Introduction
Digeneans have a complex life cycle, involving a definitive vertebrate host and at least one first intermediate host (usually an invertebrate), in which the larval stages are found. Sometimes, digeneans may present a second intermediate host (Combes et al., Reference Combes, Bartoli, Théron, Lewis, Campbell and Sukhdeo2002; Rosser et al., Reference Rosser, Alberson, Khoo, Woodyard, Pote and Griffin2016). Although the life cycle of digeneans has been commonly studied, the larval stages of numerous taxa are still unknown and their identification remains difficult due to the small number of morphologically diagnostic characteristics (Scholz et al., Reference Scholz, Aguirre-Macedo, de León, Ditrich, Salgado-Maldonato and Vidal-Matínez2000).
Digeneans are among the most common parasites identified in fish in the upper Paraná River floodplain (UPRF) (Chapell, Reference Chapell1995; Takemoto et al., Reference Takemoto, Pavanelli, Lizama, Lacerda, Yamada, Moreira, Ceschini and Bellay2009), a region important to biodiversity conservation because it maintains highly heterogeneous habitats and species richness (Filho & Ibarras, Reference Filho, Ibarras, Agostinho, Thomas, Júlio, Hahn and Rodrigues2005), consequently enabling an interesting diversity of parasites (Souza et al., Reference Souza, Machado, Dias, Yamada, Pagotto and Pavanelli2008; Takemoto et al., Reference Takemoto, Pavanelli, Lizama, Lacerda, Yamada, Moreira, Ceschini and Bellay2009). Currently, only Biomphalaria peregrina (Orbiny, 1835) has been identified as a potential invertebrate host for digeneans in the UPRF (Souza et al., Reference Souza, Machado, Dias, Yamada, Pagotto and Pavanelli2008), however, the low prevalence of parasitism indicates that other mollusc species could be acting as intermediate hosts for these organisms.
Because other mollusc species inhabit the UPRF, Aylacostoma chloroticum (Scott, 1954) is an interesting potential host candidate for digeneans because it has been registered as an intermediate host of Pseudosellacotyla lutzi (Freitas, 1941), Neocladocystis intestinalis (Vaz, 1932), Stephanoprora aylacostoma and the Echinostoma group (Quintana & Núñez, Reference Quintana and Núñez2008, Reference Quintana and Núñez2014, Reference Quintana and Núñez2016; Pinto & Melo, Reference Pinto and Melo2013a) and it is native to this environment. This mollusc is currently considered an endangered species according to the IUCN Red List of Threatened Species (Mansur, Reference Mansur2000), and in South America it is part of a conservation breeding programme called the Aylacostoma Project (Vogler et al., Reference Vogler, Beltramino, Peso and Rumi2014). In addition, it is the only species of this genus in the UPRF, a region impacted by the construction of dams in the Paraná River basin and by the introduction of species, including molluscs (Takeda et al., Reference Takeda, Fujita, Fontes Júnior, Agostinho, Rodrigues, Gomes, Thomaz and Miranda2004).
According to Abdul-Salam & Al-Khedery (Reference Abdul-Salam and Al-Khedery1992), the populations of molluscs can determine the species of digeneans present in birds and fish in a certain region and changes in the malacological community may alter the life cycle of these parasites; therefore, infections caused by trematode larvae can be used as bioindicators of environmental quality (Souza et al., Reference Souza, Machado, Dias, Yamada, Pagotto and Pavanelli2008). However, the identification of larval stages is difficult, so the use of molecular techniques is important for the correct identification of Platyhelminthes (Locke et al., Reference Locke, Al-Nasiri and Caffara2015). Therefore, molecular markers are commonly used and the fragment of the mitochondrial gene of subunit I of cytochrome c oxidase (COI) and the 28S nuclear ribosomal gene are both helpful for identification at a specific level because they represent conserved regions present in interesting variations among species of this group (Vilas et al., Reference Vilas, Criscione and Blouin2005; Moszczynska et al., Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009; Steenkiste et al., Reference Steenkiste, Locke, Castelin, Marcogliese and Abbott2015).
The identification of digenean species and their respective intermediate hosts in the UPRF are important considerations for public health policies. Some species are zoonotic (Fried et al., Reference Fried, Graczyk and Tamang2004; Pinto & Melo, Reference Pinto and Melo2013b), which is a concern for the riverside population and tourists. In addition, some species use fish as a second intermediate host, which causes damage to fish farming, making them susceptible to predation or increasing their mortality rate (Eiras et al., Reference Eiras, Takemoto, Pavanelli and Adriano2011). Therefore, the objective of this study was to characterize and identify digeneans parasitizing A. chloroticum using molecular tools.
Materials and methods
Sampling was performed during July 2017 and May 2018, in the upper Paraná River floodplain (Garças lagoon, 22°43′30.7″S, 53°13′11.6″W) in Batayporã city (Mato Grosso do Sul State, Brazil). The specimens were maintained alive in aquariums in the Laboratório de Ictioparasitologia of the NUPÉLIA (Núcleo de Pesquisa em Limnologia, Ictiologia e Aquicultura) and analysed during the study.
To identify the gastropods, the individuals were examined and identified morphologically with the support of researchers from the Laboratório de Zoobentos (NUPÉLIA) and identification was confirmed using the COI mitochondrial gene. Parasite specimens were obtained from the gastropods collected. Subsequently, the body of the gastropod was carefully removed from its shell to avoid damaging the specimen and was examined under a stereomicroscope. The digestive gland was specifically examined because this organ is generally an infection site. The digeneans obtained were identified by their morphology (Gibson et al., Reference Gibson, Jones and Bray2002; Pinto & Melo, Reference Pinto and Melo2013b) and isolated in 1.5 ml tubes containing Milli-Q water, according to morphotype.
Two specimens of parasites of each morphotype were used for DNA extraction, which was carried out using the ReliaPrep™ gDNA Tissue Miniprep System kit, following the manufacturer's instructions. The 28S gene was partially amplified using the U178 primer: 5′-GCACCCGCTGAAYTTAAG-3′ and the L1642 primer: 5′-CCAGCGCCATCCATTTTCA-3′ (Lockyer et al., Reference Lockyer, Olson and Littlewood2003). The polymerase chain reaction (PCR) conditions comprised an initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 56°C for 1 min, 72°C for 1 min and a final elongation cycle at 72°C for 5 min. The COI gene also was partially amplified using the Trem CoI: F 5′-TTTCGTTGGATCATAAGCG-3′ and e Trem CoI: R 5′-GCAGCACTAAATTTACGATCAAA-3′ primers (Bonett et al., Reference Bonett, Steffen, Trujano-Alvarez, Martin, Bursey and McAllister2011). The PCR conditions for the COI gene amplification comprised an initial denaturation at 94°C for 1 min, followed by ten cycles at 94°C for 30 s, 42°C for 40 s and 72°C for 1 min, then 30 cycles at 94°C for 30 s, 50°C for 40 s, 72°C for 1 min and a final elongation step at 72°C for 7 min. The amplicons were verified on 1% agarose gel and purified using polyethylene glycol. The sequencing reactions were prepared using BigDye™ Terminator 3.1. Cycle sequencing was performed following the manufacturer's instructions and the products were sent to the Complexo de Centrais de Apoio à Pesquisa (COMCAP) of the Universidade Estadual de Maringá for automated sequencing using an ABI3500 Applied Biosystems sequencer.
The sequences obtained were edited and aligned using BioEdit (Hall, Reference Hall1999) and MEGA 7.0 (Kumar et al., Reference Kumar1999) software, respectively. Species identification of the parasites was performed by comparing the results with previous data available in GenBank using the BLAST tool implemented in MEGA 7.0. Therefore, the sequences with high similarity were selected for further analysis (tables 1 and 2). The Kimura-2-parameter (K2P) distance among the analysed species were also calculated using MEGA 7.0. The best nucleotide substitution model was selected using jModelTest 2 (Darriba et al., Reference Darriba, Taboada, Daollo and Posada2012) and maximum-likelihood gene trees were constructed using the RAxML Black Box (Kozlov et al., Reference Kozlov, Darriba, Flouri, Morel and Stamatakis2019). Schistosoma japonicum was used as an outgroup in both analyses because this species belongs to a family distant from those of the digeneans obtained in this study – Faustulidae, Lecithodendriidae and Philophthalmidae. The novel sequences were deposited in GenBank (MK629695–MK629702).
Table 1. 28S sequences used in this study.
Table 2. COI sequences used in this study.
Results
Partial sequences of the COI gene obtained from the mollusc specimens used in this study showed 98% similarity with A. chloroticum sequences available in GenBank and a 0.7% genetic distance. A total of 26 specimens of A. chloroticum were analysed, of which 19 (73.08%) were parasitized. Three distinct digenean morphotypes were recorded in these molluscs, morphotype 1, 2 and 3. Each morphotype showed the following prevalence: morphotype 1 (31%), morphotype 2 (50%) and morphotype 3 (34%). The mean measurements of the morphotypes are presented in table 3.
Table 3. Mean measurements (min–max) (in μm) of cercariae obtained from Aylacostoma chloroticum.
For the 28S gene, morphotype 1 shared high (95–96%) genetic similarity with Paralecithodendrium parvouterus (Bhaleraeo, 1926); morphotype 2 was most similar to Pseudosellacotyla lutzi, with 99–100% similarity; and morphotype 3 was most similar to Philophthalmus gralii (Mathis and Leger, 1910), with 98% similarity. The sequences used in the analyses are shown in table 1. The mean genetic distance between the different species of Paralecithodendrium was 6.6% and ranged from 3.9% to 8.2%, with 7.1% genetic distance between these sequences and morphotype 1. The sequence obtained for morphotype 2 was identical to Pseudosellacotyla lutzi obtained from GenBank. In Philophthalmus, the mean distance between species was 1.8% and ranged from 1.7% to 2.4%, and 2.1% when Philophthalmus spp. are compared with morphotype 3. The genetic distance values between all sequences are shown in table 4. A maximum-likelihood tree was constructed from this data (fig. 1).
Fig. 1. Maximum-likelihood gene tree constructed using the 28S gene sequences analysed in this study. Schistosoma japonicum was used as an outgroup. Support values above 85 are represented by circles. (a) Cercaria morphotype 3 obtained in this study; (b) cercaria morphotype 2 obtained in this study; (c) cercaria morphotype 1 obtained in this study. Scale bars indicates the mean number of nucleotide substitutions per site.
Table 4. Genetic distances resulting from the comparison of 28S gene sequences obtained from GenBank and morphotype 1, 2 and 3 cercariae of Aylacostoma chloroticum.
The COI region of morphotypes 1 and 2 were 77% and 78% similar, respectively. However, molecular identification using this gene was not conclusive when compared with previously published data, so the results were not used for maximum-likelihood tree construction. For morphotype 3, the sequences were 88% similar to Philophthalmus gralli and P. lucipetus (Rudolphi, 1819). The sequences were aligned with the sequences of different Philophthalmus species obtained from GenBank (table 2), with a mean distance of 15.4% between the species of this genus and 14.5% divergence from morphotype 3. The genetic distance values are shown in table 5. A maximum-likelihood tree was constructed using this data (fig. 2).
Fig. 2. Maximum-likelihood gene tree constructed using the COI gene sequences of Philophthalmus species used in this study. Schistosoma japonicum was used as an outgroup. Support values above 85 are represented by circles. Scale bars indicates the mean number of nucleotide substitutions per site.
Table 5. Genetic distances resulting from the comparison of COI gene sequences obtained from GenBank and morphotype 3 cercariae of Aylacostoma chloroticum.
Discussion
This is the first molecular characterization and identification of digenean larvae obtained in an aquatic invertebrate host from the UPRF using the molecular markers 28S and cytochrome c oxidase. These results showed that A. chloroticum is an important intermediate host of digenean parasites in this region.
Based on the 28S gene partial sequence, the relationships observed in the gene tree (fig. 1) indicate that morphotypes 1 and 3 belong to Paralecithodendrium and Philophthalmus. However, morphotype 2 was identified as Pseudosellacotyla lutzi, previously recorded as parasitizing specimens of Hoplias malabaricus (Bloch, 1794) ‘traíra’ in Brazil (Pantoja et al., Reference Pantoja, Hernández-Mena, de León and Luque2018), and using A. chloroticum as an intermediate host during its life cycle (Quintana & Núñez, Reference Quintana and Núñez2014), which was validated by this study.
The COI gene provided conclusive information only for morphotype 3, indicating that this specimen belongs to Philophthalmus, a species that can use birds or mammals as definitive hosts. In Brazil, there are also records of Philophthalmus gralli parasitizing Melanoides tuberculatus (Müller, 1774), an invasive mollusc, but only under experimental conditions (Pinto & Melo, Reference Pinto and Melo2010, Reference Pinto and Melo2013a). In the UPRF, there are records of Philophthalmus lachrymosus (Braun, 1902) using Capybara, Hydrochoerus hydrochaeris (Linnaeus, 1766), as a definitive host, found in its vitreous humour (Souza et al., Reference Souza, Ribeiro, Antonuci, Ueda, Carniel, Karling, Eiras, Takemoto and Pavanelli2015). However, there have been no previous studies on the definitive host of this genus in the UPRF. Paralecithodendrium was also reported to use M. tuberculatus as a first intermediate host, insects as second intermediate hosts and bats as its definitive host (Fried et al., Reference Fried, Graczyk and Tamang2004; Santos & Gibson, Reference Santos and Gibson2015). These parasites are globally distributed, with human infections recorded in Asiatic countries, such as Indonesian and Thailand (Kumar, Reference Kumar, Stecher and Tamura1999).
In general, the molecular markers used in this study were useful for the identification of digenean parasites at the genus and species level, but the results also indicate that more than one molecular marker should be used, considering the difficulty in developing universal primers to these organisms (Moszczynska et al., Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009; Steenkiste et al., Reference Steenkiste, Locke, Castelin, Marcogliese and Abbott2015). Another difficulty is the scarcity of DNA sequences in public databases. In addition, the difficulty in morphological identification of the parasites obtained in this study may be because these specimens represent previously undescribed digenean species, thereby necessitating new molecular and morphological studies in the future.
Monitoring the life cycle of the parasites, along with the identification of their larval stages, can help in the understanding of ecosystem dynamics and environmental quality because changes in the environment are reflected in the digenean species richness and in the prevalence of infection (Souza et al., Reference Souza, Machado, Dias, Yamada, Pagotto and Pavanelli2008). Therefore, considering the high prevalence of parasitism (73.08%) in this gastropod, it is possible that this species is an important intermediate host in the life cycle of digenean and the maintenance of their populations is of great importance in terms of conservation and preservation of biodiversity. Finally, we suggest intensifying studies aimed at understanding the life cycles of digenean parasites using molecular tools because morphological identification is difficult during the initial stages of the life cycle and, perhaps more importantly, because two of the three digeneans found here present zoonotic potential.
Acknowledgements
We would like to thank the laboratories of Zoobentos of the NUPÉLIA (UEM), all researchers that provided support and materials to the conduct of this study, the COMCAP (UEM) and the Universidade Estadual de Maringá – the institution where this research was carried out.
Financial support
This work was supported by CNPq (446150/2014-2).
Conflicts of interest
None.
