Introduction
Stable-isotope signatures of carbon (δ13C) and nitrogen (δ15N) are increasingly used to study food-web architecture. In ecology, the isotopic discrimination values of δ13C and δ15N are applied as unique fingerprints, which allow for a determination of food sources and trophic interactions between organisms, respectively (Fry, Reference Fry2006; Wada, Reference Wada2009). For example, investigations of stable isotopes of nitrogen have shown that consumers are enriched in the heavy nitrogen isotope (15N) with an average Δδ15N of 3.4‰ per trophic level, and therefore it can be used to determine the position of an organism within food webs (Minagawa & Wada, Reference Minagawa and Wada1984; Vander Zanden et al., Reference Vander Zanden, Cabana and Rasmussen1997). Carbon stable-isotope composition can deliver information about the food source, such as insect larvae, molluscs, algae, vegetation and detritus (Post et al., Reference Post, Pace and Hairston2000). Accordingly, application of stable-isotope analyses (SIA) might also be helpful to elucidate trophic relationships between a parasite and its associated host (Sabadel et al., Reference Sabadel, Woodward, Van Hale and Frew2016; Nachev et al., Reference Nachev, Jochmann, Walter, Wolbert, Schulte, Schmidt and Sures2017; Yohannes et al., Reference Yohannes, Grimm, Rothhaupt and Behrmann-Godel2017).
The number of studies dealing with the stable-isotope composition of carbon and nitrogen in hosts and their parasites remains scarce, with investigations on some of the major parasite taxa, such as monogeneans, even being absent. So far, SIA of host–parasite associations has shown different patterns. Endoparasite species, such as cestodes and acanthocephalans, were found to be depleted in the heavier nitrogen isotope in comparison to the tissues of their hosts, instead of being enriched as expected for predators (Boag et al., Reference Boag, Neilson, Robinson, Scrimgeour and Handley1998; Ikken et al., Reference Ikken, Brey, Wand, Voigt and Junghans2001; Pinnegar et al., Reference Pinnegar, Campbell and Polunin2001; Deudero et al., Reference Deudero, Pinnegar and Polunin2002; Power & Klein, Reference Power and Klein2004; Persson et al., Reference Persson, Larsson and Stenroth2007; Behrmann-Godel & Yohannes, Reference Behrmann-Godel and Yohannes2013; Navarro et al., Reference Navarro, Albo-Puigserver, Coll, Saet, Forero and Kutcha2014; Nachev et al., Reference Nachev, Jochmann, Walter, Wolbert, Schulte, Schmidt and Sures2017). Also, studies on digeneans (sporocysts) provided similar results (Dubois et al., Reference Dubois, Savoye, Sauriau, Billy, Martinez and de Montaudouin2009; Doi et al., Reference Doi, Yurlova, Vodyanitskaya, Kanaya, Shikano and Kikuchi2010). In contrast, ectoparasites, such as different parasitic arthropods (Boag et al., Reference Boag, Neilson, Robinson, Scrimgeour and Handley1998; Deucett et al., Reference Doucett, Giberson and Power1999; Voigt & Kelm, Reference Voigt and Kelm2006; Schmidt et al., Reference Schmidt, Dautel, Newton and Gray2011), were 15N-enriched, similar to predators that feed on their prey.
As no information is available on stable-isotope signatures in monogeneans, the aim of the present study was to provide the first data on stable-isotope ratios of carbon and nitrogen in a monogenean with respect to its fish host. As a host–parasite system, the parasite Paradiplozoon ichthyoxanthon Avenant-Oldewage (in Avenant-Oldewage et al., Reference Avenant-Oldewage, Le Roux, Mashego and Jansen Van Vuuren2014) was selected as it occurs on the gills of two different fish species: the largemouth yellowfish, Labeobarbus kimberleyensis (Gilchrist & Thompson, 1913) and the smallmouth yellowfish, Labeobarbus aeneus (Burchell, 1822). The fish species differ in their habitat preferences and diet but both occur in the Orange-Vaal River system in South Africa. The largemouth yellowfish prefers flowing water but may also occur in dams. It is a predatory species, initially feeding on insects and small crustaceans but becoming piscivorous above a fork length of 300 mm (Skelton, Reference Skelton2001), and it can even prey on small L. aeneus. The smallmouth yellowfish frequents clear, flowing water with sandy and rocky river beds. Larvae feed on microscopic organisms but larger fish are omnivorous and may feed on benthic invertebrates, including bivalve molluscs, algae, vegetation and detritus (Skelton, Reference Skelton2001). Using these host–parasite systems, we provide the first information about the trophic interaction between a monogenean and its hosts. Moreover, as the parasite species occurs on hosts having different feeding regimes, the study attempted to understand whether the difference in biology of the fish host is reflected in the stable-isotope signatures of the hosts and eventually influences the stable-isotope composition in the monogenean.
Materials and methods
Fish and parasite sampling
During a single survey of the Vaal Dam, South Africa, in summer (January 2016), L. aeneus specimens (n = 7) were collected by means of gill nets (mesh size: 45–190 mm) around UJ Island (26°52′33.62″S; 28°10′25.76″E) (fig. 1). Incidentally, L. kimberleyensis (n = 7) that died in the nets were removed from the nets and afterwards used for sampling, whereas live specimens were released in accordance with permits from the Gauteng Department of Agriculture for collection of fish. Live and dead fish were transported back to a field laboratory on the island, and live fish were maintained in aerated plastic containers containing dam water. Thereafter, fish were euthanized by severing the spinal cord posterior to the head. The weight and the total length of each fish were determined and Fulton's fish condition factor (K) was calculated to estimate differences in nutritional status between individuals or species of fish, according to Heincke (Reference Heincke1908) (see also Nash et al., Reference Nash, Valencia and Geffen2006):
$$K = 100 \times {\rm SW}/{\rm TL}^3 $$where SW = fish weight and TL = fish total length. Subsequently, the gills were excised and assessed for P. ichthyoxanthon. The parasites were removed and frozen (−20°C) before being returned to the laboratory.
Map of the Vaal Dam and feeder rivers, showing the position of UJ Island (A) within the map of South Africa (B) The black block in (B) indicates the location of (A).
Stable-isotope analyses
For SIA, seven specimens of L. aeneus and four specimens of L. kimberleyensis were considered, as only these fish harboured sufficient parasite material for analyses. Furthermore, low parasite intensities on L. kimberleyensis necessitated pooling of monogeneans to a total of two samples. Muscle tissue from infected fishes was removed during dissection and frozen at −20°C. Thereafter, muscle samples and parasites were freeze dried to weight consistency at −77°C under negative pressure (−80 kPa). For SIA, triplicates of each sample in the range of 200–700 μg (dry weight; DW) were weighed in 4 × 6 mm tin-foil capsules for solids (IVA Analysentechnik, Meerbusch, Germany). Samples were analysed using a vario PYRO Cube elemental analyser (EA) system (Elementar Analysensysteme, Langenselbold, Germany) in C/N mode. The EA was coupled to an IsoPrime 100 isotope ratio mass spectrometer (IRMS; Elementar Analysensysteme). The EA-IRMS results were obtained following the principle of identical treatment and the experimental procedures described by Werner & Brand (Reference Werner and Brand2001) and Nachev et al. (Reference Nachev, Jochmann, Walter, Wolbert, Schulte, Schmidt and Sures2017). All isotope ratios were reported in the δ-notation as differences of the isotope ratio of the sample and isotope ratio of an international reference substance by equation (1).
$$\delta^h {\rm E}_{s,ref} = \displaystyle{R\left({}^h{\rm E} / {}^l{\rm E} \right)_s \over R\left({}^h{\rm E} /{}^l{\rm E} \right)_{ref}} - 1$$where R(hE/lE)ref denotes the ratio of the heavy and light isotope (here 13C/12C as well as 15N/14N) in the reference material, and R(hE/lE)s the ratio in the sample. As reference materials for normalization of the laboratory working standard, acetanilide, to the international scale, the USGS40 and USGS41 reference materials were used.
Statistical analyses
In order to calculate the trophic level difference (ΔTL) between the monogeneans and their hosts, the following equation was applied:
$$\left(\Delta {\rm TL} \right) = {\rm \delta}^{15} {\rm N}_{\rm parasite} - {\rm \delta}^{15} {\rm N}_{\rm host}) /{\rm TEF} $$with TEF, the trophic enrichment factor, ranging between 1.3 and 5.3‰.
The Spearman rank correlation was applied to evaluate a possible relationship between the isotopic composition and fish morphometry (length, weight, condition factor). The Wilcoxon matched pair test was used for comparisons between the isotope signatures of host tissues and parasites.
Results
The monogenean was enriched by approximately 2‰ in the heavier nitrogen isotope with respect to both fish hosts, suggesting that the monogeneans range on a 0.4–1.8 higher trophic level than the host (based on equation 2; see table 1 and fig. 2). Statistical analyses performed for the L. aeneus–P. ichthyoxanthon system (n = 7) revealed significantly higher nitrogen signatures in parasites (Wilcoxon matched pair test, Z = 2.366; P < 0.05). Due to the low number of P. ichthyoxanthon samples (n = 2), no statistical analysis could be performed for the L. kimberleyensis–P. ichthyoxanthon system. The carbon signatures of both parasite and host tissues showed similar values, as can be expected for a parasite that feeds on its host tissues. The low differences observed in the carbon signatures suggest that the fish species use largely overlapping food sources, with only small nutritional variations. These trends are mirrored in the stable-isotope signatures of the monogeneans collected from the respective fish species. No significant relationships between the stable-isotope composition of the fish host and monogeneans, nor between the fish morphometric parameters and isotope composition of parasites, were found.
Means and standard deviations of δ13C vs. δ15N for muscle tissue of selected fish hosts (L. aeneus and L. kimberleyensis) and their monogenean parasite P. ichthyoxanthon.
Data on fish morphometry and stable-isotope composition of carbon and nitrogen in selected host–parasite systems.
TL, total length; W, weight; K, Fulton's condition factor.
Discussion
In the present study, signatures of stable isotopes for carbon and nitrogen for a monogenean–host system are presented for the first time. Regarding nitrogen, the monogenean P. ichthyoxanthon was enriched by approximately 2‰ with respect to both of its host species. This difference corresponds to an average isotope fractionation of one trophic level, as consumers are δ15N enriched in the range from 1.3 to 5.3‰ with respect to their diet (Minagawa & Wada, Reference Minagawa and Wada1984). Other flatworms, such as adult cestodes, have shown contrasting patterns, in being depleted in the heavier nitrogen isotope compared to the host (Boag et al., Reference Boag, Neilson, Robinson, Scrimgeour and Handley1998; Ikken et al., Reference Ikken, Brey, Wand, Voigt and Junghans2001; Pinnegar et al., Reference Pinnegar, Campbell and Polunin2001; Deudero et al., Reference Deudero, Pinnegar and Polunin2002; Power & Klein, Reference Power and Klein2004; Persson et al., Reference Persson, Larsson and Stenroth2007; Behrmann-Godel & Yohannes, Reference Behrmann-Godel and Yohannes2013; Navarro et al., Reference Navarro, Albo-Puigserver, Coll, Saet, Forero and Kutcha2014; McGrew et al., Reference McGrew, O'Hara, Stricker, Castellini, Beckmen, Salman and Ballweber2015). However, studies on adult digeneans and other monogeneans are currently lacking and therefore it is not possible to provide any pattern for the Platyhelminthes. A reasonable explanation for the different stable-isotope signatures between P. ichthyoxanthon and its host species could be its nutritional mode. Diplozoids feed on blood, similar to a predator feeding on its prey. Interestingly, other parasite taxa, such as nematodes, that feed on host tissues are also enriched in the heavier nitrogen isotope (Boag et al., Reference Boag, Neilson, Robinson, Scrimgeour and Handley1998; Neilson et al., Reference Neilson, Boag and Hartley2005; O'Grady & Dearing, Reference O'Grady and Dearing2006; Nachev et al., Reference Nachev, Jochmann, Walter, Wolbert, Schulte, Schmidt and Sures2017). More specifically, our results corroborate isotope fractionation in other blood-sucking ectoparasites, such as parasitic insects and ticks (Boag et al., Reference Boag, Neilson, Robinson, Scrimgeour and Handley1998; Voigt & Kelm, Reference Voigt and Kelm2006; Schmidt et al., Reference Schmidt, Dautel, Newton and Gray2011), where the parasites act as micropredators. The active feeding mode combined with the ectoparasitic lifestyle determines the isotopic fractionation of monogeneans and other parasites with a similar nutrition strategy. In contrast, parasites such as cestodes and acanthocephalans have no digestive system and synthesize no digestive enzymes. They are unable to catabolize macromolecules, such as lipids and proteins, or to synthesize several macromolecules, such as fatty acids, nucleic acids, amino acids and many others. They therefore rely entirely on an appropriate supply from their hosts (Barrett, Reference Barrett1981). Accordingly, they assimilate metabolically reprocessed molecules derived from the host, which are depleted in 15N and therefore show opposite patterns to those found for monogeneans, which feed directly on their hosts. Regarding nutritional relationships, monogeneans therefore have more similarities to ectoparasitic arthropods than to other endoparasitic flatworms.
Comparing the fish species, the difference in carbon isotope signatures was rather low, with a ∆δ13C of 0.5‰. This corroborates the suggestion that the diet of the species largely overlaps, with probably a small preference for an omnivorous feeding strategy in L. aeneus versus a predatory lifestyle in L. kimberleyensis (Skelton, Reference Skelton2001). However, these principal nutritional differences do not lead to differences in the trophic level of the fishes, as their ∆δ15N was found to be 0.4‰. This probably indicates that in both fish species a large proportion of the diet consists of macroinvertebrates. Given their different habitat preferences the specific macroinvertebrate species they prey on are likely to be different. Moreover, L. aeneus also feeds on detritus and algae, whereas the diet of L. kimberleyensis includes fish, which may occasionally be L. aeneus. The partly overlapping diet might be due to the size of the fish. The piscivorous mode of nutrition develops fully in L. kimberleyensis when it grows bigger than 300 mm (Skelton, Reference Skelton2001). Given the fact that the average size of L. kimberleyensis was even smaller than the average size of L. aeneus in our study indicates that piscivority in L. kimberleyensis was not fully developed.
The difference between the fish species can be explained by differences in their diet and habitat preferences. Although the difference is rather small, it seems to be sufficient to influence the carbon stable-isotope composition of the parasite, as the carbon signatures of the parasites mirror those of their fish hosts. This can be explained by the micropredatory way of feeding of P. ichthyoxanthon. When fish feed on different food sources the associated differences in carbon isotopes will be present in the blood that carries nutrients. As the monogeneans feed on their host's blood, they are therefore provided with these differences in carbon isotopes. Given the overlap in the hosts' diet, the resulting difference concerning the nitrogen isotopes is ∆δ15N of 0.6‰, which is higher than that of the host species. This specifically stresses the fact that the same parasite species may display different stable-isotope signatures depending on its particular host species. Therefore, the trophic position of a parasite with a broad species specificity may change, depending on the trophic position of its hosts. Although most monogeneans are known to be strictly species specific (Rohde, Reference Rhode1993), diplozoons display varying stenospecificity (Le Brun et al., Reference Le Brun, Renaud and Lambert1988), with P. ichthyoxanthon occurring in two species of the genus Labeobarbus (Avenant-Oldewage & Milne, Reference Avenant-Oldewage and Milne2014).
In conclusion, our data clearly show that the stable-isotope signatures of micropredatory parasites mirror the feeding strategy of their hosts. Care should be taken in future studies to consider the feeding regime of the hosts when interpreting stable-isotope data. This may even refer to a host species changing diet due to migration, seasonal changes or availability of food.
Financial support
Our thanks are due the National Research Foundation South Africa and UJ Faculty Research Committee for funding the fieldwork (NRF grant no. 80789).
Conflict of interest
None.
Ethical standards
The capture of fish was in accordance with permits from the Gauteng Department of Agriculture for collection of fish (permit number: CPE3000123). Slaughter of fish was in accordance with guidelines set out by the South African National Animal Ethics Council and approval by the University of Johannesburg Ethics committee (Protocol number: 9 April 2013).
