Introduction
Platyhelminth parasites of fish comprise the Trematoda (Digenea and Apidogastrea), the Monogenea and Cestoda (tapeworms and allies), with monogeneans and trematodes being responsible for the majority of infections (Cribb et al. Reference Cribb, Chisholm and Bray2002). Of these taxa, the majority of monogeneans and larvae of some digeneans are ectoparasites on fins, skin, or gills. Monogenea have a direct life cycle without intermediate hosts (Bakke et al. Reference Bakke, Cable and Harris2007), with Gyrodactylus von Nordmann, 1832, being one of the most common genera infecting freshwater fishes, while digenean trematodes have complex life cycles involving a series of hosts (Klinger and Floyd Reference Klinger and Floyd1998). Diplostomidae Poirier, 1866, is a family of digenean trematodes infecting freshwater snails and fish as intermediate hosts, and mammals and fish-eating birds as definitive hosts (Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002). Metacercarial stages of some diplostomid species are known for inducing black spot disease, a common syndrome among freshwater fish (Post Reference Post1983), easily recognized by melanin cysts formed around the parasite (Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002). With the introduction of allochthonous fishes to European freshwater systems, their parasites have also been introduced, including taxa that induce black spot disease in fishes (Stoyanov et al. Reference Stoyanov, Georgieva, Pankov, Kudlai, Kostadinova and Georgiev2017). So far, helminthological studies of freshwater fish in the Adriatic drainage have been focused on mainland freshwater ecosystems (Benovics et al. Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021a, Reference Benovics, Francová, Volta, Dlapka and Šimková2021b, Reference Benovics, Gettová and Šimková2021c; Dezfuli et al. Reference Dezfuli, Franchella, Bernacchia, De Bastiani, Lorenzoni, Carosi, Lorenzoni and Bosi2024; Mladineo et al. Reference Mladineo, Zrnčić and Oraić2009; Řehulková et al. Reference Řehulková, Benovics and Šimková2020; Stojanovski et al. Reference Stojanovski, Blazhekovikj-Dimovska and Smiljkov2020; Zrnčić et al. Reference Zrnčić, Oraić, Šoštarić, Ćaleta, Buj, Zanella and Šurmanović2009) and coastal lagoons (Dezfuli et al. Reference Dezfuli, Giari, Castaldelli, Lanzoni, Rossi, Lorenzoni and Kennedy2014; Giari et al. Reference Giari, Ruehle, Fano, Castaldelli and Poulin2020), with a single record of a black spot disease parasite detected in Rutilus prespensis (Karaman, 1924) (Stojanovski et al. Reference Stojanovski, Blazhekovikj-Dimovska and Smiljkov2020).
Islands in the eastern Adriatic are a part of a larger Outer Dinaric range, characterized by intense karstification processes (Terzić et al. Reference Terzić, Peh and Marković2010), and are built out of limestone and dolomite (Bonacci Reference Bonacci2015). Because of extreme permeability of the karst terrain, infiltration of water is fast. Therefore, surface flow on Adriatic islands is rare and appears only after heavy precipitation events (Bonacci Reference Bonacci2015). Consequently, only few freshwater fishes are found on these islands, and next to nothing is known about their biology or parasite fauna. On Krk Island, in the northeastern Adriatic, a population of the minnow Phoxinus lumaireul (Schinz, 1840) (Actinopterygii: Leuciscidae) inhabits the Suha Ričina stream. Genetic data – lack of haplotype sharing between the island and neighboring mainland populations (Reier et al. Reference Reier, Bogutskaya and Palandačić2022) – indicate that this population is an autochthonous population that got separated from the mainland populations in the course of the sea level rise following the last glacial maximum and was not introduced by humans. In the present study, we aimed at characterizing the helminth ectoparasite fauna of this unique island population of P. lumaireul from Krk Island to get first insights into the ectoparasite diversity these fish are exposed to.
Materials and methods
A total of 38 P. lumaireul was collected (Supplementary Figure S1) at three locations along a 5-km stretch of the Suha Ričina stream on Krk Island, Croatia, in the northeastern Adriatic (Supplementary Figure S2), in April and October 2022 (44°59’57.66’’ N, 14°42’32.94’’ E; 45°0’49.86’’ N, 14°41’56.16’’ E; 45°01’16.76’’ N, 14°40’59.09’’ E). Fish were caught using backpack electrofishing (AGK, model: IG200-1, power: 5 kW). Suha Ričina is the longest (12 km) freshwater stream on any of the Adriatic islands with a basin area of 26 km2 (Rubinić and Ožanić Reference Rubinić and Ožanić1998). During dry season, as the water level lowers, there is almost no surface flow, and the stream primarily flows underground, where its flow is maintained throughout the year (Figure 1) (Valković et al. Reference Valković, Obhođaš and Kutle2014). Suha Ričina stream and the majority of Krk Island are a part of Natura 2000 areas (HR2001357 and HR1000033). Sampling was conducted by licensed operators with a collecting permit (UP/I-324-02/22-01/1) from the government and local level authorities.
a) Suha Ričina flowing through Baška valley. b) Metacercariae cyst (black spot) on the caudal fin of a P. lumaireul seen through a stereomicroscope. c) Maximum likelihood tree (1,000 bootstrap replicates; TPM3u+F+R2 model; 785 base pairs) of the digenean dataset. d) Maximum likelihood tree (1,000 bootstrap replicates; TVM+F+R2 model; 489 base pairs) of the monogenean dataset. ML bootstrap values (cutoff at 50%) are depicted above branches and BI posterior probabilities (cutoff at 0.7) below branches. Haplotypes of specimens collected on Krk Island are marked with a triangle.
Fish samples were initially stored in 1:1 mixture of distilled water and un-denatured 96% ethanol in the field, and afterwards transferred to un-denatured 96% ethanol. Standard length of each fish was measured in millimeters and weight in grams prior to fixation. Fish were visually screened for ectoparasites using a stereomicroscope (Figure 1). To determine helminth infection parameters following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997), the number of metacercariae cysts (black spots) was counted to calculate prevalence (percentage of infected hosts), mean abundance (mean of the number of individuals of a particular parasite species per host examined), and mean intensity (mean of the number of individuals of a particular parasite species per infected host in a sample). The relationship between the number of parasites and host parameters (standard length and weight) was evaluated through the non-parametric Spearman’s rs correlation test using PAST 4.10 (Hammer and Harper Reference Hammer and Harper2001). Parasites were removed using forceps and scalpel and individually stored in 96% ethanol until further processing.
Genomic DNA was extracted from individual worms, three digenean and one monogenean, using a Quick-DNATM MiniPrep Plus Kit (Zymo Research) following the manufacturer’s Solid Tissues protocol with minor modifications; specifically, elution was repeated two times after initial incubation for 10 minutes instead of five. PCR amplification and sequencing of parts of the 28S ribosomal RNA for digenean parasites (primers C1: 5’-ACC CGC TGA ATT TAA GCA T-3’ and D2: 5’-TGG TCC GTG TTT CAA GAC-3’ (Chisholm et al. Reference Chisholm, Morgan, Adlard and Whittington2001)), and the internal transcribed spacer 2 (ITS2) for Gyrodactylus (primers ITS4.5: 5’-CAT CGG TCT CTC GAA CG-3’ and ITS2: 5’-TCC TCC GCT TAG TGA TA-3’ (Matejusová et al., Reference Matejusová, Gelnar, McBeath, Collins and Cunningham2001)) followed Koblmüller et al. (Reference Koblmüller, Resl, Klar, Bauer, Zangl and Hahn2024), with the exception of the exact PCR cycling conditions (Supplementary Figure S3). Sequences were visualized on an ABI 3500 capillary sequencer (Applied Biosystems).
Sequences were checked and edited manually in MEGA 11.0 (Tamura et al. Reference Tamura, Stecher and Kumar2021). BLAST search against GenBank was used for tentative species identification. The newly generated DNA sequences have been deposited at GenBank under the accession numbers PP918946 (Uvulifer sp.; all samples that worked had identical sequences) and PP918958 (Gyrodactylus sp.). For phylogenetic placement of our samples, ITS2 sequences of monogenean and 28S sequences of digenean parasites from GenBank (Supplementary Table S1) were added to the alignments. Sequences were aligned with MUSCLE and p-distances calculated (Supplementary Table S3; Supplementary Table S4) using MEGA 11.0. The best-fit models of evolution were determined with ModelFinder (Kalyaanamoorthy et al. Reference Kalyaanamoorthy, Minh, Wong, Von Haeseler and Jermiin2017) using the BIC criterion. Phylogenetic trees were inferred in Phylosuite v1.2.2. (Zhang et al. Reference Zhang, Gao, Jakovlić, Zou, Zhang, Li and Wang2020) using maximum likelihood (ML) with IQTREE (Nguyen et al. Reference Nguyen, Schmidt, Von Haeseler and Minh2015) and Bayesian inference (BI) with MrBayes 3.2.6. (Ronquist et al. Reference Ronquist, Teslenko, Van Der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). For both sets of alignments (groups of parasites), BI analyses were run with six Markov chain Monte Carlo (MCMC) chains in two parallel runs for 3,000,000 generations and sampled every 1,000 generations. The initial 25% of sampled data was discarded as burn-in after ensuring that the average standard deviation of split frequencies was <0.01 (indicating run convergence). Node support in the ML tree was assessed with 1,000 standard bootstrap replicates for both groups of parasites.
Results and discussion
A total of 38 specimens of P. lumaireul was collected, with a standard length of 30–68 mm and weighing 0.411–5.53 g (Table 1, Supplementary table S2). In addition, three specimens of European eel, Anguilla anguilla (Linnaeus, 1758) were also recorded at two of the sites (one at 45°0’49.86’’ N, 14°41’56.16’’ E and two at 45°01’16.76’’ N, 14°40’59.09’’ E). Of the 38 individuals screened, 17 P. lumaireul were found with metacercariae cysts on the fins and subepidermal tissue (207 metacercariae cysts in total; Table 1), while a single monogenean (genus Gyrodactylus) was found on the skin. We found no significant correlation between intensity of the black spot disease and host parameters (standard length: Spearman’s rs = 0.1349, p = 0.4194; weight: Spearman’s rs = 0.1403, p = 0.4007).
Mean standard length and mean mass of the host Phoxinus lumaireul (N=38) from Krk Island (Croatia), prevalence, mean abundance, and mean intensity of metacercarial cysts of Uvulifer sp
Based on BLAST search, maximum likelihood, and Bayesian inference analysis, the 28S sequences indicate that the metacercariae cysts (the three samples had identical sequences) found on P. lumaireul from Krk Island belong to Uvulifer Yamaguti, 1934, a genus of diplostomid digeneans (Figure 1). The Uvulifer sample from Krk Island was resolved as the sister group of Uvulifer from South Africa, but the large genetic distances (Figure 1, Supplementary Table S3) suggest that these samples are unlikely to belong to the same species. Currently, a single species, U. denticulatus Rudolphi, 1819, is recognized in Europe (López-Jiménez et al. Reference López-Jiménez, De León and García-Varela2018), and based on our phylogenetic analysis, there is no clear match with another Uvulifer species; therefore, it can be speculated that the specimens from Krk Island belong to this particular species. Unfortunately, no 28S sequence data of unambiguously identified U. denticulatus are available on GenBank, preventing robust species-level identification. By employing molecular and morphological methods, recent studies (Achatz et al. Reference Achatz, Curran, Patitucci, Fecchio and Tkach2019; López-Jiménez et al. Reference López-Jiménez, De León and García-Varela2018) have uncovered previously unknown species richness within Uvulifer in the Neotropics. Similarly, molecular data indicate cryptic diversity in European Apophallus Lühe, 1909, another genus of diplostomid digeneans inducing black spot disease in cyprinids (Sándor et al. Reference Sándor, Molnár, Gibson, Székely, Majoros and Cech2017). Only a few records of Uvulifer parasites in Europe have been documented thus far in the literature. Most of these records are from definitive hosts (kingfishers) (Baylis Reference Baylis1939; Sitko et al. Reference Sitko, Faltýnková and Scholz2006) and first intermediate hosts (snails) (Faltýnková et al. Reference Faltýnková, Našincová and Kablásková2008) with a single record from second intermediate hosts (fish) (Knaack Reference Knaack1971), which is why the taxon is considered as rare (Faltýnková et al. Reference Faltýnková, Našincová and Kablásková2008). Hitherto, only Posthodiplostomum cuticola (von Nordmann, 1832) Dubois, 1936 has been reported as a black spot disease parasite from the Adriatic drainage (Stojanovski et al. Reference Stojanovski, Blazhekovikj-Dimovska and Smiljkov2020). Thus, the Uvulifer specimens from Krk represent one of the first records of the parasite found on fish in Europe, a first record from a Phoxinus host, a first record for the Adriatic drainage and Croatia, and a first record for Southern Europe in general, since all the other records originate from Central Europe (Faltýnková et al. Reference Faltýnková, Našincová and Kablásková2008; Knaack Reference Knaack1971; Sitko et al. Reference Sitko, Faltýnková and Scholz2006) and the UK (Baylis Reference Baylis1939).
The metacercarial stages of the Uvulifer and other black spot disease genera are known to be pathogenic to their fish intermediate hosts. In cases of high infection intensities, deleterious effects and mortalities are possible (Hoffman and Putz Reference Hoffman and Putz1965; Hunter and Hunter Reference Hunter and Hunter1938; Lemly and Esch Reference Lemly and Esch1984; McAllister et al. Reference McAllister, Tumlison, Robison and Trauth2013), especially in juvenile fish, which can experience blood loss, physiological stress, and mortalities (Krull Reference Krull1934; Lemly and Esch Reference Lemly and Esch1984). To estimate the effect of Uvulifer parasites on P. lumaireul in an isolated habitat such as Suha Ričina stream on Krk Island, detailed population based studies are required.
Based on BLAST search, maximum likelihood, and Bayesian inference analysis, the ITS2 sequence of the single monogenean (Gyrodactylus) found on P. lumaireul from Krk Island is most closely related (p-distances: 1.25–1.88%; Supplementary Table S4) to Gyrodactylus pannonicus Molnár, 1968; G. albolacustris Lumme, Ziętera and Lebedeva, Reference Lumme, Ziętara and Lebedeva2017; G. botnicus Lumme, Ziętera and Lebedeva, Reference Lumme, Ziętara and Lebedeva2017; and G. danastriae Lumme, Ziętera and Lebedeva, Reference Lumme, Ziętara and Lebedeva2017, all of which parasitize on Phoxinus. As genetic distances between the Gyrodactylus from Krk Island and any of these other four species exceed the genetic distance between G. albolacustris and G. pannonicus (Supplementary Table S4), it can be argued that the Gyrodactylus from Krk Island could represent a distinct, undescribed species following the logic of Lumme et al. (Reference Lumme, Ziętara and Lebedeva2017). Several more species of Gyrodactylus, belonging to different genetic lineages, have been reported from Phoxinus in Europe (Pettersen et al. Reference Pettersen, Østbye, Holmen, Vøllestad and Atle2016; and references therein). In most of the previous studies on Phoxinus parasites, the host was identified as P. phoxinus. A recent revision of the genus, however, revealed that it comprises a complex of several originally largely allopatrically distributed species (Palandačić et al. Reference Palandačić, Naseka, Ramler and Ahnelt2017). The majority of monogeneans are highly host-specific (Bakke et al. Reference Bakke, Harris and Cable2002), and even though one host species might be parasitized by several Gyrodactylus species, this large number of Gyrodactylus species apparently specialized on P. phoxinus might actually indicate that different Gyrodactylus species are specific to different Phoxinus species. Hence, host–parasite relationships in the Phoxinus-Gyrodactylus system need to be reevaluated based on recent taxonomic changes/additions in both the hosts and parasites.
Characterizing host–parasite interactions, especially those of vulnerable host species, is crucial not just for estimating biodiversity loss, but also because of the key roles in the ecosystem functioning that parasite species may serve (Ingelbrecht et al. Reference Ingelbrecht, Lear, Martin, Lymbery, Norman, Boxshall and Morgan2024). In this study, we present important new data on the ectoparasitic helminth diversity in southern Europe. We provide the first record for the genus Uvulifer in the Adriatic drainage, and in southern Europe in general, and find indication for a hitherto undescribed Gyrodactylus species parasitizing Phoxinus lumaireul on Krk Island. Detailed morphological work is needed to clarify the species identity of our new records. Further, whether these two species are common and widely distributed in southern Europe or have, especially in the case of Gyrodactylus sp., a restricted distribution is still unclear and requires further studies focusing on these taxa.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000889.
Acknowledgements
We would like to thank the Division of Zoology within the Department of Biology, University of Zagreb and the Biology Students Association-BIUS for providing equipment and means to conduct field research. We further thank Toni Kraljić and the Termen Association for providing accommodation in the field throughout the duration of the research-educational project Freshwater ichthyofauna of Krk Island 2022.
Financial support
The authors acknowledge the financial support by the University of Graz, the Student Center of the University of Zagreb (T.Š., grant number: 402-09/22-01/01, 475-02-01-22-46), and Oikon Ltd. – Institute of Applied Ecology (T.Š., grant number: 29/2022) for the research-educational project Freshwater ichthyofauna of Krk Island 2022.
Competing interest
None
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