Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil

Caracterização morfológica e molecular de metacercárias de Austrodiplostomum compactum em olhos e cérebros de peixes do Rio Ivaí, Brasil

Abstract

Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Keywords:
COI gene tree; Diplostomidae; endoparasites; freshwater fish; Loricariidae

Resumo

Austrodiplostomum spp. (Platyhelminthes: Digenea) são endoparasitos com uma ampla distribuição geográfica na América do Sul. Durante a fase larval, parasitam os olhos, cérebros, músculos, brânquias, rins e bexiga natatória de uma grande variedade de peixes. As metacercárias de Austrodiplostomum spp. apresentam várias características morfológicas durante o desenvolvimento, as quais são muito semelhantes entre as espécies, o que torna necessário o uso de ferramentas moleculares para contribuir para a elucidação durante a fase larval. O objetivo deste estudo foi realizar análises morfológicas e moleculares de Austrodiplostomum sp. encontradas em espécimes de Hypostomus provenientes do rio Ivaí, no Paraná, Brasil. Dos 93 espécimes analisados (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], e Hypostomus sp. 2 [n = 10]), 60 foram parasitados. Um total de 577 metacercárias de Austrodiplostomum foram coletadas dos hospedeiros infectados; o DNA de sete dessas amostras foi extraído, amplificado e sequenciado. Os dados morfológicos, associados aos valores de distância genética e as relações observadas na árvore gênica do COI, indicam que todas as metacercárias são A. compactum. Este é o primeiro registo de A. compactum parasitando H. hermanni, H. albopunctatus, Hypostomus sp. 1, e Hypostomus sp. 2 no rio Ivaí.

Palavras-chave:
Árvore gênica COI; Diplostomidae; endoparasitos; peixes de água doce; Loricariidae

Introduction

Austrodiplostomum Szidat & Nani, 1951 (Digenea: Diplostomidae) are digenetic trematodes belonging to the phylum Platyhelminthes (Travassos et al., 1928; Takemoto et al., 2009). This genus included only two species: A. mordax Szidat & Nani, 1951, found in South America, and A. compactum Lutz, 1928 (syn. A. ostrowskiae Dronen, 2009) reported in the United States, Mexico, El Salvador, Honduras, Costa Rica, Venezuela, Peru and Brazil (García-Varela et al., 2016; De Núñez, 2017; Sereno-Uribe et al., 2019a).

Digenetic trematodes have a complex life cycle in which they can parasitize three hosts (Kohn et al., 2013). In the adult stage, they are found in the digestive tract of piscivorous birds, such as Nannopterum brasilianus (Gmelin, 1789), the definitive hosts of this parasite (Kennedy & Spencer, 2014; García-Varela et al., 2016). During reproduction, the parasite produces eggs that are released into the aquatic environment through the feces of the birds (Travassos et al., 1928). The miracidia emerge from the eggs and penetrate the integument of mollusks of the genus Biomphalaria (Preston, 1910): B. straminea (Dunker, 1848), B. glabrata (Say, 1818), B. prona (Martens, 1873), and B. havanensis (Pfeiffer, 1839) (Pinto & Melo, 2013; Rosser et al., 2016; De Núñez, 2017).

After morphological transformations, the miracidium gives rise to free-swimming forms of the parasite called cercariae (Travassos et al., 1928). Cercariae can actively infect fish and evolve into metacercariae. The metacercariae of Austrodiplostomum spp. are usually found in the eyes, brains, and muscles of fishes (Travassos et al., 1928; Lehun et al., 2020). However, can also be found in the gill, renal parenchyma and swim bladder (Monteiro et al., 2016). The presence of this parasite in the eyes can impair vision, which makes the infected fishes susceptible to predation and facilitates transmission of the parasite to the definitive host, piscivorous birds (Affonso et al., 2017).

In Brazil, A. compactum metacercariae are most abundantly found in fishes from the Cichlidae and Sciaenidae families, but they have also been found in Anostomidae, Auchenipteridae, Characidae, Curimatidae, Erythrinidae, Pimelodidae, and Loricariidae (Santos et al., 2012; Ramos et al., 2013). Despite the wide distribution and increasing number of occurrences of Austrodiplostomum spp. in fishes (Pelegrini et al., 2019; Acosta et al., 2020; Campos et al., 2020; Ramos et al., 2013, 2020) in Brazilian rivers, there are still sites where studies are scarce, such as the Ivaí River, located entirely in the state of Paraná, Brazil, and is an important tributary of the Paraná River basin.

Austrodiplostomum spp. metacercariae have morphological structures at the developmental stage, including an oral sucker, pseudosuckers, pharynx, intestinal caeca, tribocytic organ, and gonads (De Núñez, 2017). However, at this stage, morphological identification is hampered by the similarity of structures between species and by some morphological features that may be absent (De Núñez, 2017). In these cases, the use of molecular techniques is beneficial in the identification process (Sereno-Uribe et al., 2019b; Onaca et al., 2019). The molecular marker cytochrome c oxidase subunit 1 (COI) is a useful tool for identifying digeneans. Moszczynska et al. (2009) stated that COI sequences showed good resolution at the species level, making it a practical target for digeneans; the same is true in a study by Locke et al. (2015), in which COI sequences were suitable for the discrimination of Diplostomum von Nordmann, 1832 species.

Thus, the aim of this study was to perform morphological and molecular analyses using the COI mitochondrial gene to aid the identification of the larval stage of Austrodiplostomum sp., collected from four species of the Hypostomus Lacépède, 1803 sourced from the Ivaí River.

Materials and Methods

Specimens of Hypostomus (93) were collected at three sites on the left bank of the Ivaí River, located in the municipality of Engenheiro Beltrão-Paraná, Brazil (23°40’15.4″S, 52°09’36.6″W; 23°40’06.3″S, 52°09’31.5″W; and 23°38’57.7″S, 52°09’52.7″W). The collection was authorized by the Instituto Chico Mendes de Biodiversidade (ICMBio), permit number 66135-3, and occurred in May, June, and September 2019 and March 2020. Fishes were captured by gillnets (3.0/5.0/6.0/7.0 cm measured between opposing stretched nodes) that were placed at the stated locations at dusk and collected the following morning, totaling a 12-h exposure. The captured fish were anesthetized with benzocaine and killed according to the Euthanasia Practice Guidelines of the Conselho Nacional de Controle de Experimentação Animal (CONCEA) with permission from the Comissão de Ética no Uso de Animais of the Universidade Estadual de Maringá (CEUA– no. 5073090620). Fishes were identified according to the protocols by Frota et al. (2016) and Zawadzki et al. (2020). During the identification of the hosts, two species were found that were not yet identified at specific level (Hypostomus sp.1 and Hypostomus sp.2).

After collecting the biometric data of the fishes, the eyes and brains were removed and placed into Petri dishes containing saline solution. Subsequently, they were observed under a stereomicroscope to search for digenetic parasites. For morphological analysis, metacercariae were collected from the eyes of the following fishes: Hypostomus sp. 1 (five specimens), Hypostomus sp. 2 (five specimens), H. albopunctatus (Regan, 1908) (five specimens with an additional three specimens collected from the brains) and H. hermanni (Ihering, 1905) (five specimens with an additional four specimens collected from the brains).

The specimens were fixed and stained with carmine according to the method reported by Eiras et al. (2006). Representative specimens of Austrodiplostomum compactum were deposited in the Helminthological Collection of the Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, Brazil (CHIOC: 39707), and the host fishes were deposited in the Ichthyological Collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia), under the numbers: H. hermanni (NUP 22655), H. albopunctatus (NUP 22659), Hypostomus sp.1 (NUP 22656) and Hypostomus sp. 2 (NUP 22657).

The measurements recorded for the length and width of the body, as well as structures such as the oral sucker, pharynx, trybocytic organ, and metacercaria gonads, were expressed in millimeters. The images were captured using the optical photographic equipment OPTHD 3.7, attached to a Nikon Eclipse e200 microscope. The prevalence, mean abundance, and mean intensity of parasitic infections in the hosts were calculated according to Bush et al. (1997).

DNA was extracted from metacercariae (n = 7) of Austrodiplostomum sp. found in the following host organs: brain (1) and eyes (1) of H. albopunctatus, eyes (3) of H. hermanni, eyes (1) of Hypostomus sp. 1, and eyes (1) of Hypostomus sp. 2. The extraction was carried out using the ReliaPrep™ gDNA Tissue Miniprep System kit, following the manufacturer’s instructions. COI gene was partially amplified using primers for Plat-diploCOXF:5′-CGTTTRAATTATACGGATCC-3′ and bR:5′AGCATAGTAATMGCAGCAGC-3′ (Moszczynska et al., 2009).

Polymerase chain reaction (PCR) conditions comprised an initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min, and a final elongation cycle at 72 °C for 10 min. The amplicons were verified on a 1% agarose gel by comparing with a 100 bp ladder Invitrogen (lot no. 765323) and purified using a polyethylene glycol protocol (Rosenthal et al., 1993). The samples were sequenced by ACTGene Análises Moleculares Ltda, using the ABI-3500 automated sequencer. Access to genetic heritage was authorized by the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (register no. A29B419).

The sequences obtained were edited manually and aligned using the BioEdit 7.2 (Hall, 1999) and MEGA 7.0 (Kumar et al., 2016) software, respectively. The sequence similarity values of the parasites were obtained by comparing the sequences with GenBank data sets using the BLASTn tool. The novel sequences were deposited in GenBank (accession numbers: MT627211; MT632470–MT632475).

To construct the gene tree using the COI gene, sequences of Austrodiplostomum, Diplostomum, and Tylodelphys Diesing, 1850 were added from GenBank, and Australapatemon mclaughlini Gordy, Locke, Rawlings, Lapierre, Hanington, 2017 was used as an outgroup (Table 1). The gene tree was constructed using the maximum likelihood statistical method with the Randomized Axelerated Maximum Likelihood (RaxML) program (Kozlov et al., 2019). The best nucleotide substitution model (HKY+I+G) was selected based on the Bayesian information criterion using jModelTest 2 (Darriba et al., 2012).

Table 1
COI sequences available in GenBank used in this study.

Parasite species	Location	Haplotype	GenBank accession number	Reference
Austrodiplostomum compactum	-------	H1	MH378899-MH378901	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H16	MH378902	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378903- MH378905	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H5	MH378906	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378907	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H17	MH378908	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H18	MH378909	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H7	MH378910	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	Honduras	H2	MH378911	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378912- MH378914	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H19	MH378915	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378916- MH378919	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H8	MH378920- MH378921	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	Mexico	H2	MH378922	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378923	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H7	MH378924	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378925- MH378927	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H2	MH378940	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378941- MH378949	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H8	MH378950	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	-------	H1	MH378951- MH378953	Sereno-Uribe et al. (2019a)
Austrodiplostomum compactum	Brazil	H21	MN179321	Lopez-Hernandez et al. (2019)
Austrodiplostomum compactum	Brazil	H20	MN179320	Lopez-Hernandez et al. (2019)
Austrodiplostomum compactum	Brazil	H1	MZ323246	Achatz et al. (2022)
Austrodiplostomum ostrowskiae	Brazil	H1	KR271025	Locke et al. (2015)
Austrodiplostomum ostrowskiae	United States	H4	KR271026	Locke et al. (2015)
Austrodiplostomum ostrowskiae	Peru	H15	KR271027	Locke et al. (2015)
Austrodiplostomum ostrowskiae	United States	H2	KR271028	Locke et al. (2015)
Austrodiplostomum ostrowskiae	-------	H1	KM115890-KM115899	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	-------	H4	KM115884-KM115889	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	United States	H1	KT728783	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H6	KT728787- KT728788	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H6	KT728798	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H1	KT728799	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H6	KT728791	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H1	KT728793	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H4	KT728794	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H9	KT728795	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	United States	H2	KT728786	Rosser et al. (2016)
Austrodiplostomum ostrowskiae	-------	H1	KM115883	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H2	KM115944	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	El Salvador	H7	KM115967	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	El Salvador	H1	KM115966	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H3	KM115965	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H5	KM115964	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115962-KM115963	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115900--KM115901	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115902- KM115909	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Venezuela	H1	KM115910- KM115914	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H4	KM115915	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115916- KM115917	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H4	KM115918- KM115920	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115921-KM115933	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H4	KM115934	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115935	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H4	KM115936- KM115937	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115938	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H10	KM115939	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H11	KM115940	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115941- KM115943	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H12	KM115945	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H5	KM115946	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115947	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H8	KM115948	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115949- KM115950	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H13	KM115951	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H7	KM115952- KM115953	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H1	KM115954- KM115961	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	Mexico	H14	KM115882	García-Varela et al. (2016)
Austrodiplostomum ostrowskiae	United States	H2	MF124271	Blasco-Costa & Locke (2017)
Austrodiplostomum ostrowskiae	United States	H2	JX468066	O’Hear et al. (2014)
Austrodiplostomum mordax	Argentina	-------	MH378895	Sereno-Uribe et al. (2019a)
Austrodiplostomum mordax	Argentina	-------	MH378896	Sereno-Uribe et al. (2019a)
Austrodiplostomum mordax	Argentina	-------	MH378897	Sereno-Uribe et al. (2019a)
Austrodiplostomum mordax	Argentina	-------	MH378898	Sereno-Uribe et al. (2019a)
Austrodiplostomum sp.1	Mexico	-------	MH378928	Sereno-Uribe et al. (2019a)
Austrodiplostomum sp.1	United States	-------	KR271029	Locke et al. (2015)
Austrodiplostomum sp.1	Mexico	-------	MH378929	Sereno-Uribe et al. (2019a)
Austrodiplostomum sp.1	Mexico	-------	MH378930	Sereno-Uribe et al. (2019a)
Austrodiplostomum sp.2	United States	-------	KU707940	Rosser et al. (2016)
Austrodiplostomum sp.2	United States	-------	KR271030	Locke et al. (2015)
Austrodiplostomum sp.2	Mexico	-------	MH378938	Sereno-Uribe et al. (2019a)
Austrodiplostomum sp.2	Mexico	-------	MH378939	Sereno-Uribe et al. (2019a)
Diplostomum sp. 15	China	-------	KR271126	Locke et al. (2015)
Diplostomum sp. 9	Canada	-------	KR271410	Locke et al. (2015)
Diplostomum sp. 10	Canada	-------	KR271096	Locke et al. (2015)
Diplostomum sp. 17	Canada	-------	KR271131	Locke et al. (2015)
Diplostomum ardeae	Canada	-------	KR271033	Locke et al. (2015)
Diplostomum ardeae	Porto Rico	-------	MT324592	Locke et al. (2020)
Diplostomum ardeae	Porto Rico	-------	MT324592	Locke et al. (2020)
Diplostomum lunaschiae	Brazil	-------	MT324620	Locke et al. (2020)
Diplostomum lunaschiae	Brazil	-------	MT324621	Locke et al. (2020)
Diplostomum lunaschiae	Brazil	-------	MT324623	Locke et al. (2020)
Diplostomum lunaschiae	Brazil	-------	MT324626	Locke et al. (2020)
Tylodelphys sp. 6	Mexico	-------	MK172796	Sereno-Uribe et al. (2019a)
Tylodelphys sp. 6	Mexico	-------	MK172797	Sereno-Uribe et al. (2019a)
Tylodelphys sp. 6	Mexico	-------	MK172798	Sereno-Uribe et al. (2019a)
Tylodelphys sp. 6	Mexico	-------	MK172799	Sereno-Uribe et al. (2019a)
Australapatemon mclaughlini	Canada	-------	KY587406	Gordy et al. (2017)
Austrodiplostomum sp. (A1)	Brazil	H3	MT627211	Present study
Austrodiplostomum sp. (A2)	Brazil	H1	MT632471	Present study
Austrodiplostomum sp. (A3)	Brazil	H1	MT632473	Present study
Austrodiplostomum sp. (A4)	Brazil	H1	MT632474	Present study
Austrodiplostomum sp. (A5)	Brazil	H1	MT632475	Present study
Austrodiplostomum sp. (A6)	Brazil	H1	MT632470	Present study
Austrodiplostomum sp. (A7)	Brazil	H2	MT632472	Present study

The Kimura-2-parameter (K2P) distance was calculated between the analyzed species obtained in this study and sequences available in GenBank using MEGA 7.0 software. The groups were formed according to the species identified in the database. The haplotype network was generated using the PopArt program (Leigh & Bryant, 2015), and all sequences available in GenBank for A. compactum and A. ostrowskiae were used, totaling 155 sequences. Although they were presented as synonyms (De Núñez, 2017), in our analyses, the name A. ostrowskiae was kept as it is in the deposit record, precisely to confirm that they are genetically the same species.

Results

Of the total specimens analyzed (n = 93), 60 were infected and 577 metacercariae were collected from infected eyes and brains. In the eyes, metacercariae were found free in the aqueous and vitreous humor. In the brain, metacercariae were found free, close to the optic nerve and at a lower quantity when compared to the eyes of the same hosts. Only H. hermanni and H. albopunctatus fishes presented metacercariae in the brain (Table 2).

Table 2
Austrodiplostomum compactum in Hypostomus spp. from the Ivaí River, Engenheiro Beltrão, Paraná, Brazil.

Hosts	N/I	N/E	N/B	P (%)	MA ± SE	MI ± SE
H. hermanni	50/38	500	4	76	10. ± 2.43	13.2 ± 2.64
H. albopunctatus	09/08	41	7	88.8	4.55 ± 0.98	5.1 ± 0.86
Hypostomus sp.1	24/10	19	0	41.6	0.79 ± 0.20	1.9 ± 0.11
Hypostomus sp.2	10/04	06	0	40	0.60 ± 0.26	1.5 ± 0.18

• (N/I) number of fishes analyzed and number of infected fishes; (N/E) number of metacercariae collected in eyes; (N/B) number of metacercariae collected in brain; (P%) prevalence expressed as a percentage; (MA) mean abundance; (MI) mean intensity; (SE) standard error.

The biometric data of the hosts showed that H. albopunctatus had the largest weight and size (Table 3). However, there was no morphological variation between metacercariae of different species, between hosts of the same species or between different collection sites. The amount of metacercariae collected from the eyes and brains of fishes’ species demonstrates that infection can occur at random. Some hosts, regardless of size, may have a greater amount of metacercariae than others, as occurred in the eyes of H. hermanni, where 72 metacercariae were collected, while in other species less than 20 metacercariae were found.

Table 3
Biometric data of Hypostomus spp. (Siluriformes, Loricariidae) collected from the Ivaí River, Engenheiro Beltrão, Paraná, Brazil.

Host	N	SL ± SE (cm)	W ± SE (g)
H. hermanni	50	16.15 ± 0.23	128.65 ± 6.16
H. albopunctatus	9	23.33 ± 1.75	342.66 ± 61.46
Hypostomus sp.1	24	12.95 ± 0.31	67.03 ± 5.47
Hypostomus sp.2	10	12.02 ± 0.44	50.53 ± 5.31

• Mean values are expressed followed by ± standard error. (N) number of hosts analyzed; (SL) standard length; (W) weight.

The main morphological characteristics and structure measurements of 20 metacercariae specimens collected from the eyes of Hypostomus were: Elongated body, slightly concave in the ventral face 1.63 (1.4-1.97) long, 0.59 (0.5-0.75) wide; in relation to the body has a small conical segment in the posterior region; subterminal oral sucker 0.06 (0.04-0.09) long, 0.06 (0.05-0.09) wide; have two pseudosuckers, one on each side of the oral sucker; pharynx 0.05 (0.05-0.07) long, 0.054 (0.04-0.08) wide; esophagus short; intestinal caeca ending near the posterior region; oval tribocytic organ in the posterior half of the body 0.3 (0.1- 0.35) long, 0.17 (0.08-0.22) wide; anterior gonad 0.048 (0.03-0.06) long, 0.046 (0.04-0.06) wide; posterior gonad 0.045 (0.03-0.07) long, 0.045 (0.04-0.06) wide; width glandular cells scattered throughout the body (Figure 1). The metacercariae found in the hosts brains showed no differences in morphological or structural measurements.

Figure 1
Metacercariae of Austrodiplostomum compactum collected in eyes and brains Hypostomus hermanni from the Ivaí River - Paraná - Brazil. Metacercariae body (a), forebody; OS = oral sucker, PS = pseudosuckers, P = pharynx (b), hind body; TO = tribocytic organ, G = gonads (c).

Sequences of the COI region (405 bp) were obtained for seven specimens, totaling three distinct haplotypes in the Ivaí River. Overall, 21 haplotypes were obtained by the analysis of A. compactum sequences from GenBank. Considering the sequences obtained in this study, five were allocated to the most common haplotype (H1) (Figure 2). This haplotype was shared by 64.52% of the total analyzed sequences, including samples from other regions of Brazil, Mexico, Venezuela, El Salvador, and the United States. The A1 specimen, which parasitized the eye of H. albopunctatus, constituted a different haplotype (H3), and this was shared with one specimen of A. ostrowskiae from México. The A7 specimen shared the H2 haplotype with A. compactum and A. ostrowskiae specimens from México, United States, and Honduras. Haplotypes H1 and H2 were common in both analyzed species, A. compactum and A. ostrowskiae, thereby, confirming that it is the same species.

Figure 2
Haplotype network of Austrodiplostomum compactum (syn. A. ostrowskiae) obtained from GenBank and the sequences obtained in this study.

Comparisons made between the sequences obtained from the metacercariae with genetic sequences available in GenBank, resulted in values between 99.01% and 100% similarity with A. compactum (K2P distance of 0.1% and 0.4%) and A. ostrowskiae (K2P distance of 0.2% and 0.4%), respectively (Table 4). In relation to the other Austrodiplostomum species available in GenBank, the values of genetic distance ranged from 10.1% to 11.5%. For Diplostomum species, the values ranged from 13.6% to 16.6%, whereas those for Tylodelphys sp. ranged from 10.6% to 10.9%.

Table 4
Values of genetic distance (K2P) between the sequences of the COI region obtained from GenBank (8-16), grouped according to the identified species, and sequences of Austrodiplostomum sp. obtained in the present study (1-7).

	1	2	3	4	5	6	7	8	9	10	11	12	13	14		15
1. A1
2. A2	0.0 02
3. A3	0.002	0.000
4. A4	0.002	0.000	0.000
5. A5	0.002	0.000	0.000	0.000
6. A6	0.002	0.000	0.000	0.000	0.000
7. A7	0.005	0.002	0.002	0.002	0.002	0.002
8. Austrodiplostomum sp. 1	0.104	0.101	0.101	0.101	0.101	0.101	0.104
9. Austrodiplostomum sp. 2	0.108	0.105	0.105	0.105	0.105	0.105	0.108	0.108
10. Austrodiplostomum compactum*	0.004	0.001	0.001	0.001	0.001	0.001	0.002	0.103	0.106
11. Austrodiplostomum ostrowskiae	0.004	0.002	0.002	0.002	0.002	0.002	0.002	0.102	0.105	0.002
12. Autrodiplostomum mordax	0.109	0.112	0.112	0.112	0.112	0.112	0.115	0.134	0.132	0.113	0.114
13. Diplostomum sp.	0.156	0.156	0.156	0.156	0.156	0.156	0.153	0.165	0.184	0.154	0.154	0.167
14. Diplostomum ardeae	0.136	0.139	0.139	0.139	0.139	0.139	0.136	0.160	0.181	0.138	0.137	0.161	0.129
15. Diplostomum lunaschiae	0.163	0.166	0.166	0.166	0.166	0.166	0.163	0.180	0.187	0.165	0.164	0.174	0.141	0.081
16. Tylodelphys sp.	0.106	0.109	0.109	0.109	0.109	0.109	0.106	0.139	0.138	0.108	0.108	0.144	0.153	0.137	0.145

• ^*
  A. compactum (syn. A. ostrowskiae).

The gene tree presented in Figure 3 shows that the sequences derived from the samples extracted from the Ivaí River (highlighted by lozenge) as well as, sequences from A. compactum (syn. A. ostrowskiae), were allocated to a single clade. Interestingly, the clade was close to another cluster formed by a different species of Austrodiplostomum, whereas the species of Tylodelphys and Diplostomum formed distinct clusters.

Figure 3
Maximum-likelihood gene tree constructed using the COI gene sequences. Australapatemon mclaughlini was used as an outgroup. ♦ = Sequences obtained from Austrodiplostomum sp. collected in fishes from the Ivaí River.

Discussion

The morphological data associated with the partial sequences of the COI gene (the distance values and the relationships observed in the gene tree) indicate that all metacercariae in this study were A. compactum. Moreover, the data corroborate with the morphological data described by De Núñez (2017) that assumes synonymy between A. compactum and A. ostrowskiae. COI gene provided important information for the identification of digenean at the species level, and this led to the generation of seven new sequences. Among the Hypostomus species analyzed, three parasite haplotypes were found, the most frequent (H1) being recorded in all species and occurring in both eyes and brains.

The genetic distance values generated between the analyzed sequences were similar to those found in A. compactum. In contrast, the values found in relation to the other species analyzed were higher than 10%, and similar to the values found among individuals belonging to different genera. For example, the distance value obtained between A3 and A. mordax was 11%, whereas between Diplostomum lunaschiae Locke, Drago, Núñez, Souza, Takemoto, 2020 and Tylodelphys was 14%. According to Hebert et al. (2003), when comparing COI gene sequences in the case of animals, values greater than 2% of the distance are indicative of different species.

The metacercariae of A. compactum are considered generalists and have a wide geographical distribution (Ramos et al., 2013). In Brazil, metacercariae have been reported in hydrographic basins belonging to the states of Tocantins, Amazonas, Minas Gerais, São Paulo, Santa Catarina, and Paraná, and are found in the eyes of many different fishes that have been recorded in lists of parasite-host interactions (Ramos et al., 2013; Lehun et al., 2020). However, few studies have addressed the presence of A. compactum in Hypostomus. This digenean has been detected in the eyes of the following fishes: H. regani in the upper Paraná River floodplain (Yamada et al., 2008); H. affinis (Steindachner, 1877) in the Guandu River, state of Rio de Janeiro (Azevedo et al., 2010); and H. ancistroides (Ihering, 1911), H. iheringii (Regan, 1908), H. margaritifer (Regan, 1908), H. strigaticeps (Regan, 1908), and Hypostomus sp. in the Paranapanema River, state of São Paulo (Zica et al., 2011).

In addition to the wide distribution, A. compactum can also co-occur sharing the eye lens of fish, with A. mordax and Diplostomidae gen. sp. as reported in the studies by Pelegrini et al. (2021) carried out with H. regani collected in the Tietê-Batalha drainage basin in the state of São Paulo. Despite this, we found only A. compactum in the Hypostomus of the Ivaí River.

In the present study, the prevalence of A. compactum in H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 fishes sourced from the Ivaí River ranged from 40% to 88.8%, which is higher than the prevalence of the parasite in Hoplias malabaricus (Bloch, 1794), which was shown to be 11.11%, furthermore the prevalence of A. compactum found in Plagioscion squamosissimus (Heckel, 1840) sourced from the upper Paraná River floodplain, was found to be 95.06% (Machado et al., 2005). In the present study, the mean intensity showed that 13.26 parasites and 72 metacercariae were found in the eyes of a single individual of the H. hermanni species. Therefore, the infection rate by A. compactum metacercariae is frequently high as stated by Zica et al. (2009); however, our results show that the infection rate of this parasite in Loricariidae fishes may vary in different geographic locations.

In conclusion, this study contributes to the knowledge of the diversity of the parasitic fauna present in the Ivaí River, especially regarding the geographical distribution of Austrodiplostomum. In addition, new hosts have been found, such as Hypostomus sp. 1 and Hypostomus sp. 2. Further studies are required to monitor the occurrence of this digenean in fishes.

Acknowledgements

We would like to thank the encouragement and support of materials used in this research, provided by the Programa de pós-graduação em Biologia Comparada, Departamento de Biotecnologia, Genética e Biologia Celular, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura – NUPÉLIA, and the Programa de pós-graduação em Ecologia de Ambientes Aquáticos Continentais of Universidade Estadual de Maringá. This research relied on a doctoral scholarship provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES: 88882.449210/2019-01), through the Programa de pós-graduação em Biologia Comparada at the Universidade Estadual de Maringá.

• How to cite: Cracco AF, Scorsim B, de Oliveira AV, Takemoto RM. Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil. Braz J Vet Parasitol 2022; 31(2): e021421. https://doi.org/10.1590/S1984-29612022021

References

• Achatz TJ, Martens JR, Kostadinova A, Pulis EE, Orlofske SA, Bell JA, et al. Molecular phylogeny of Diplostomum, Tylodelphys, Austrodiplostomum and Paralaria (Digenea: Diplostomidae) necessitates systematic changes and reveals a history of evolutionary host switching events. Int J Parasitol 2022; 52(1): 47-63. http://dx.doi.org/10.1016/j.ijpara.2021.06.002 PMid:34371018.
  » http://dx.doi.org/10.1016/j.ijpara.2021.06.002
• Acosta AA, Smit NJ, Da Silva RJ. Diversity of helminth parasites of eight siluriform fishes from the Aguapeí River, upper Paraná basin, São Paulo state, Brazil. Int J Parasitol Parasites Wildl 2020; 11: 120-128. http://dx.doi.org/10.1016/j.ijppaw.2020.01.003 PMid:32025487.
  » http://dx.doi.org/10.1016/j.ijppaw.2020.01.003
• Affonso IP, Karling LC, Takemoto RM, Gomes LC, Nilsson PA. Light-induced eye-fluke behavior enhances the parasite life cycle. Front Ecol Environ 2017; 15(6): 340-341. http://dx.doi.org/10.1002/fee.1513
  » http://dx.doi.org/10.1002/fee.1513
• Azevedo RK, Abdallah VD, Luque JL. Acanthocephala, Annelida, Arthropoda, Myxozoa, Nematoda and Platyhelminthes parasites of fishes from the Guandu River, Rio de Janeiro, Brazil. Check List 2010; 6(4): 659-667. http://dx.doi.org/10.15560/6.4.659
  » http://dx.doi.org/10.15560/6.4.659
• Blasco-Costa I, Locke SA. Life History, Systematics and Evolution of the Diplostomoidea Poirier, 1886: Progress, promises and challenges emerging from molecular studies. Adv Parasitol 2017; 98: 167-225. http://dx.doi.org/10.1016/bs.apar.2017.05.001 PMid:28942769.
  » http://dx.doi.org/10.1016/bs.apar.2017.05.001
• Bush AO, Lafferty KD, Lotz JM, Shostak AW. Parasitology meets ecology on its own terms: Margolis et al. revisited. J Parasitol 1997; 83(4): 575-583. http://dx.doi.org/10.2307/3284227 PMid:9267395.
  » http://dx.doi.org/10.2307/3284227
• Campos DWJ, Manoel LO, Franceschini L, Veríssimo-Silveira R, Delariva RL, Ribeiro CS, et al. Occurrence of metacercariae of Austrodiplostomum compactum (Lutz, 1928) (Trematoda, Diplostomidae) in Pimelodus platicirris in the Ilha Solteira Reservoir, São Paulo, Brazil. An Acad Bras Ciênc 2020; 92(Suppl 2): e20180649. http://dx.doi.org/10.1590/0001-3765202020180649
  » http://dx.doi.org/10.1590/0001-3765202020180649
• Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 2012; 9(8): 772. http://dx.doi.org/10.1038/nmeth.2109 PMid:22847109.
  » http://dx.doi.org/10.1038/nmeth.2109
• de Núñez MO. Redescription of Austrodiplostomum compactum (Trematoda: Diplostomidae) from its type host and locality in Venezuela, and of Austrodiplostomum mordax from Argentina. J Parasitol 2017; 103(5): 497-505. http://dx.doi.org/10.1645/16-128 PMid:28604263.
  » http://dx.doi.org/10.1645/16-128
• Eiras JC, Takemoto RM, Pavanelli GC. Métodos de estudo e técnicas laboratoriais em parasitologia de peixes Maringá: EDUEM; 2006.
• Frota A, Deprá GC, Petenucci LM, Graça WJ. Inventory of the fish fauna from Ivaí River basin, Paraná State, Brazil. Biota Neotrop 2016; 16(3): e20150151. http://dx.doi.org/10.1590/1676-0611-BN-2015-0151
  » http://dx.doi.org/10.1590/1676-0611-BN-2015-0151
• García-Varela M, Sereno-Uribe AL, Pinacho-Pinacho CD, Domínguez-Domínguez O, Pérez-Ponce de León G. Molecular and morphological characterization of Austrodiplostomum ostrowskiae Dronen, 2009 (Digenea: Diplostomatidae), a parasite of cormorants in the Americas. J Helminthol 2016; 90(2): 174-185. http://dx.doi.org/10.1017/S0022149X1500005X PMid:25735970.
  » http://dx.doi.org/10.1017/S0022149X1500005X
• Gordy MA, Locke SA, Rawlings TA, Lapierre AR, Hanington PC. Molecular and morphological evidence for nine species in North American Australapatemon (Sudarikov, 1959): a phylogeny expansion with description of the zygocercous Australapatemon mclaughlini n. sp. Parasitol Res 2017; 116(8): 2181-2198. http://dx.doi.org/10.1007/s00436-017-5523-x PMid:28623502.
  » http://dx.doi.org/10.1007/s00436-017-5523-x
• Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98.
• Hebert PDN, Cywinska A, Ball SL, De Waard JR. Biological identifications through DNA barcodes. Proc Biol Sci 2003; 270(1512): 313-321. http://dx.doi.org/10.1098/rspb.2002.2218 PMid:12614582.
  » http://dx.doi.org/10.1098/rspb.2002.2218
• Kennedy M, Spencer HG. Classification of the cormorants of the world. Mol Phylogenet Evol 2014; 79: 249-257. http://dx.doi.org/10.1016/j.ympev.2014.06.020 PMid:24994028.
  » http://dx.doi.org/10.1016/j.ympev.2014.06.020
• Kohn A, Cohen SC, Justo MCN, Fernandes BMM. Digenea. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013. p. 301-316.
• Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 2019; 35(21): 4453-4455. http://dx.doi.org/10.1093/bioinformatics/btz305 PMid:31070718.
  » http://dx.doi.org/10.1093/bioinformatics/btz305
• Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33(7): 1870-1874. http://dx.doi.org/10.1093/molbev/msw054 PMid:27004904.
  » http://dx.doi.org/10.1093/molbev/msw054
• Lehun AL, Hasuike WT, Silva JOS, Ciccheto JRM, Michelan G, Rodrigues AFC, et al. Checklist of parasites in fish from the upper Paraná River floodplain: an update. Rev Bras Parasitol Vet 2020; 29(3): e008720. http://dx.doi.org/10.1590/s1984-29612020066 PMid:32935771.
  » http://dx.doi.org/10.1590/s1984-29612020066
• Leigh JW, Bryant D. POPART: full-feature software for haplotype network construction. Methods Ecol Evol 2015; 6(9): 1110-1116. http://dx.doi.org/10.1111/2041-210X.12410
  » http://dx.doi.org/10.1111/2041-210X.12410
• Locke SA, Al-Nasiri FS, Caffara M, Drago F, Kalbe ARM, Lapierre JD, et al. Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes. Int J Parasitol 2015; 45(13): 841-855. http://dx.doi.org/10.1016/j.ijpara.2015.07.001 PMid:26276524.
  » http://dx.doi.org/10.1016/j.ijpara.2015.07.001
• Locke SA, Drago FB, Núñez V, Souza GTR, Takemoto RM. Phylogenetic position of Diplostomum spp. from New World herons based on complete mitogenomes, rDNA operons, and DNA barcodes, including a new species with partially elucidated life cycle. Parasitol Res 2020; 119(7): 2129-2137. http://dx.doi.org/10.1007/s00436-020-06713-4 PMid:32472382.
  » http://dx.doi.org/10.1007/s00436-020-06713-4
• López-Hernández D, Locke SA, De Assis JCA, Drago FB, de Melo AL, Rabelo ÉML, et al. Molecular, morphological and experimental-infection studies of cercariae of five species in the superfamily Diplostomoidea (Trematoda: Digenea) infecting Biomphalaria straminea (Mollusca: Planorbidae) in Brazil. Acta Trop 2019; 199: 105082. http://dx.doi.org/10.1016/j.actatropica.2019.105082 PMid:31351070.
  » http://dx.doi.org/10.1016/j.actatropica.2019.105082
• Machado PM, Takemoto RM, Pavanelli GC. Diplostomum (Austrodiplostomum) compactum (Lutz, 1928) (Platyhelminthes, Digenea) metacercariae in fish from the floodplain of the Upper Paraná River, Brazil. Parasitol Res 2005; 97(6): 436-444. http://dx.doi.org/10.1007/s00436-005-1483-7 PMid:16151731.
  » http://dx.doi.org/10.1007/s00436-005-1483-7
• Monteiro CM, Martins AN, Albuquerque MC, Clapp MDS, Duarte R, Sabas CSS, et al. Austrodiplostomum compactum Szidat & Nani (Digenea: Diplostomidae) in final and second intermediate hosts from upper São Francisco River in the State of Minas Gerais, Brazil. Braz J Vet Med 2016; 38(Suppl 3): 146-150.
• Moszczynska A, Locke SA, McLaughlin JD, Marcogliese DJ, Crease TJ. Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths. Mol Ecol Resour 2009; 9(Suppl s1): 75-82. http://dx.doi.org/10.1111/j.1755-0998.2009.02634.x PMid:21564967.
  » http://dx.doi.org/10.1111/j.1755-0998.2009.02634.x
• O’Hear M, Pote L, Yost M, Doffitt C, King T, Panuska C. Morphologic and molecular identifications of digenetic trematodes in double-crested cormorants (Phalacrocorax auritus) from the Mississippi Delta, USA. J Wildl Dis 2014; 50(1): 42-49. http://dx.doi.org/10.7589/2012-10-249 PMid:24171572.
  » http://dx.doi.org/10.7589/2012-10-249
• Onaca FMT, Graça RJ, Fabrin TMC, Takemoto RM, Oliveira AV. Molecular characterization and identification of digenean larval stages in Aylacostoma chloroticum (Prosobranchia: Thiaridae) from a neotropical floodplain. J Helminthol 2019; 94: e73. http://dx.doi.org/10.1017/S0022149X19000725 PMid:31412968.
  » http://dx.doi.org/10.1017/S0022149X19000725
• Pelegrini LS, Leite LAR, Gião T, Bueno RMR, Serrano TD, Silva RJ, et al. Diversity, similarity, and host–parasite relationships in parasitic infracommunities of Hypostomus spp from the Tietê-Batalha River basin, southeastern Brazil.Stud Neotrop Fauna Environ 2021; 55: 1-13. https://doi.org/10.1080/01650521.2020.1870033
  » https://doi.org/10.1080/01650521.2020.1870033
• Pelegrini LS, Gião T, Vieira DHMD, Müller MI, Silva RJ, León GP, et al. Molecular and morphological characterization of the metacercariae of two species of diplostomid trematodes (Platyhelminthes, Digenea) in freshwater fishes of the Batalha River, Brazil. Parasitol Res 2019; 118(7): 2169-2182. http://dx.doi.org/10.1007/s00436-019-06362-2 PMid:31183598.
  » http://dx.doi.org/10.1007/s00436-019-06362-2
• Pinto HA, Melo AL. Biomphalaria straminea and Biomphalaria glabrata (Mollusca: Planorbidae) as new intermediate hosts of the fish eyefluke Austrodiplostomum compactum (Trematoda: Diplostomidae) in Brazil. J Parasitol 2013; 99(4): 729-733. http://dx.doi.org/10.1645/12-13.1 PMid:23360402.
  » http://dx.doi.org/10.1645/12-13.1
• Ramos IP, Franceschini L, Zago AC, Zica ÉOP, Wunderlich AC, Carvalho ED, et al. New host records and a checklist of fishes infected with Austrodiplostomum compactum (Digenea: Diplostomidae) in Brazil. Rev Bras Parasitol Vet 2013; 22(4): 511-518. http://dx.doi.org/10.1590/S1984-29612013000400010 PMid:24473875.
  » http://dx.doi.org/10.1590/S1984-29612013000400010
• Ramos IP, Pagliarini CD, Franceschini L, Silva RJ. Metacercariae of Austrodiplostomum compactum (Trematoda, Diplostomidae) in non-native fish species in Brazil: a possible explanation for the high rate of parasitic infection. An Acad Bras Cienc 2020; 92(Suppl 2): e20180984. http://dx.doi.org/10.1590/0001-3765202020180984 PMid:32935738.
  » http://dx.doi.org/10.1590/0001-3765202020180984
• Rosenthal A, Coutelle O, Craxton M. Large-scale production of DNA sequencing templates by microtitre format PCR. Nucleic Acids Res 1993; 21(1): 173-174. http://dx.doi.org/10.1093/nar/21.1.173 PMid:8441614.
  » http://dx.doi.org/10.1093/nar/21.1.173
• Rosser TG, Alberson NR, Khoo LH, Woodyard ET, Pote LM, Griffin MJ. Characterization of the life cycle of a fish eye fluke, Austrodiplostomum ostrowskiae (Digenea: Diplostomidae), with Notes on Two Other Diplostomids Infecting Biomphalaria havanensis (Mollusca: Planorbidae) from Catfish Aquaculture Ponds in Mississipi, USA. J Parasitol 2016; 102(2): 260-274. http://dx.doi.org/10.1645/15-850 PMid:26741049.
  » http://dx.doi.org/10.1645/15-850
• Santos RS, Marchiori N, Santarem VA, Takahashi HK, Mourino JLP, Martins ML. Austrodiplostomum compactum (Lutz, 1928) (Digenea, Diplostomidae) in the eyes of fishes from Paraná River, Brazil. Acta Sci Biol Sci 2012; 34(2): 225-231. http://dx.doi.org/10.4025/actascibiolsci.v34i2.9337
  » http://dx.doi.org/10.4025/actascibiolsci.v34i2.9337
• Sereno-Uribe AL, Gómez LA, Núñez MO, León GP-P, García-Varela M. Assessing the taxonomic validity of Austrodiplostomum spp. (Digenea: Diplostomidae) through nuclear and mitochondrial data. J Parasitol 2019a; 105(1): 102-112. http://dx.doi.org/10.1645/18-51 PMid:30807727.
  » http://dx.doi.org/10.1645/18-51
• Sereno-Uribe AL, Andrade-Gómez L, De León GPP, García-Varela M. Exploring the genetic diversity of Tylodelphys (Diesing, 1850) metacercariae in the cranial and body cavities of Mexican freshwater fishes using nuclear and mitochondrial DNA sequences, with the description of a new species. Parasitol Res 2019b; 118(1): 203-217. http://dx.doi.org/10.1007/s00436-018-6168-0 PMid:30552574.
  » http://dx.doi.org/10.1007/s00436-018-6168-0
• Takemoto RM, Pavanelli GC, Lizama MAP, Lacerda ACF, Yamada FH, Moreira LHA, et al. Diversity of parasites of fish from the Upper Paraná River floodplain, Brazil. Braz J Biol 2009;69(2 Suppl 2): 691-705. http://dx.doi.org/10.1590/S1519-69842009000300023 PMid:19738975.
  » http://dx.doi.org/10.1590/S1519-69842009000300023
• Travassos L, Artigas P, Pereira C. Fauna helmintológica dos peixes de água doce do Brasil. Arch Inst Biol 1928; 1: 5-68.
• Yamada FH, Moreira LHA, Ceschini TL, Takemoto RM, Pavanelli GC. Novas ocorrências de metacercária de Austrodiplostomum compactum (Lutz, 1928) (Platyhelminthes: Digenea) parasito de olhos de peixes da bacia do rio Paraná. Rev Bras Parasitol Vet 2008; 17(3): 163-166. http://dx.doi.org/10.1590/S1984-29612008000300010 PMid:19245765.
  » http://dx.doi.org/10.1590/S1984-29612008000300010
• Zawadzki CH, Tencatt LF, Britski HA. Taxonomic revision of Hypostomus albopunctatus (Siluriformes: Loricariidae) reveals a new piece of the Hypostomus jigsaw in the upper Rio Paraná basin. J Fish Biol 2020; 96(1): 230-242. http://dx.doi.org/10.1111/jfb.14209 PMid:31749168.
  » http://dx.doi.org/10.1111/jfb.14209
• Zica EOP, Brandão H, Zawadzki CH, Nobile AB, Carvalho ED, Da Silva RJ. The occurrence of Austrodiplostomum compactum (Lutz, 1928) (Digenea: Diplostomidae) metacercariae in the eyes of loricariid fish (Siluriformes: Osteichthyes: Loricariidae) from Brazil. J Helminthol 2011; 85(1): 73-79. http://dx.doi.org/10.1017/S0022149X10000271 PMid:20459879.
  » http://dx.doi.org/10.1017/S0022149X10000271
• Zica EOP, Santos KR, Ramos IP, Zanatta AS, Carvalho ED, Silva RJ. First case of an infection of the metacercariae of Austrodiplostomum compactum (Lutz, 1928) (Digenea, Diplostomidae) in Hypostomus regani (Ihering, 1905) (Siluriformes: Loricariidae). Pan-Am J Aquat Sci 2009; 4(1): 35-38.

Publication Dates

• Publication in this collection
  22 Apr 2022
• Date of issue
  2022

History

• Received
  02 Dec 2021
• Accepted
  10 Mar 2022