Molecular identification of <i>Austrodiplostomum</i> sp., an eye parasite among farmed tambaqui in Amazonia

Farias, Eduardo Freitas de; Silva, Hallana Cristina Menezes da; Carvalho, Ana Paula Costa de; Martins, Rodrigo de Melo; Belem-Costa, Andrea; Duncan, Wallice Paxiúba; Gomes, Ana Lúcia Silva; Matoso, Daniele Aparecida

doi:10.1590/1678-4685-GMB-2021-0345

Abstract

Austrodiplostomum Szidat & Nani, 1951 is a genus of parasitic digenetic trematodes widely distributed in the Neotropical region. Infects a wide variety of species, families and requests for freshwater fish. We identify samples of Austrodiplostomum sp, based on metacercariae isolates from eyes of tambaqui (Colossoma macropomum), a fish of high commercial importance in Brazil, and widely consumed by the population of the northern region. The sequences obtained clustered with A. compactum. This is the first report of the occurrence of diplostomids in farmed tambaqui in Amazonia.

Keywords:
Metacercariae; parasitism; Colossoma macropomum

Introduction

Parasites use other organisms as support for food, shelter, and transportation. Their developmental stages may involve more than one host species in a number of higher taxa. AustrodiplostomumSzidat and Nani 1951Szidat, L and Nani A (1951) Diplostomiasis cerebrales del pejerrey. Rev Inst Nac Invest Ciencias Nat 1:324-384. is a genus of parasitic trematodes in the family Diplostomidae (order Diplostomida). The genus is widely distributed in the Neotropical region and has molluscs and fish as intermediate hosts (Monteiro et al., 2016Monteiro CM, Martins NA, Albuquerque MC, Clapp MDS, Duarte R, Sabas CSS and Brasil-Sato MC (2016) 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 38:146-150.). On entering the host bloodstream, these parasites eventually reach the eyes and transform into metacercariae. During this life stage, they can compromise host development by additionally infecting a number of organs, including gills, muscle, swimming bladder and brain. Freshwater diplostomides colonize fish of different species, families and orders (Martins et al., 1999Martins ML, Fujimoto RY, Nascimento AA and Moraes FR (1999) Occurrence of Diplostomum sp Nordmann, 1832 (Digenea: Diplostomatidae) in Plagioscion squamosissimus Heckel, 1840, from Volta Grande Reservoir, MG, Brazil. Acta Sci Biol Sci 21:263-266.; Ramos et al., 2013Ramos IP, Francheschini L, Zago AC, Zica EOP, Wunderlich AC, Carvalho ED and Silva RJ (2013) New host records and a checklist of fishes infected with Austrodiplostomum compactum (Digenea: Diplostomidae) in Brazil. Rev Bras Parasitol Vet 22:511-518.). Taxonomic studies of parasitic organisms in fish help define which type of parasite is present in a given host, allowing for searches to be made for mitigating measures and the development of good management practices that will prevent infestation proliferation (Florindo et al., 2017Florindo MC, Jerônimo GT, Steckert LD, Acchile M, Figueredo AB, Gonçalves ELT, Cardoso L, Marchiori NC, Assis GC and Martins ML (2017) Metazoan parasites of freshwater ornamental fishes. Lat Am J Aquat Res 45:992-998.). Approaches to the study and definition of groups of fish parasites based on purely morphological analysis may encounter difficulties because the identification of larval stages, in many cases, can only be clarified with molecular analyses (Moszczynska et al., 2009Moszczynska A, Locke SA, McLaughlin JD, Marcogliese DJ and Crease TJ (2009) 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 9:75-82.). The use of molecular markers in studies to study genetic variability and provide species-specific diagnosis for different forms of parasitic organisms provides an alternative approach to those more traditional studies using only morphological and etiological characters (Sperling et al., 1994Sperling F, Anderson G and Hickey D (1994) A DNA-based approach to the identification of insect species for post mortem interval estimation. J Forensic Sci 39:418-427.; Otranto and Stevens, 2002Otranto D and Stevens JR (2002) Molecular approaches to the study of myiasis-causing larvae. Int J Parasitol 32:1345-1360.).

The information generated in this study will provide extra input and can be used as primary data to combat and control of these pathogenic organisms, by improvement of zootechnical methods for this commercially important fish species, widely cultivated in the Amazon region.

Material and Methods

Sample collection and morphological analysis

The fish were collected at the Balbina Fish Farm Station, the largest fingerling producing unit in the State of Amazonas. It is located in the coordinates (2˚4'5'' S; 60˚31'58'' W) in a hydroelectric area in the Presidente Figueiredo city. Metacercariae of the genus Austrodiplostomum, parasitizing Colossoma macropomum (tambaqui), were carefully removed from the eyes of infected fish, after anaesthetized by benzocaine. Some of the specimens (6), were preserved in alcohol while others (7) were preserved in formalin (36%), for, respectively, analysis with scanning electron (SEM) and optical microscopy. A series of 12 metric dimensions were taken from 13 metacercariae specimens. Morphological characterization followed the methods of Santos et al. (2002Santos RS, Pimenta FDA, Martins ML, Takahashi HK and Marengoni NG (2002) Metacercariae of Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomatidae) in fishes of Paraná River, Brazil. Prevalence, seasoning and intensity of infection. Acta Sci Biol Sci 24:475-480. ), Kohn et al. (1995Kohn A, Fernandes BMM and Baptista-Farias MFD (1995) Metacercariae of Diplostomum (Austrodiplostomum) compactum (Trematoda, Diplostomidae) in the eyes of Plagioscion squamosissimus (Teleostei, Sciaenidae) from the reservoir of the Hydroelectric Power Station of Itaipu, Brazil. Mem Inst Oswaldo Cruz 90:341-344.), Novaes et al. (2006Novaes JLC, Ramos IP, Carvalho ED and Silva RJ (2006) Metacercariae of Diplostomum compactum Lutz, 1928 (Trematoda: Diplostomidae) Quoy and Gaimard 1824 (Teleostei, Cichlidae) from Barra Bonita reservoir - São Paulo, Brazil. Arq Bras Med Vet Zootec 58:1229-1231.) and Albuquerque et al. (2017Albuquerque NB, Morey GAM, Morais AM and Malta JCO (2017) Metacercariae of Austrodiplostomum compactum (Lutz, 1928) (Trematoda, Diplostomidae) infecting the eyes of Plagioscion squamosissimus (Heckel, 1840) (Perciformes, Scienidae) from Lake Catalão, Amazonas, Brazil. Acta Amaz 47:141-146. ). Samples were deposited in the Helminthological Collection of the Amazon National Research Institute (INPA). All procedures described were approved by the UFAM Ethics Committee for Animal Experimentation, under permit number 002/14-CEUA/UFAM.

DNA sequence analysis

Total metacercarian DNA was extracted using a PureLink™ Genomic DNA Mini Kit, Invitrogen (Thermo Fisher Scientific), and following the manufacturer’s instructions. This was followed by a PCR treatment using the GoTaq® Hot Start Polymerase kit, (Promega). Primers were as described by Moszczynska et al. (2009Moszczynska A, Locke SA, McLaughlin JD, Marcogliese DJ and Crease TJ (2009) 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 9:75-82.) for the amplification of the Cytochrome Oxidase Subunit I (COI) region of the mitochondrial DNA of metacercaria. PCR reactions had a final volume of 20 µL, with: 10.8 µL of ultra-pure water, 0.2 µL of dNTP, 1.2 µL of MgCl2, 0.8 µL of forward primer and 10 mM of reverse primer, 0.2 µL of GoTaq® Hot Start Polymerase and 2 µL of 50 ng DNA. In the thermal cycler, samples were submitted to the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 denaturation cycles at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. To cross-check the PCR technique, a 1.5% agarose gel electrophoresis was performed, immersed in TBE1X, with 80 V electric current, for 40 min. The gel was then observed under UV light.

Following the positive PCR reaction result, samples were purified using the ExoSap protocol. Sequencing reactions used the BigDye™ kit Terminator v3.1 Cycle Sequencing, Applied Biosystems (Thermo Fisher Scientific), following the manufacturer’s instructions. Samples were purified using the NG purification protocol. Sequencing was performed in an automatic sequencer (Sanger ABI 3130, Applied Biosystems). For molecular analyses of Maximum Likelihood (ML) and Bayesian Inference (BI) were analysed 34 sequences (1 of D. huronense HM064670, 1 of D. spathaceum KR271467, 8 of A. compactum MH378944-MH378951, 4 of Austrodiplostomum sp2 MH378931-MH378934, 3 of A. sp1 MH378928-MH378930, 3 of A. mordax MH378896-MH378898, 2 A. ostrowskiae KT728794-KT728795, 12 new sequences of A. compactum MT271131- MT271142). Partial sequences containing 416 bases corresponding to the COI gene, were edited and aligned using the BioEdit editor Clustal W program (Hall, 1999Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95-98.). We used the PAUP* program (Swofforf, 1998Swofford DL (1998) PAUP*. Phylogenetics Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachussets. ), for obtain the best evolutionary model by using Modeltest 3.7 (Posada and Crandall, 1998Posada D and Crandall KA (1998) MODELTEST: Testing the model of DNA substitution. Bioinformatics 14:817-818.) under the Akaike Information Criterion (AIC). The best model was GTR+G+I, which was used for Maximum Likelihood (ML) analyses. Bootstrap Phylogeny test with 1050 replications and heuristic search to assess branch support was performed in ML analyses using the MEGA 10.1.7 program (Kumar et al., 2018Kumar S, Stecher G, Li M, Knyaz C and Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Bio Evol 35:1547-1549.). Species relationships within Austrodiplostomum were assessed using Bayesian inference (BI). BI analysis was carried out with MrBayes v. 3.2.7 (Ronquist et al., 2012Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA and Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539-542.) on the using Markov chain Monte Carlo searches on two simultaneous runs of four chains for 10⁶ generations, sampling trees every 10³ generations. The burn-in was set for the 25% of the trees sampled. A consensus topology and nodal support estimated as posterior probability values (Huelsenbeck et al., 2001Huelsenbeck JP, Ronquist F, Nielsen R and Bollback JP (2001) Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294:2310-2314.) were calculated from the remaining trees. Phylogenetic trees were visualised and finalised in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Results and Discussion

Metacercariae collected from C. macropomum vitreous humor were measured and characterized morphologically as follows: body foliaceous, bisegmented, anterior region with ventral portion with a slight depression; integument papillaceous; subterminal oral suction cup small, flanked by two well-developed pseudoventae; tribocytic organ present, with a large number of glandular cells spread throughout the anterior region; posterior region consisting of a reduced tapering process (Figure 1). Morphometric data recorded from the metacercariae samples appears in Tables 1 and 2.

Figure 1 -
Austrodiplostomum compactum metacercariae scanning electron micrograph. (a,c) Whole specimen, (b) anterior region evidencing oral suction cup and pseudoventer, (d) tribocytic organ.

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Table 1 -
Morphometric data of Austrodiplostomum compactum metacercariae, (fish from Brazil), with respective localities and hosts and sample size.

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Table 2 -
Morphometric data of Austrodiplostomum compactum metacercariae (fish from the Amazon basin), with measurements, localities, hosts and sample size.

Molecular analyses indicated that sequences obtained from the eyes of tambaqui belong to the species A. compactum (Figure 2). The Austrodiplostomum genus was considered by García-Varela et al. (2016García-Varela M, Sereno-Uribe AL, Pinacho-Pinacho CD, Domínguez-Domínguez O and León GPP (2016) Molecular and morphological characterization of Austrodiplostomum ostrowskiae Dronen, 2009 (Digenea: Diplostomatidae), a parasite of cormorants in the Americas. J Helminthol 90:174-185.) to contain three species: A. mordax, A. compactum and A. ostrowskiae. However, studies by Sereno-Uribe et al. (2019Sereno-Uribe AL, Gómez LA, Ostrowski-Núñez M, León GPP and García-Varela M (2019) Assessing the taxonomic validity of Austrodiplostomum spp. (Digenea: Diplostomidae) through nuclear and mitochondrial data. J Parasitol 105:102-112.) of the three forms of Austrodiplostomum concluded that A. ostrowskiae was synonymous with A. compactum.

Figure 2 -
Bayesian inference (BI) tree inferred with partial sequences of COI mtDNA. The tree was reconstructed using 12 news A. compactum sequences, marked by asterisk (present study). In the analysis are observed 4 main subclades for Austrodiplostomum. Bootstrap support and posterior probability values are given next to each branch. The bar indicates the expected number of substitutions per site

The records collected by the current study represent the first for the State of Amazonas of an outbreak of diplostomids in farmed fish stock. It is also the first report of the genus Austrodiplostomum in the eyes (aqueous humor) of tambaqui, C. macropomum, the most important species for commercial fish farming in northern Brazil. An infective triad formed by mollusks, piscivorous birds (Cormorant) and tambaqui fry was recorded at the Fry Production Technology and Training Center, Vila Balbina/Presidente Figueiredo in the Metropolitan Region of Manaus, Central Amazon, Brazil. Parasitic helminths are important agents in the etiology of different fish diseases and can harm their hosts in a variety of ways (Eiras, 1994Eiras JC (1994) Elements of ichthyo parasitology. Fundação Eng. António de Almeida, Porto, 339 p.). The literature on the occurrence of A. compactum indicates a marked preference for the eye region (aqueous humor/retina/crystalline) (Santos et al., 2002Santos RS, Pimenta FDA, Martins ML, Takahashi HK and Marengoni NG (2002) Metacercariae of Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomatidae) in fishes of Paraná River, Brazil. Prevalence, seasoning and intensity of infection. Acta Sci Biol Sci 24:475-480. ; Martins et al., 2002Martins ML, Mello A, Paiva AMFC, Fujimoto RY, Schalch SHC and Colombano NC (2002) Prevalence, seasonality and infection intensity by Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomidae) in fish of the Volta Grande Reservoir, state of Minas Gerais, Brazil. Acta Sci Biol Sci 24:469-474. ; Abdallah et al., 2005Abdallah VD, Azevedo RK and Luque JL (2005) Community ecology of metazoan parasites of Cyphocharax gilbert (Quoy e Gaimard, 1824) (Characiformes:Curimatidae) from Guandu river, State of Rio de Janeiro, Brazil. Rev Bras Parasitol Vet 14:154-159. ; Azevedo et al., 2006Azevedo RK, Abdallah VD and Luque JL (2006) Ecology of the metazoan parasite community of the Acar Geophagus brasiliensis (Quoy and Gaimard, 1824) (Perciformes: Cichlidae) of the Guandu River, State of Rio de Janeiro, Brazil. Acta Sci Biol Sci 28:403-411.; Bachmann et al., 2007Bachmann F, Greinert JA, Bertelli PW, Silva Filho HH, Lara NOT, Ghiraldelli L and Martins ML (2007) Parasitic fauna of Pimelodus maculatus (Osteichthyes: Pimelodidae) from the Itajaí-Açu river in Blumenau, state of Santa Catarina, Brazil. Acta Sci Biol Sci 29:109-114. ; Yamada et al., 2008Yamada FH, Moreira LHA, Ceschini TL, Takemoto RM and Pavanelli GC (2008) New occurrences of metacercariae ofAustrodiplostomum compactum(Lutz, 1928) (Platyhelminthes: Digenea) eye flukes of fish from the Paraná Basin. Rev Bras Parasitol Vet 17:163-166.; Santos et al., 2012Santos RS, Pimenta FDA, Martins ML, Takahashi HK and Marengoni NG (2002) Metacercariae of Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomatidae) in fishes of Paraná River, Brazil. Prevalence, seasoning and intensity of infection. Acta Sci Biol Sci 24:475-480. ). However, metacercariae of this species may also occur in other organs, such as the brain and gills (Brasil-Sato and Santos, 2005Brasil-Sato MC and Santos MD (2005) Metazoan parasites of Conorhynchos conirostris (Valenciennes, 1840) an endemic siluriform fish of the São Francisco basin, Brazil. Rev Bras Parasitol Vet 14:160-166.; Machado et al., 2005Machado PM, Takemoto RM and Pavanelli GC (2005) Diplostomum (Austrodiplostomum) compactum (Lutz, 1928) (Platyhelminthes, Digenea) metacercariae in fish from the floodplain of the Upper Paraná River, Brazil. Parasitol Res 97:436-444.). The presence of metacercaria in the eyes of farmed fish can cause mild optical damage, or have more severe impacts such as cataracts, blood vessel obstruction, retinal detachment and blindness (Monteiro et al., 2016Monteiro CM, Martins NA, Albuquerque MC, Clapp MDS, Duarte R, Sabas CSS and Brasil-Sato MC (2016) 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 38:146-150.; Lima et al., 2019Lima MJS, Veiga RP, Sousa LF, Santana MB, Oliveira MSB, Tavares-Dias M and Corrêa LL (2019) Metacercariae of Austrodiplostomum spp. (Digenea: Diplostomidae) infecting the eyes and brains of fish in Brazilian Amazon. Arq Inst Biol 86:e0932018.).

In the current study, corneal opacity was observed in 30% of the sampled individuals. However, it is unlikely that damage percentage could have been much higher because the parasitic intensity was low, ranging from one to eight larvae per analysed eye. There was also a certain lethargy on the part of the fish at the time of capture in the tanks. This aspect of parasitism makes the fish more easily caught by cormorants.

The registration and identification of a parasite’s biodiversity in a given locality are the first steps in carrying out control measures. It is very important to collect these data to guide technical professionals in the diagnosis of diseases. In this case, the lack of knowledge about the occurrence of pathogens and the absence of sanitary measures in the transport of fingerlings between properties can trigger dissemination processes that later are difficult to control. Therefore, the results of this study indicate that diplostomid samples collected in the vitreous humor of C. macropomum belong to the species A. compactum. These data put an alert because they include the State of Amazonas as an occurrence region of this species in addition to recording for the first time the existence of diplostomids in Amazon pisciculture. This discovery may be important because many aquaculture properties located in the Presidente Figueiredo region present conditions for the development of this parasite life cycle. In addition, many fish farms in the surroundings are supplied with fingerlings from Balbina Station.

Acknowledgements

The current study was supported in part by INCT ADAPTA II funded by CNPq - Brazilian National Research Council (465540/2014-7). FAPEAM - Projeto Controle de Acantocéfalo em Piscicultura - CAP, Edital Amazonas Estratégico - Edital 004/2018 Universidade Federal do Amazonas (UFAM/LABICA). FAPEAM/SEPLANCTI/Governo do Estado do Amazonas - POSGRAD Res. No. 002/2016. Adrian Barnett helped with the English.

References

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Azevedo RK, Abdallah VD and Luque JL (2006) Ecology of the metazoan parasite community of the Acar Geophagus brasiliensis (Quoy and Gaimard, 1824) (Perciformes: Cichlidae) of the Guandu River, State of Rio de Janeiro, Brazil. Acta Sci Biol Sci 28:403-411.
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Edited by

Associate Editor: Louis Bernard Klaczko

Publication Dates

Publication in this collection
30 Jan 2023
Date of issue
2023

History

Received
01 Nov 2021
Accepted
21 Sept 2022

This is an open-access article distributed under the terms of the Creative Commons Attribution License

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Author/Locality/Host/sample size	Body		Oral sucker		Pharynx		Tribocytic organ
	length	width	length	width	length	width	length	width
*Khon et al., 1995Kohn A, Fernandes BMM and Baptista-Farias MFD (1995) Metacercariae of Diplostomum (Austrodiplostomum) compactum (Trematoda, Diplostomidae) in the eyes of Plagioscion squamosissimus (Teleostei, Sciaenidae) from the reservoir of the Hydroelectric Power Station of Itaipu, Brazil. Mem Inst Oswaldo Cruz 90:341-344. Hidreletrica de Itaipu - PR Plagioscion squamosissimus* N = (14)	2170 (1470-2740)	970 (600-1180)	77 (41-97)	79 (56-116)	83 (64-94)	60 (45-79)	507 (326-650)	370 (251-500)
Santos et al., 2002Santos RS, Pimenta FDA, Martins ML, Takahashi HK and Marengoni NG (2002) Metacercariae of Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomatidae) in fishes of Paraná River, Brazil. Prevalence, seasoning and intensity of infection. Acta Sci Biol Sci 24:475-480. Paraná River - SP Plagioscion squamosissimus N = (20)	1434 (880-1840)	611 (400-792)	65 (44-90)	52 (40-64)	62 (50-64)	49 (40-60)	285 (200-600)	182 (160-232)
Santos et al., 2002Santos RS, Pimenta FDA, Martins ML, Takahashi HK and Marengoni NG (2002) Metacercariae of Diplostomum (Austrodiplostomum) compactum Lutz, 1928 (Digenea, Diplostomatidae) in fishes of Paraná River, Brazil. Prevalence, seasoning and intensity of infection. Acta Sci Biol Sci 24:475-480. Paraná River - SP Cichla ocellaris N = (20)	1462 (069-2480)	711 (560-960)	68 (40-98)	56 (30-98)	68 (44-98)	52 (38-78)	308 (200-496)	180 (120-320)
Paes et al., 2003Paes JVK, Santos KR, Carvalho ED and Silva RJ (2003) Ocorrência de metacercária de Diplostomum compactum (Trematoda, Diplostomidae) parasitando Plagioscion squamosissimus (Teleostei, Sciaenidae) proveniente do reservatório de Nova Avanhandava, Buritama, São Paulo. Arq Inst Biol 70:383-387. Tiête River - SP Plagioscion squamosissimus N = (21)	1911 (1301-2386)	678 (482-854)	71 (51-87)	73 (51-92)	69 (49-84)	58 (45-74)	401 (205-554)	246 (127-347)
Novaes et al., 2006Novaes JLC, Ramos IP, Carvalho ED and Silva RJ (2006) Metacercariae of Diplostomum compactum Lutz, 1928 (Trematoda: Diplostomidae) Quoy and Gaimard 1824 (Teleostei, Cichlidae) from Barra Bonita reservoir - São Paulo, Brazil. Arq Bras Med Vet Zootec 58:1229-1231. Reservatório Barra Bonita - SP Geophagus brasiliensis N = (05)	1880 (1584-1947)	642 (537-706)	59 (45-83)	68 (54-77)	61 (53-73)	56 (50-64)	428 (422-434)	258 (220-319)
Zica et al., 2009Zica EOP, Santos KR, Ramos IP, Zanatta AS, Carvalho ED and Silva RJ (2009) Frist case of n infection of the metacercariae of Austrodiplotomum compactum (Lutz, 1928) (Digenea, Diplostomidae) in Hypostomus regain (Ihering, 1905) (Siluriformes: Loricariidae). Pan-Am J Aquat Sci 4:35-38. Reservatório de Chavantes - SP Hypostomus regani N = (10)	1988 (1570-2281)	756 (543-864)	91 (69-102)	84 (75-99)	73 (57-85)	64 (57-80)	373 (287-414)	243 (178-310)
*Lepera et al., 2017Lapera IM, Silva AC, Canônico BM, Perezin GF, Tebaldi JH, Pala G, MAnrique WG and Hoppe EGL (2017) Metazoan parasites of Plagioscion squamosissimus, na invasive species in the Tietê River, São Paulo, Brazil. Rev Bras Parasitol Vet 26:143-151. Tietê River - SP Plagioscion squamosissimus* N = 50	1789 (1450-2166)	683 (566-902)	72 (57-86)	76 (49-97)	80 (61-99)	58 (41-75)	405 (302-492)	256 (193-319)

Author/Locality/Host/sample size	Body		Oral sucker		Pharynx		Tribocytic organ
	length	width	length	width	length	width	length	width
Dumbo, 2014Dumbo JC (2014) Espécies de metazoários parasitos de Acestrorhynchus falcirostris (Cuvier, 1819) (Characiformes: Acestrorhynchidae) de lagos de várzea da AmazôniaD. Sc. Thesis, Instituto Nacional de Pesquisas da Amazônia, Manaus, 201 p. Varzea lakes/Am Acestrorhynchus falcirostris N = 34	1665 (1330-2466)	-	59 (28-77)	57 (35-77)	49 (21-84)	39 (20-90)	309 (112-420)	248 (150-396)
Pereira, 2016Pereira NRB (2016) As espécies parasitas com potencial zoonótico em peixes amazônicos. D. Sc. Thesis, Universidade Federal do Amazonas, Manaus, 152 p. Amazon basin Cichla monoculus N = 07	1438 (1178-1956)	_	63 (53-66)	67 (52-74)	61 (56-64)	50 (45-54)	335 (279-460)	194 (147-256)
Pereira, 2016Pereira NRB (2016) As espécies parasitas com potencial zoonótico em peixes amazônicos. D. Sc. Thesis, Universidade Federal do Amazonas, Manaus, 152 p. Amazon basin Hoplias malabaricus N = 01	1253	_	53	61	42	49	218	181
Albuquerque et al., 2017Albuquerque NB, Morey GAM, Morais AM and Malta JCO (2017) Metacercariae of Austrodiplostomum compactum (Lutz, 1928) (Trematoda, Diplostomidae) infecting the eyes of Plagioscion squamosissimus (Heckel, 1840) (Perciformes, Scienidae) from Lake Catalão, Amazonas, Brazil. Acta Amaz 47:141-146. Lake Catalão Plagioscion squamosissimus N = 15	1783 (148-2024)	662 (589-782)	60 (44-72)	70 (48-82)	63 (47-79)	57 (41-69)	345 (230-425)	181 (122-238)
Morais and Malta, 2015Morais AM and Malta JCO (2015) Biodiversity of monogenoideans from red piranha Pygocentrus nattereri (Kner, 1958) (Characiformes: Serrasalmidae) in Central Amazonia: Occurrence and taxonomy. Neotrop Helminthol 9:265-276. Amazonian lakes Pygocentrus nattereri N = (02)	2050 (1370-2880)	-	66 (43-110)	-	73 (59-92)		-	280 (260-370)
Present study Igarapé, Balbina-Am Colossoma macropomum N = 13	1619 (1274-1844)	662 (554-902)	81 (66-89)	68 (64-77)	79 (79-80)	65 (63-78)	348 (312-392)	177 (135-227)

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