Metacercariae of Austrodiplostomum spp. (Digenea: Diplostomidae) infecting the eyes and brains of fish in Brazilian Amazon

Arq. Inst. Biol., v.86, 1-7, e0932018, 2019 RESUMO: O objetivo deste trabalho foi relatar a ocorrência de Austrodiplostomum spp. nos olhos e cérebros de Acaronia nassa, Caquetaia spectabilis, Satanoperca acuticeps, Curimatella sp. e Crenicichla marmorata em um lago do rio Amazonas no estado do Pará (Brasil). Dos 49 peixes examinados, 10,2% foram parasitados por metacercárias de Austrodiplostomum spp., A. nassa sendo o hospedeiro com maior número de metacercárias. Em cinco espécies de peixes examinadas, foram coletadas 51 metacercárias, sendo 45 encontradas nos olhos e 6 na caixa craniana. Nos olhos, as metacercárias estavam livres e ativas no humor vítreo, mas nenhuma opacificação foi observada. No cérebro, as metacercárias também eram livres e ativas, localizadas principalmente abaixo do encéfalo, no assoalho do crânio, na altura dos lobos oftálmicos e próximo ao nervo óptico. Em observações laboratoriais, no entanto, o peixe infectado não apresentou distúrbios comportamentais, o que pode estar relacionado ao baixo nível de parasitismo. Este foi o primeiro relato de Austrodiplostomum spp. em C. spectabilis, S. acuticeps, Curimatella sp., C. marmorata e A. nassa.


INTRODUCTION
The Amazon River system is formed by various orders of rivers, lakes, and other geographic events. It contains around 20% of world's freshwater and a tremendous richness of fish, and its approximately 3,000 species (JUNK, 2013) make it one of the main resources for human food and the local economy. These fish are hosts for a variety of groups of Digenea from the Diplostomidae family, including Austrodiplostomum spp. LOCKE et al. (2010) have suggested that the identification of these metacercariae may not be based only on morphology, and the use of molecular markers is required. So, in this paper we considered the name Austrodiplostomum spp.
Austrodiplostomum spp. has been responsible for verminous cataracts, also known as diplostomosis, in freshwater fish. The metacercariae of these digeneans that infect the eyes of fish can be found in the retina, vitreous, aqueous, and/or crystalline humor, and also in the brains of hosts. The presence of these metacercariae at heightened levels of abundance can cause exophthalmos, displacement of the retina, opacity of the crystalline lens, blindness, and even the death of host fish (HECKMANN, 1992;BULLARD;OVESTREET, 2008;MONTEIRO et al., 2016;VITAL et al., 2016). The formation of cataracts in infected fish is more intense after the parasites complete their larval development and are ready to infect the definitive host, augmenting the susceptibility of intermediate host fish to predation (SANTOS et al., 2002;PINTO;MELO, 2013;CORRÊA et al., 2014). This is therefore an efficient transmission method for these digeneans (MOURITSEN; POULIN, 2003;VITAL et al., 2016).
Austrodiplostomum spp. occurs in fish in North and South America (BULLARD; OVESTREET, 2008). In Brazil, it has been registered infecting the eyeballs of Geophagus brasiliensis (NOVAES et al., 2006), Geophagus surinamensis (SANTOS et al., 2002), Cichla ocellaris (SANTOS et al., 2002), Cichla sp. (SANTOS et al., 2002), Cichla monoculus (MACHADO et al. 2005;VITAL et al. 2016), Satanoperca papaterra (MACHADO et al. 2005), Hoplias malabaricus (SANTOS et al., 2002;CORRÊA et al., 2014), Hypostomus regani (YAMADA et al., 2008;ZICA et al., 2009), Schizodon borellii, Auchenipterus osteomystax, Serrasalmus maculatus (YAMADA et al., 2008), and Plagioscion squamosissimus (KOHN et al. 1995;SANTOS et al., 2002;MACHADO et al., 2005;SANTOS et al., 2002;ALBUQUERQUE et al., 2017), all of which are fish from different ecosystems. Despite the wide distribution and the increasing number of cases of occurrence of these eye fluke infections in Brazilian fish, the possible impacts on wild fish species remain unknown or neglected. Furthermore, despite the large number of fish in the Brazilian Amazon, infection has been described in only nine species in the region (VITAL et al., 2016). The objective of the present study was therefore to report the occurrence of metacercariae of Austrodiplostomum spp. in the eyes and brain of five species of fish from a lake in the Amazon River system in the north of Brazil, and to evaluate the possible disorders in the behavior of infected hosts.

MATERIALS AND METHODS
In October 2016, specimens of Acaronia nassa Heckel, 1840 (Cichlidae); Caquetaia spectabilis Steindachner, 1875 (Cichlidae); Satanoperca acuticeps Heckel, 1840 (Cichlidae); Curimatella sp. Eigenmann & Eigenmann, 1889 (Curimatidae) and Crenicichla marmorata Pellegrin, 1904 (Cichlidae) were collected in Lake Maicá in the state of Pará, Brazil (Fig. 1). This lake is located in the east of the municipal region of Santarém in the state of Pará, beginning in the Amazon River and extending as far as the Paraná do Ituqui River. It is connected to smaller lakes and contains various species of flora and fauna typical of the Amazon lowlands along its length.
The fish were euthanized for parasitological analysis using the transection of the cervical cord method for parasitological analysis. The total length (cm) and weight (g) were measured, and the organs were examined to verify the occurrence of metacercariae of Austrodiplostomum spp. The metacercariae found were compressed between a slide and a cover slip, fixed in AFA (70% alcohol, formalin and acetic acid) and preserved in 70% alcohol. The metacercariae were subsequently stained with Carmine de Langeron, clarified with Methyl Salicylate, and mounted on permanent slides using Canada balsam (EIRAS et al., 2006). The taxonomic identification of the parasites was carried out in accordance with KOHN et al. (1995) and GIBSON et al. (2002). The morphometric analysis of the metacercariae was performed using an optical microscope (Zeiss Axioplan), the images were obtained with a digital camera (coupled Axiocam ERc 5s), and the measurements (μm) were performed using the Zen program. The ecological terms used (prevalence, mean intensity and abundance) were in accordance with the recommendations of BUSH et al. (1997).

RESULTS
Of a total of 49 fish specimens from five species necropsied (Table 1 and Fig. 2), 10.2% were parasitized by metacercariae of Austrodiplostomum spp. A total of 51 metacercariae were recovered, being 45 from the eyes and 6 from the brain. In A. nassa were recovered 18 metacercariae and S. acuticeps 15 metacercariae (Table 2). In the host eyes, the metacercariae were free and active in the vitreous humor, but no sign of opacification was observed.   In the cerebral vault, the metacercariae were also free and active, and located mainly below the brain, on the cranial floor, at the height of the ophthalmic lobes, near the optic nerve. It was observed that the ophthalmic lobes, especially the veins that irrigate this region, were dilated and highly visible in the parasitized fish, a characteristic not observed in non-parasitized fish and/or those parasitized only in the eyes. However, in laboratory observations, the fish showed no behavioral changes. Austrodiplostomum spp. exhibited a foliaceous body, slightly concave on the ventral surface, small conical segments in the posterior region, an oral suction cup located at the anterior extremity, pseudo-suckers located laterally to the oral suction cup, and an oval pharynx (Fig. 3). The morphometric measurements of the metacercariae were compared with those described in the literature (Table 3).

DISCUSSION
Although metacercariae of Austrodiplostomum spp. have been found infecting the eyes, gills, swimming bladder, and musculature of several South American fish species due to their low parasitic specificity (ZICA et al., 2011) they infect the brains of hosts less frequently. In this study, we found lower intracranial abundance than in the eyes of infected fish. HECKMANN (1992) reported that infections of metacercariae of Diplostomum mordax in the cranial cavity of Orestias agasii, O. olivaceus, O. luteus and Basilichthys bonariensis induced compression of the neural tissue and migration of the metacercariae to the brain, leading to hemorrhage; cell necrosis; inflammation; fibrosis and the rupture of nerve fibers. However, such alterations were not observed in fish in the present study infected by Austrodiplostomum spp., due to the low levels of parasitism, a finding corroborated by SIEGMUND et al. (1997) in Basilichthys austrais infected by metacercariae of Diplostomum mordax and Tylodelphys destructor. CORRÊA et al. (2014) reported that H. malabaricus, with widespread infection by Austrodiplostomum spp. in the eyes and cranial cavity, presented alterations in swimming capacity such as imbalance and a lack of coordination. Host size, duration of transmission period, and host growth rate are important factors in brain disorders    GOATER, 2001) and in the behavior of infected fish. Such brain and visual changes expose infected fish to predators such as aquatic piscivorous birds (BULLARD; OVESTREET, 2008), definitive hosts of this parasite. Species of Curimatella are detritivorous fish that consume algae, debris, and associated microorganisms. Acaronia nassa, C. espectabilis and S. acuticeps are omnivorous fish that feed on aquatic invertebrates, algae, insects, and fish. Crenichla marmorata is a carnivorous fish that feeds on aquatic invertebrates and fish (SANTOS et al. 2004;FROESE;PAULY, 2017

CONCLUSIONS
Due to the low specificity of Austrodiplostomum spp., it has been found in several hosts of different watersheds. In this study, we extended the record of its geographic distribution to the eastern Amazon region, as well as added C. espectabilis, S. acuticeps, Curimatella sp., C. marmorata, and A. nassa as secondary hosts of Austrodiplostomum spp., increasing to 14 the number of hosts for this digenean in the Brazilian Amazon.