplankton as hosts (e.g. Stunkard, Reference Stunkard1969, Reference Stunkard1980a; Køie, Reference Køie1975; Bray et al., Reference Bray, Waeschenbach, Cribb, Weedall, Dyal and Littlewood2009), it is almost certain that jellyfish act as key intermediate hosts for C. warehou. The first intermediate host of C. warehou is unknown.
The use of jellyfish as hosts by species of Cephalolepidapedon is supported by knowledge of its only congener, Cephalolepidapedon saba Yamaguti, 1970. Metacercariae of C. saba have been recorded in the scyphozoan jellyfish Aurelia aurita sensu lato (Ohtsuka et al., Reference Ohtsuka, Kondo and Sakai2010; Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016), Chysaora pacifica and Cyanea nozakii (Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016). Adults and metacercariae of C. saba have been found in the intestines of the Japanese butterfish, Psenopsis anomala (Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016). This fish species associates with and feeds upon jellyfish (Masuda et al., Reference Masuda, Yamashita and Matsuyama2008; Ohtsuka et al., Reference Ohtsuka, Koike, Lindsay, Nishikawa, Miyake, Kawahara, Mujiono, Hiromi and Komatsu2009). Cephalolepidapedon saba has been identified in specimens of C. pacifica, and C. nozakii captured simultaneously with the associated fish P. anomala. Nematocysts in the intestines of the fish, supported by stable isotope studies, support the conclusion of Kondo et al. (Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016) that transmission of C. saba to P. anomala occurs through medusivory. Adult C. saba have also been recorded from Scomber japonicus (Bray & Gibson, Reference Bray and Gibson1990; Bartoli & Bray, Reference Bartoli and Bray2004) and S. australasicus (Korotaeva, Reference Korotaeva1974). These species are known to feed upon gelatinous zooplankton (Takano, Reference Takano1954) and the congener Scomber scombrus feeds on the hydromedusa Aglantha digitale (Runge et al., Reference Runge, Pepin and Silvert1987).
Opechona cf. kahawai
Opechona kahawai is a little-studied parasite that has been recorded from only two host species, Seriola lalandi in Victoria (Hutson et al., Reference Hutson, Ernst, Mooney and Whittington2007) and Arripis (either trutta or truttacea) in Tasmania (Bray & Cribb, Reference Bray and Cribb2003). Therefore, our discovery of O. cf. kahawai parasitizing Scomber australasicus may be a new definitive host for O. kahawai. No intermediate hosts are known for O. kahawai, and our study represents the first record of O. cf. kahawai from an intermediate host, A. eurodina. The high degree of similarity between the DNA sequences of metacercariae from A. eurodina and adult digeneans from S. australasicus suggest that A. eurodina is an additional host for this species. Although one sequence from a digenean from A. eurodina exactly matched one sequence from a digenean from a S. australasicus, there were also differences of one and three base pairs relative to the other sequences. Intraspecific variation in ITS2 sequences is typically low, so it is possible that more than one species was present. However, the differences are more likely to be sequencing error, due to the difficulty of obtaining fresh digenean specimens from S. australasicus (the fish had been frozen and then defrosted for dissection). Further sequencing with a greater number of replicates from both hosts (and ideally also from the other known hosts of O. kahawai) are necessary to resolve this issue. While the life history of O. kahawai is poorly known, the life cycles of other species within the genus Opechona have been elucidated experimentally: Opechona bacillaris (Molin, 1859) (see Køie, Reference Køie1975), Opechona cablei Stunkard, 1980 (see Stunkard, Reference Stunkard1980b) and Opechona pyriforme (Linton, 1900) (see Stunkard, Reference Stunkard1969). These species all use hydrozoan or scyphozoan medusae and/or ctenophores as intermediate hosts. Unencysted metacercariae of these and other Opechona species have been found in a wide range of gelatinous zooplankton, including hydrozoan jellyfish (Bray & Gibson, Reference Bray and Gibson1990; Gómez del Prado-Rosas et al., Reference Gómez del Prado-Rosas, Segura-Puertas, Álvarez-Caden and Lamothe-Argumedo2000; Martorelli, Reference Martorelli2001; Martell-Hernández et al., Reference Martell-Hernández, Ocaña-Luna and Sánchez-Ramírez2011; Diaz Briz et al., Reference Diaz Briz, Martorelli, Genzano and Mianzan2012), scyphozoan jellyfish (Bray & Gibson, Reference Bray and Gibson1990; Morandini et al., Reference Morandini, Martorelli, Marques and da Silveira2005; Ohtsuka et al., Reference Ohtsuka, Kondo and Sakai2010; Nogueira Júnior et al., Reference Nogueira Júnior, Diaz Briz and Haddad2014; Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016) and ctenophores (Yip, Reference Yip1984; Martorelli, Reference Martorelli2001; Morandini et al., Reference Morandini, Martorelli, Marques and da Silveira2005).
In addition to gelatinous zooplankton, metacercariae of Opechona species are recorded from planktonic polychaetes (Reimer et al., Reference Reimer, Berger, Heuer, Lainka, Rosenthal and Scharnweber1971), chaetognaths (Lebour, Reference Lebour1917; Reimer et al., Reference Reimer, Berger, Heuer, Lainka, Rosenthal and Scharnweber1971; Køie, Reference Køie1975; Øresland & Bray, Reference Øresland and Bray2005), a pelagic heteropod mollusc (Morales-Ávila et al., Reference Morales-Ávila, Saldierna-Martínez, Moreno-Alcántara and Violante-González2018) and free in the plankton (Nicoll, Reference Nicoll1910; Franc, Reference Franc1951).
This study is the second report of an Opechona species from S. australasicus, the previous being O. bacillaris from the Great Australian Bight (Korotaeva, Reference Korotaeva1974). High prevalences (45–100%) of O. bacillaris were found in S. australasicus (45.2% of 42 fish) (Korotaeva, Reference Korotaeva1974). It is quite likely that the O. bacillaris identified by Korotaeva were, in fact, O. austrobacillaris or O. kahawai/O. cf. kahawai, as the species are morphologically similar (Bray & Cribb, Reference Bray and Cribb1998) and those species had not yet been described in 1974.
Scomber australasicus are omnivores that feed primarily upon pelagic ascidians, pyrosomes and salps, and, to a lesser degree, on fish and crustaceans (Bulman et al., Reference Bulman, Althaus, He, Bax and Williams2001). Scomber japonicus and S. australasicus also feed on the siphonophore Chelophyes appendiculata (Takano, Reference Takano1954) and S. scombrus feeds on the hydromedusan A. digitale (Runge et al., Reference Runge, Pepin and Silvert1987). Given the broad diet range (including gelatinous zooplankton) of S. australasicus and its congeners, it seems likely that S. australasicus also feeds upon A. eurodina. This information suggests that A. eurodina is a genuine intermediate host for O. cf. kahawai. The range of animals in which Opechona metacercariae and sexual adults have been found suggests there may be other second intermediate (and definitive) hosts in the Pacific. Gastropods from the superfamily Buccinoidea, Costoanachis avara, Nassarius pygmaeus and Astyris lunata are the first intermediate hosts of O. pyriforme, O. bacillaris and O. cablei (Stunkard, Reference Stunkard1969; Køie, Reference Køie1975; Stunkard, Reference Stunkard1980b, respectively). Species of Nassarius are found along much of the Australian coastline (ABRS, 2020), so would be appropriate targets for further investigation into the life cycle of O. cf. kahawai.
Adult O. kahawai have been found in the guts of Arripis (Bray & Cribb, Reference Bray and Cribb2003) and S. lalandi (Hutson et al., Reference Hutson, Ernst, Mooney and Whittington2007). These are large, predatory fish that may seem unlikely to prey upon gelatinous zooplankton. Thus, it seems likely that they become parasitized by feeding upon fish that eat gelatinous zooplankton so that the life cycle involves four hosts. In Australian waters, the diet of S. lalandi includes Trachurus sp. and S. australasicus (Ward et al., Reference Ward, Goldsworthy and Rogers2008). Trachurus species are definitive hosts of many Opechona species, including O. bacillaris (Kovaleva, Reference Kovaleva1963 and Nikolaeva & Kovaleva, Reference Nikolaeva, Kovaleva and Delyamure1966, both cited in Bray & Gibson, Reference Bray and Gibson1990; MacKenzie et al., Reference MacKenzie, Campbell, Mattiucci, Ramos, Pereira and Abaunza2004), Opechona species (Machida & Uchida, Reference Machida and Uchida1990), O. pyriforme (MacKenzie et al., Reference MacKenzie, Campbell, Mattiucci, Ramos, Pereira and Abaunza2004) and also host metacercariae of O. bacillaris (Gaevskaya & Kovaleva, Reference Gaevskaya and Kovaleva1982, cited in Bray & Gibson, Reference Bray and Gibson1990). Trachurus species are a main prey item of Arripis trutta in south-eastern Australia (Hughes et al., Reference Hughes, Stewart, Lyle, McAllister, Stocks and Suthers2013). Trachurus species are known to associate with jellyfish (Masuda et al., Reference Masuda, Yamashita and Matsuyama2008; Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016) and, thus, seem a likely pathway for O. kahawai to reach S. lalandi and species of Arripis.
The definitive hosts of Opechona species in the north-east Atlantic are predominately fishes of the family Scombridae: S. australasicus, S. japonicus, S. scombrus, Rastrelliger brachysoma and Rastrelliger kanagurta (see Bray & Gibson, Reference Bray and Gibson1990 for references); but also include fishes from at least 13 other families (see Bray & Gibson, Reference Bray and Gibson1990). Interestingly, the other Opechona species found in Australia, O. austrobacillaris, is known from the predatory fish Pomatomus saltatrix (Bray & Cribb, Reference Bray and Cribb1998). Whilst gelatinous zooplankton have been found in their stomach contents, stable isotopes have shown their diet to be dominated by fish and squid (Cardona et al., Reference Cardona, Álvarez de Quevedo, Borrell and Aguilar2012) and that their main prey are Scomber species. As scombrids have been categorized as a major definitive host of Opechona species, it is plausible that adult O. austrobacillaris and O. kahawai from Scomber species eaten by predatory fish have survived and thrived in the intestines of the predators P. saltatrix and S. lalandi, respectively.
Implications of findings
There have been no studies of the effects of O. cf. kahawai or C. warehou on their fish hosts. However, trematodes such as these, found in the intestines of their definitive hosts, are generally not considered significant pathogens (Cribb, Reference Cribb and Rohde2005). These digeneans are small in comparison to the fish studied and occurred in low intensities. Although intestinal digeneans may feed on food within the host's intestines, and not directly damage the host, they may still reduce fitness of the host fish through energy loss and increased feeding effort (Bartoli & Boudouresque, Reference Bartoli and Boudouresque2007). The effects of the digenean parasites may be greater upon hosts other than fish within their life cycle.
No obvious effects of the digeneans were observed on the jellyfish. This is consistent with observations by Køie (Reference Køie1975) on most ctenophores and hydromedusae infected by O. bacillaris. However, she found that very small hydromedusae Hydractinia carnea were unable to swim when penetrated by four cercariae. Sizes of the hydromedusae were not provided, but even large H. carnea medusae have a bell diameter of only 2.4 mm (Schuchert, Reference Schuchert2008) – much smaller than the jellyfish in this study. Populations of the ctenophore Pleurobrachia pileus declined after heavy infection by O. bacillaris and didymozoid trematodes (Yip, Reference Yip1981, Reference Yip1984), suggesting that heavy infections can negatively affect second intermediate jellyfish hosts. The hosts most affected by O. bacillaris and C. warehou are likely to be the first intermediate hosts, which are typically castrated by digeneans (Cribb, Reference Cribb and Rohde2005). While there are no studies on first intermediate hosts of C. warehou, male gastropods (N. pygmaeus) infected with O. bacillaris cercariae had a highly reduced mating organ, and infected males and females had reduced and non-functional gonads (Køie, Reference Køie1975). Similarly, gastropods parasitized by lepocreadiid cercariae believed to be Opechona sp. were completely castrated (Averbuj & Cremonte, Reference Averbuj and Cremonte2010). As prevalences of infected snails may be high (e.g. 7.4% of N. pygmaeus in Køie's study, up to 54.2% of Buccinanops cochlidium in Averbuj and Cremonte's), infections could have effects on a population level.
Conclusion
This study identified new jellyfish hosts for the lepocreadiid trematodes C. warehou and O. cf. kahawai. Sequences of ITS2 proved to be an effective tool in identifying digenean metacercariae from jellyfish. Results from morphology, ecological data and previous studies suggest that these jellyfish act as second intermediate hosts of these digenean species. New definitive hosts for C. warehou and O. cf. kahawai have been identified (Seriolella brama and Scomber australasicus, respectively) and previously reported definitive host of C. warehou confirmed (Seriolella punctata).
Acknowledgements
We thank Rod Watson and Dr Ricky Gleeson for assistance in the field, and Dr Jo Sumner, Dr Scott Cutmore and Dr Adnan Moussalli for DNA sequencing assistance.
Financial support
This study was funded by the Griffith School of Environment, Museums Victoria (J.G.B, 1854 scholarship) and the Linnean Society Systematics Research Fund (J.G.B.).
Conflicts of interest
None.
Ethical standards
Animals were collected under permits from the Victorian Department of Primary Industries (RP699) and the Queensland Department of Primary Industries and Fisheries (90306).
