Introduction
Biological invasions are among the main threats to global biodiversity and represent a major problem for the integrity and functioning of ecosystems (Molnar et al. Reference Molnar, Gamboa, Revenga and Spalding2008; Poulin Reference Poulin2017), including the potential to disrupt host-parasite dynamics (Chalkowski et al. Reference Chalkowski, Lepczyk and Zohdy2018). These invasions commonly result from human-mediated introductions of species outside their original distribution, which establish self-sustainable populations throughout space and time (Jeschke et al. Reference Jeschke, Liu, Saul, Seebens, Mehner and Tockner2021; Olden et al. Reference Olden, Kai, García-Berthoud, Kinge, South, Vitule, Mehner and Tockner2021; Richardson et al. Reference Richardson, Pysek, Rejmanek, Barbour, Dane Panetta and West2000). Thus, negative impacts on biodiversity emerge due to invasive species arising in the new area, and native species do not have enough time to adjust to the presence of the invading organism (Poulin, Reference Poulin2017).
In Brazil, the upper Paraná River basin drains the southeast region, which represents South America’s most developed and environmentally degraded region, threatened by anthropic activities such as pollution, urbanization, deforestation, and invasive species (Dagosta et al. Reference Dagosta, Monção, Nagamatsu, Pavanelli, Carvalho, Lima, Langeani, Dutra, Ota, Seren, Tagliacollo, Menezes, Britski and de Pinna2024). Approximately 128 non-native fish species have been recorded in this basin (Dagosta et al. Reference Dagosta, Monção, Nagamatsu, Pavanelli, Carvalho, Lima, Langeani, Dutra, Ota, Seren, Tagliacollo, Menezes, Britski and de Pinna2024). In 2015, the giant Arapaima gigas (Schinz 1822) ‘pirarucu’ was recorded in the Água Vermelha reservoir, Rio Grande (Carvalho et al. Reference Carvalho, Casatti, Manzotti and Ravazzi2015). This marks the most recent record of a large non-native fish species in this basin.
Arapaima gigas, the largest species of freshwater bony fish in South America, can grow over three meters in length and weigh around 200 kg (Castello and Stewart Reference Castello and Stewart2010; Ferreira Reference Ferreira, Queiroz, Torrente-Vilara, Ohara, Pires, Zuanon and Doria2013; Nelson et al. Reference Nelson, Grande and Wilson2016). The natural distribution of the species encompasses river floodplains across two major basins (Amazon and Essequibo), five countries (Brazil, Colombia, Peru, Ecuador, and Guyana), and a variety of freshwater wetlands, ecoregions, and habitat types (Fricke et al. Reference Fricke, Eschmeyer and Van der Laan2024; Gurdak et al. Reference Gurdak, Arantes, Castello, Stewart, Watson, Krueger, Taylor and Youn2019). Due to its economic potential, A. gigas has also started to be produced through aquaculture (Valenti et al. Reference Valenti, Barros, Moraes-Valenti, Bueno and Cavalli2021), and commercialized for jumbo aquarium farming, contributed to the rapid expansion of their distribution in recent years (Sousa et al. Reference Sousa, Pereira, Cintra, Freitas, Mereles, Zacardi, Faria-Júnior, Castello and Vitule2022).
Pirarucus are carnivorous and voracious predators (Doria et al. Reference Doria, Catâneo, Torrente-Vilara and Vitule2020). Based on other studies about the introduction and invasive potential of A. gigas in Brazil (Carvalho et al. Reference Carvalho, Casatti, Manzotti and Ravazzi2015; Doria et al. Reference Doria, Catâneo, Torrente-Vilara and Vitule2020; Latini et al. Reference Latini, Resende, Pombo and Coradin2016), and other countries such as Bolivia (Carvajal-Vallejos et al. Reference Carvajal-Vallejos, Van Damme, Cordova, Coca, Van Damme, Carvajal-Vallejos and Carpio2011; Miranda-Chumacero et al. Reference Miranda-Chumacero, Wallace, Calderón, Calderón, Willink, Guerrero, Siles, Lara and Chuqui2012; Van Damme et al. Reference Van Damme, Méndez, Zapata, Carvajal-Vallejos, Carolsfeld and Olden2015), Peru (Ortega et al. Reference Ortega, Guerra, Ramírez and Bert2007; Van Damme et al. Reference Van Damme, Carvajal-Vallejos, Pouilly, Perez, Carpio, Van Damme, Carvajal-Vallejos and Carpio2011), India (Kumar et al. Reference Kumar, Raj, Arjun, Katwate and Raghavan2019), Thailand (FAO 2021), and Indonesia (Fadjar et al. Reference Fadjar, Islamy and Herawati2019; Marková et al. Reference Marková, Jerikho, Wardiatno, Kamal, Magalhães, Bohatá, Kalous and Patoka2020), a high potential to cause changes in species diversity and high per capita effect in the receiving area is expected. Specifically considering the upper Paraná River basin, the effects of its introduction are still unknown, including those related to its host-parasite relationships.
In this context, species invasions, introductions, and range shifts can alter community composition and lead to novel host-parasite interactions (Telfer and Bown Reference Telfer and Bown2012). Parasites have been recognized as important components of global biodiversity, by regulating the abundance or density of host populations, stabilizing food webs, and contributing to the structuring of animal communities (Poulin and Morand Reference Poulin and Morand2004; Timi and Poulin Reference Timi and Poulin2020). Thus, parasites directly interfere with the processes of biological diversification and the composition of the host community and may determine the final success of biological introductions and/or invasions of their respective hosts (Dunn and Hatcher Reference Dunn and Hatcher2015; Gómez and Nichols Reference Gómez and Nichols2013; Perkins et al. Reference Perkins, White and Gillingham2017), altering the interaction forces between native and introduced/invasive species (Dunn and Hatcher Reference Dunn and Hatcher2015). In a scenario of biological invasions, non-native hosts may acquire native parasites in the new area of distribution. Two different outcomes can be expected: (1) If the invasive species is a competent host for the native parasite, a potential consequence is ‘parasite spillback’ (Wilson et al. Reference Wilson, Saunders and Hutson2019). Parasite spillback entails an increase in the abundance of the parasite in native hosts as the invasive host acts as a new reservoir of infection, and (2) if the introduced species is not a competent host for the parasite, the new host can act as an ‘ecological sink’, resulting in a dilution effect that reduces the abundance of the parasite in native hosts (Keesing et al. Reference Keesing, Holt and Ostfeld2006; Kelly et al. Reference Kelly, Paterson, Townsend, Poulin and Tompkins2009; Llopis-Belenguer et al. Reference Llopis-Belenguer, Blasco-Costa, Balbuena, Sarabeev and Stouffer2020).
Spillback events were suggested involving metacercariae of Austrodiplostomum compactum (Lutz 1928) (syn. Austrodiplostomum ostrowski) (Trematoda, Diplostomidae), which widely occurs in native and non-native fish species in the upper Paraná River basin (Lacerda et al. Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012; Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020). This diplostomid requires a three-host lifecycle involving a first intermediate host (Biomphalaria spp. gastropods), a second intermediate host (fish) (or rarely observed paratenic host, as amphibians), and piscivorous birds act as definitive hosts (Violante González et al. Reference Violante-González, García-Varela, Rojas-Herrera and Guerrero2009). Due to its low specificity, metacercariae of Au. compactum can infect various species of freshwater fish (Campos et al. Reference Campos, Manoel, Franceschini, Veríssimo-Silveira, Delariva, Ribeiro and Ramos2020 and references therein; Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020) and parasitize various organs (= diplostomiasis), such as the gills, swim bladder, brain, musculature, and most commonly the eyes (vitreous humor) (Ramos et al. Reference Ramos, Franceschini, Zago, Zica, Wunderlich and Carvalho2013).
Because of the high morphological similarity among Austrodiplostomum spp. metacercariae, molecular techniques have been used to support taxonomic and systematic studies and as a means of elucidating the relationship between these parasites, their hosts, and the relationship between them (Achatz et al. Reference Achatz, Martens, Kostadinova, Pulis, Orlofske, Bell, Fecchio, Oyarzún-Ruiz, Syrota and Tkach2022; Ostrowski de Núñez, Reference De Núñez M2017; Sereno-Uribe et al. Reference Sereno-Uribe, Gómez, de Núñez, de León and García-Varela2019). One of the tools commonly used in these studies is the mitochondrial cytochrome oxidase subunit I (COI mtDNA), a molecular marker that has proved useful for identifying digeneans at the species level (Cracco et al. Reference Cracco, Scorsim, Oliveira and Takemoto2022).
As part of our studies on the possible effects on the A. gigas introduction in the Água Vermelha reservoir, upper Paraná River basin, states of São Paulo/Minas Gerais, some representatives of the parasitic fauna of this species were evaluated in the non-natural distribution area. We reported the parasite-host relationship involving Au. compactum metacercariae parasitizing the eyes of A. gigas, supported by morphological and molecular data (COI mtDNA). Furthermore, new insights about a possible ‘ecological sink’ involving this host-parasite relationship in this non-natural distribution area are discussed.
Materials and methods
Host sampling and parasitological procedures
All applicable institutional, national, and international guidelines for the ethical handling of animals were followed licenses for collection of zoological material (SISBio # 64763-5) and the Ethics Committee for Animal Experimentation (CEUA-IBILCE/UNESP # 241/2023). According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (A7F643D).
Sixty specimens of A. gigas were collected in the lower portion of the course of the Grande River (upper Paraná River basin), on the border of the states of São Paulo and Minas Gerais, in the Água Vermelha Hydroelectric Power Plant reservoir, Brazil (Figure 1) – denoting that this area is outside the natural distribution of the species. Fish were obtained through artisanal fisherman, using fishing rod and live bait, between January 2023 and March 2023.
Map with the approximate natural distribution of Arapaima gigas in South America (grey shadow), with detail of the study sample area (filled red rectangle), located in the Água Vermelha reservoir, Grande River, upper Paraná River basin – state of São Paulo, Brazil.
All fish were individually stored in plastic bags, frozen, and placed in a styrofoam box with ice for transportation to the laboratory for necropsy, where they were weighed (total weight in grams of fish with viscera) and measured (standard length in centimeters measured from the tip of the snout to the final vertebra). The eyes of the fish were removed and examined using a stereomicroscope. Some detected metacercariae were preserved in 96% ethanol for molecular analyses; others were fixed in hot water (~60 °C), stained with chloride carmine or Mayer’s carmalumen, dehydrated in an ethanol series, cleared in eugenol, and mounted on permanent slides using Canada balsam (Amato et al. Reference Amato, Boeger and Amato1991).
To identify the metacercaria, morphological and morphometrical analyses were performed using a phase contrast microscope with a computerized image analysis system (Leica X, Leica Microsystems, Wetzlar, Germany). The morphological measurements followed the recommendations of Ostrowski de Núñez (Reference De Núñez M2017). All measurements based on 10 specimens are given in micrometers (μm) and are expressed as means, followed by the range in parentheses. The prevalence (P, in percentage), mean intensity of infection (MII), and mean abundance of infection (MA) (expressed as mean ± standard error followed by the range) of metacercariae were calculated according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Voucher specimens are deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro State, Brazil (CHIOC 39692). Voucher specimens of the fish hosts were fixed in 10% formalin, preserved in 70% ethanol, and deposited in the Ichthyological Collection of the Três Lagoas (CITL 1509), Federal University of Mato Grosso do Sul. The scientific name of the host follows Fricke et al. (2024).
DNA extraction, amplification, sequencing, and alignments
To confirm the morphological identification of the metacercariae to species, some specimens were mounted on slides with 96% alcohol, covered with a coverslip, and photographed. Conspecific specimen vouchers (paragenophores, according to Pleijel et al. Reference Pleijel, Jondelius, Norlinder, Nygren, Oxelman, Schander, Sundberg and Thollesson2008) were mounted in Canada balsam medium.
Genomic DNA was extracted from the two metacercaria specimens, using the Pure LinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Fragments of COI mtDNA were amplified and sequenced using the following primers: MplatCOX1dF (5′-TGTAAAACGACGGCCAGTTTWCITTRGATCATAAG-3′) and MplatCOX1dR (5′-CAGGAAACAGCTATGACTGAAAYAAYAIIGGATCICCACC-3′) (Moszczynska et al. Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009). The thermocycling profile applied was as follows: an initial denaturation of 1 min at 94°C, followed by 5 amplification cycles of 40 s at 90°C, 40 s at 45°C for annealing, and 1 min at 72°C; then, 35 cycles were performed at 94°C for 40 s, 51°C for annealing temperature for 40 s, 72°C for 1 min, and 5 min at 72°C for final extension.
Conventional polymerase chain reaction (PCR) amplifications were performed using the following reagent concentrations: 1.25 μl of 1xPCR buffer (20mM Tris-HCl, pH 8.4 and 50mM KCl); 0.75 μl of 1.5 mM MgCl2; 0.25 μl of 150 μM each dNTP; 0.25 μl of each PCR primer; 0.1 μl of Taq Polimerase; and l μl of the extracted DNA.
The PCR products were run on an agarose gel (1%) using GelRed™ (Biotium, Hayward, USA) fluorescent nucleic acid dye, loading buffer, and visualized under UV light to confirm amplicon size and yield. The size of the amplicons was estimated by comparison with the 1 kb Plus DNA Ladder (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA). PCR amplicons were purified using the Exo-SAP IT® enzyme (USB Corporation). Automated sequencing was performed directly on purified PCR products using the BigDye® Terminator v.3.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) for cycle sequencing. Sequences were run on an ABI 3500 DNA genetic sequencer (Applied Biosystems). The new sequences were assembled and edited using Geneious version 7.1.3. The resulting consensus sequences were queried against other deposited sequences in Genbank (https://www.ncbi.nlm.nih.gov/genbank/) using a BLASTn search.
Phylogenetic analyses
To establish the phylogenetic position of our species, we compared the two newly generated COI mtDNA sequences of Au. compactum, with 55 sequences of diplostomids retrieved from GenBank that were previously reported in hosts from the American continent. The digenean Australapatemon mclaughlini Gordy, Locke, Rawlings, Lapierre & Hanington Reference Gordy, Locke, Rawlings, Lapierre and Hanington2017 was used as the outgroup for phylogenetic reconstruction (see Table 1 for details). The alignments were performed using MUSCLE implemented on Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012) with default settings. The presence of stop codons and indels for the COI mtDNA alignment was verified by amino acid translation using the trematode mitochondrial code table on Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012). To evaluate the occurrence of substitution saturation, the Iss indices were estimated using DAMBE 5 (Xia Reference Xia2013).
List of digeneans included in the phylogenetic analyses, with details of the host, locality, and GenBank accession numbers of sequences from the mitochondrial cytochrome c oxidase I (COI mtDNA) genes. New sequences obtained for the present study are in bold. (*) Specific host unclear; (**) Species used as outgroup
The best-fitting model for nucleotide substitution selected by jModelTest (Posada Reference Posada2008) using the Akaike information criterion was HKY+I+G. Phylogenies were reconstructed for each alignment under Bayesian inference (BI) using MrBayes v. 3.2 (Ronquist and Huelsenbeck Reference Ronquist and Huelsenbeck2003) and maximum-likelihood (ML) on PhyML 3.1 software (Guindon et al. Reference Guindon, Dufayard, Lefort, Anisimova, Hordijk and Gascuel2010). The BI was run using four Markov chain Monte Carlo searches with 10,000,000 generations and sampling tree topologies every 100 generations. The burning was set to the first 25% of generations; the consensus trees were estimated using the remaining topologies. The nodes with posterior probabilities greater than or equal to 0.90 were considered well-supported. The ML analyses were estimated using random starting trees and 1,000 bootstrap replicates; nodes with bootstrap values greater than or equal to 70 were considered well-supported. The final trees were visualized and edited using FigTree v. 1.3.1 (Rambaut Reference Rambaut2009) and CorelDraw X6.
Pairwise genetic distances within and among sequences were calculated using the p-distance model and a bootstrap procedure with 1,000 replicates in MEGAX (Kumar et al. Reference Kumar, Stecher and Tamura2016).
Results
Characterization of hosts and parasites
The mean standard length ± SD (range) of the 60 hosts analyzed was 56.5 ± 23.4 (29.5–116.0) cm, and the body mass values were 3.8 ± 5.1 (0.301–23.0) kg. A total of 159 metacercariae were collected from the eyes (vitreous humor). Only five hosts with a standard length between 69 and 116 cm were infected (P=8.33%), and showed a mean intensity of infection of 31.8 ± 17.2 (1–93) and a mean abundance of 2.6 ± 1.7 (0–93).
The morphology of the metacercariae found corresponded to the redescription of Au. compactum proposed by Ostrowski de Núñez (Reference De Núñez M2017). Our specimens presented bipartite body, total length 1,298 (1,100–1,627; n = 9) by 410 (276–581; n = 10) wide at midforebody level; forebody spatulate, slightly concave ventrally; hindbody very short, with a small conical segment, 103 (87–126; n = 7) long. Oral sucker subterminal 51 (42–69; n = 7) long by 63 (44–78; n = 7) wide, two well-developed lateral pseudosuckers, symmetricals, on each side of oral sucker; pseudosuckers 101 (86–127; n = 7) long by 107 (88–127; n = 7) wide; ventral sucker absent. Pharynx small, 50 (43–61; n = 6) long by 39 (31–48; n = 6) wide, esophagus short, 58 (46–67; n = 3) long, ceca simple, reaching level of genital primordia. Tribocytic organ (= holdfast organ) elliptical and bilobed, 269 (210–347; n = 7) long by 194 (152–262; n = 7) wide. Glandular cells most spread in the anterior region, extending from pseudosuckers to before the tribocytic organ region. Genital primordia poorly developed, differentiated in two small testes; ovary not distinct.
Molecular analysis
Two fragments of the COI-mtDNA gene were amplified from two metacercariae, 517 bp and 434 bp in length (GenBank accession PQ861092 and PQ861093, respectively). The BLAST search using our newly generated COI-mtDNA sequences showed higher similarity with three sequences of Au. compactum (and those deposited as the synonym Au. ostrowski) previously available in GenBank (see details in Table 1 and Supplementary Table S1). When edited and aligned, the new partial sequences had a similarity of 100% to the partial sequences of Au. compactum previously deposited in Genbank.
The final COI mtDNA alignment was 377 bp long. The Iss indicated no saturation in either the transitions or transversions, and the critical index of substitution saturation (Iss.c) values were above the Iss values. Both ML and BI phylogenetic analyses of COI mtDNA alignment converged with similar topologies. In the final COI mtDNA phylogenetic reconstruction (Figure 2), all Au. compactum sequences grouped in a well-supported monophyletic clade (pp=1; bootstrap= not supported) and were positioned as a sister group of Austrodiplostomum mordax Szidat & Nani 1951 (pp = 1; bootstrap = 82). Tylodelphys spp. clade was recovered as a sister group of Austrodiplostomum spp. clade (pp = 0.99; bootstrap = not supported).
Consensus Bayesian topology based on COI mtDNA of digenean parasites. GenBank accession numbers precede species names. New sequences obtained for the present study are highlighted in bold. Australapatemon mclaughlini (Strigeidae) was used as an outgroup. Support values are shown at nodes: BI posterior probabilities and ML bootstrap scores greater than or equal to 0.90 and 70, respectively, were considered well supported. The clade with sequences of Austrodiplostomum compactum (Diplostomidae) is highlighted with a red block. The branch length scale bar indicates the number of substitutions per site (information about the sequences is shown in the Table 1).
For the mtCOI gene, the genetic divergences between all Austrodiplostomum spp. varied 11% to 15% (40–41 bp) (Supplementary Table S1). No intraspecific genetic divergence was observed between the two new Au. compactum sequences.
Discussion
The morphology of the metacercariae analyzed in the present study conformed to Ostrowski de Núñez (Reference De Núñez M2017), and the new fragments of the COI-mtDNA gene are most close to another Au. compactum (and those deposited as the synonym Au. ostrowski) sequences available in GenBank. Both ML and BI topologies obtained herein corroborate other studies on phylogenetic relationships of diplostomids (Achatz et al. Reference Achatz, Martens, Kostadinova, Pulis, Orlofske, Bell, Fecchio, Oyarzún-Ruiz, Syrota and Tkach2022; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Locke et al. Reference Locke, Drago, Núñez, Souza and Takemoto2020; Sereno-Uribe et al. Reference Sereno-Uribe, Gómez, de Núñez, de León and García-Varela2019). Therefore, this evidence allows us to assume that these metacercariae belong to Au. compactum. This is the first scientific record of Au. compactum metacercariae parasitizing the eyes of A. gigas, independently of the distribution (native or non-native) and origin (farmed or wild) of the host specimens.
Austrodiplostomum compactum is widely distributed in the Neotropical region and uses migratory fish-eating birds Nannopterum (syn. Phalacrocorax) spp. as definitive hosts, which reduces or eliminates geographic barriers for the parasite dispersion (Lacerda et al. Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012; Sereno-Uribe et al. Reference Sereno-Uribe, Gómez, de Núñez, de León and García-Varela2019). The known distribution of Au. compactum includes eight countries: United States, Mexico, El Salvador, Honduras, Costa Rica, Venezuela, Peru, and Brazil (García-Varela et al. Reference García-Varela, Sereno-Uribe, Pinacho-Pinacho, Domínguez-Domínguez and Pérez Ponce De León2016; Locke et al. Reference Locke, Al-Nasiri, Caffara, Drago, Kalbe, Lapierre, McLaughlin, Nie, Overstreet, Souza, Takemoto and Marcogliese2015; O’Hear et al. Reference O’Hear, Pote, Yost, Doffitt, King and Panuska2014; Sereno-Uribe et al. Reference Sereno-Uribe, Gómez, de Núñez, de León and García-Varela2019). In Brazil, the occurrence of these metacercariae has been recorded in at least 45 freshwater fish species, belonging to 14 families and five orders, including the new data reported herein (Campos et al. Reference Campos, Manoel, Franceschini, Veríssimo-Silveira, Delariva, Ribeiro and Ramos2020; Cracco et al. Reference Cracco, Scorsim, Oliveira and Takemoto2022; and present study). Thus, in terms of intermediate fish host, Au. compactum can be regarded as a generalist species. A parasite’s degree of specialization or generalization can influence its ability to acquire new hosts (Schatz and Park Reference Schatz and Park2021). Generalists are expected to switch hosts more readily than specialists, and this host switching can involve taxonomically distant hosts, as observed in Au. compactum.
Spillback events were suggested based on the high infection rates of metacercariae of Au. compactum in eyes of non-native fish species in the Paraná River basin (Lacerda et al. Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012; Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020). Some of these events involved Plagioscion squamosissimus (Heckel 1840) as a highly susceptible second intermediate host for this parasite (Lacerda et al. Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012; Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020). This non-native host stands out in the Paraná River basin for the high infection rates of Au. compactum metacercariae, showing prevalence ranging from 36 to 98%, and mean intensity of infection ranging from four to 98 metacercariae per host (Lacerda et al. Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012; Kohn et al. Reference Kohn, Fernandes and Baptista-Farias1995; Machado et al. Reference Machado, Takemoto and Pavanelli2005; Paes et al. Reference Paes, Carvalho and Silva2010; Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020; Silva-Souza, Reference Silva-Souza1998; Souza-Santos et al. Reference Souza-Santos, Pimenta, Martins, Takahashi and Marengoni2002).
Since parasites can exhibit different levels of infection across host species, and this variation may influence the spread of parasite species to new hosts, the transmission opportunities of a parasite increase proportionally to the number of host species it infects (Schatz and Park Reference Schatz and Park2021). Non-native hosts are more likely to acquire parasites that already have a high prevalence in the established host populations of the new distribution area, regardless of whether those established populations are invasive or native. Plagioscion squamosissimus represents one of the most abundant fish species in the Água Vermelha reservoir (AES Tietê, 2015 apud AES Tietê, 2018). Although there is no published data on infection by Au. compactum metacercariae in fish from the Água Vermelha reservoir, previous studies suggest that P. squamosissimus may act as a parasitic amplifier, increasing the number of infective propagules in the surrounding area and favoring its life cycle in the Paraná River basin (Ramos et al. Reference Ramos, Pagliarini, Franceschini and Silva2020). It could increase the chance of occurrence of accidental encounter and alternative host species infection (Lootvoet et al. Reference Lootvoet, Blanchet, Gevrey, Buisson, Tudesque and Loot2013).
Although the present study showed that A. gigas may act as a second intermediate host for Au. compactum metacercariae in the upper Paraná River basin, we found that only hosts with a standard length between 69 and 116 cm long were infected. Given the preferential definitive hosts to Au. compactum are cormorants Nannopterum spp. (García-Varela et al. Reference García-Varela, Sereno-Uribe, Pinacho-Pinacho, Domínguez-Domínguez and Pérez Ponce De León2016) which shows medium-sized with around 50–60 cm long (Monteiro et al. Reference Monteiro, Amato and Amato2011), predation on such large fish (>29 cm) seems unlikely, which eventually prevents the completion of the parasite’s life cycle involving hosts of large size.
Therefore, host competence alone is not always a good predictor of the impact of non-native hosts on parasite transmission in the invaded area (Gendron et al. Reference Gendron and Marcogliese2017). Demographic factors such as host density (Lootvoet et al. Reference Lootvoet, Blanchet, Gevrey, Buisson, Tudesque and Loot2013) may determine the outcome of native parasite acquisition by a non-native host species. Although the population of A. gigas in this reservoir has increased exponentially in recent years, evidenced by the frequent sightings and captures by fishermen, its density is possibly relatively lower than that of native areas. However, due to the scientific report about introducing this species into the Água Vermelha reservoir is relatively recent (less than 10 years), there is no published data about the density of A. gigas. Moreover, there are no previous studies on parasitic fauna of native and other non-native hosts from this area, limiting our inferences about the dynamic of transmission involving Au. compactum metacercariae. Even though the density of A. gigas could be relatively low in the Água Vermelha reservoir, in areas where the species is native and with higher population density, previous studies on the parasitic fauna of natural populations of A. gigas have not reported the occurrence of metacercariae of Au. compactum (see Baylis Reference Baylis1927; Cajiao-Mora et al. Reference Cajiao-Mora, Brule, Warren, Ksepka, Dutton and Bullard2024 and references therein; Kritsky et al. Reference Kritsky, Boeger and Thatcher1985; Santos et al. Reference Santos, Ceccarelli and Luque2008a; Santos et al. Reference Santos, Moravec and Venturieri2008b; Santos and Moravec Reference Santos and Moravec2009; Thatcher Reference Thatcher1981; Thatcher Reference Thatcher2006).
Possible explanations for the low prevalence observed herein may be the synergistic effect of factors such as the relatively low density of A. gigas in this reservoir, accidental infection, or its role as a paratenic host. The assumption that smaller (< 69 cm) infected specimens of A. gigas may have been preyed upon and were therefore not sampled in our study is made difficult by parental care. The juveniles are cared for by the males, while the females defend the shoal against possible predators (Farias et al. Reference Farias, Leão, Almeida, Verba, Crossa, Honczaryk and Hrbek2015). The juveniles show rapid growth and are cared for until their independence at 35–40 cm long and 4–5 months of age (Farias et al. Reference Farias, Leão, Almeida, Verba, Crossa, Honczaryk and Hrbek2015). Therefore, even if specimens of A. gigas were infected when smaller than 29 cm and not preyed, they are expected to accumulate metacercariae over time of exposure, and these metacercariae could be found in larger hosts, possibly in higher infection rates.
For parasites with heteroxenous life cycles, it is also essential to consider how interspecific interactions (e.g., competition and predation) between invasive non-native and native hosts evolve over time. Given that large infected A. gigas specimens are unlikely to be a significant prey item for the definitive hosts, these large and infected specimens could act as an ‘ecological sink’ in the upper Paraná River basin, disrupting the transmission of Au. compactum metacercariae to the definitive hosts. However, this relationship is expected to have a low ecological effect on the native parasite life cycle due to its low prevalence in A. gigas. Future studies, including more specimens of A. gigas with shorter lengths (< 29 cm), will be conducted to elucidate their role in transmitting Au. compactum.
In the present study, just five hosts greater than 69 cm were infected by Au. compactum metacercariae, suggesting that the infection rates in A. gigas may be influenced by the host body size and age. A larger body surface may increase the possibility of active penetration of cercariae (Luque et al. Reference Luque, Amato and Takemoto1996). Moreover, commonly larger hosts are also older and have been exposed to parasite colonization longer (Albuquerque et al. Reference Albuquerque, Morey, Morais and Malta2017). Since displostomid metacercariae are presumably long-lived, their numbers tend to increase with host age (Karvonen and Marcogliese Reference Karvonen, Marcogliese, Woo, Leong and Buchmann2020). A similar relationship has been observed between P. squamosissimus and Au. compactum in native and non-native areas (Albuquerque et al. Reference Albuquerque, Morey, Morais and Malta2017; Machado et al. Reference Machado, Takemoto and Pavanelli2005; Paes et al. Reference Paes, Carvalho and Silva2010).
Fishes with high rates of infection caused by metacercariae of diplostomids in eyes (> 20 metacercariae per eye) (Karvonen et al. Reference Karvonen, Sepällä and Valtonen2004; Seppälä et al. Reference Seppälä, Karvonen and Tellervo-Valtonen2004; Seppälä et al. Reference Sepällä, Karvonen and Valtonen2011) can show retinal detachment, lens opacity (cataracts), exophthalmos, and blindness (Silva-Souza Reference Silva-Souza1998), resulting in loss of visual acuity. It can make fish hosts more susceptible to predation, facilitating the transmission of the parasite to the definitive hosts (Affonso et al. Reference Affonso, Karling, Takemoto, Gomes and Nilsson2017; Seppälä et al. Reference Seppälä, Karvonen and Tellervo-Valtonen2004) and can reduce its ability to capture prey (Voutilainen et al. Reference Voutilainen, Figueiredo and Huuskonen2008). Although the mean intensity of infection observed herein was 31.8 ± 17.2 (1–93), there is an outlier among analyzed hosts (a single host with 93 metacercariae). Among five infected hosts, only two presented more than 20 metacercariae in the eyes.
Arapaima gigas is a visual predator (Bezerra et al. Reference Bezerra, Soares, Carvalho and Coelho2013), and its natural diet is mainly composed of items of animal origin (carnivorous), with juvenile individuals preferentially consuming aquatic invertebrates (insects, mollusks, and crustaceans), and adults primarily consuming fish (Rodrigues et al. Reference Rodrigues, Moro and Santos2015). So, in the present study, the low infection rates of Au. compactum in A. gigas possibly does not act as a biological filter to hinder or contain the invasion because it probably has a low negative effect on the visual acuity of the hosts. It could be advantageous to this introduced predator if it competes for prey with other carnivorous fish species in the reservoir. These tentative suggestions require further analysis of the diet and parasites of native and non-native hosts from the same reservoir. This possible beneficiation was proposed by Lacerda et al. (Reference Lacerda, Takemoto, Tavares-Dias, Poulin and Pavanelli2012) in a study that compared parasite infections of native and introduced species in the same basin (upper Paraná River basin). The authors observed that P. squamosissimus exhibited lower levels of abundance of metacercariae of diplostomids compared to the native Hoplias malabaricus (Bloch, 1794), suggesting that the invader may gain some advantage if competition for prey.
Although our study represents the first scientific report on the occurrence of these metacercariae in A. gigas, it does not exclude the possibility of this parasite-host relationship occurring in the area where this host is native, given that this parasite is generalist, presents a widespread distribution, and is dispersed by piscivorous birds (final hosts). Considering the population of A. gigas introduced into the Água Vermelha reservoir, a broader investigation is necessary to provide information about how the relative density and competency of native and non-native hosts can influence the population dynamics of Au. compactum metacercariae in this reservoir, including changes in the density of preferential hosts by predation after A. gigas introduction. Moreover, it is necessary to evaluate whether infection rates by these metacercariae will increase over time of exposure and if higher rates of infection can negatively affect the food intake of these hosts.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25000045.
Acknowledgements
We acknowledge the anonymous reviewers for their suggestions and remarks that improved the manuscript. We are also very thankful to the respective curators of the helminthological collections CHIOC and CHIBB and the ichthyological collections DZSJRP and CITL for receiving our material (parasites and fish, respectively) to deposit. We are thankful to Aymar Orlandi-Neto for being helpful and improving the map.
Author contribution
All authors (L. Franceschini; F.G.C. Dias; B.S. Miguel; A.C. Zago; M.L. Orsi; J.R.S. Vitule; F.S. Almeida; F. Porto-Foresti; J.A. Balbuena; I.P. Ramos; L. Casatti) contributed to the study conception and design. The data preparation, curation, and analysis were performed by L. Franceschini. The manuscript was written by L. Franceschini and revised by the co-authors. All authors read and approved the final manuscript.
Availability of data and materials
Available upon request.
Financial support
This study was financed, in part, by the São Paulo Research Foundation (FAPESP), Brazil, Process Number 2022/05857-6 (LF); 2023/05883-0 (FGCD); and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (National Council for Scientific and Technological Development) – Process Number 304403/2021 (LC); 302367/2018–7 and 310471/2023-0 (JRSV).
Competing interest
The authors declare none competing interests.
Ethical standard
All applicable institutional, national, and international guidelines for the ethical handling of animals were followed licenses for the collection of zoological material (SISBio # 64763-5) and the Ethics Committee for Animal Experimentation (CEUA-IBILCE/UNESP # 241/2023). According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (A7F643D).
