Metacercariae of Tylodelphys sp . ( Trematoda : Diplostomidae ) parasite of Brochis multiradiatus and Corydoras splendens ( Siluriformes : Callichthyidae ) from the Peruvian Amazon

Corydoras splendens (Castelnau, 1855) and Brochis multiradiatus (Orcés V., 1960) are highly demanded for exporters in the Peruvian Amazon and importers all over the world. The exportation of this fish species may represent a gateway for endoparasites to enter in a new environment with serious effects, so, the present study reports the presence of Tylodelphys sp. infecting C. splendens and B. multiradiatus. Fish were acquired from fishermen of the city of Iquitos, Peru. After the necropsy and analyses of the fish, metacercariae of Tylodelphys sp. were found in the liver and intestine. This finding represents the first report of Tylodelphys Diesing, 1850 infecting a fish in the Peruvian Amazon. Considering the possible damages caused by species of Tylodelphys, highlighting the nutritional and respiratory ones, prophylactic measures must be taken into consideration for exporters to avoid some damages in the fish that can deteriorate their health and also to avoid the introduction of this parasite to other countries.


Introduction
The ornamental fishery is a major activity in the Amazon Region and constitutes an important economic resource, since these fish are exported to other countries in large quantities (Chao, 2001).Species of Callichthyidae have benthic habits and are considered ornamental fish with high appreciation in the international market due to its rusticity and color patterns.In the Amazon Region, they are captured in rivers and streams (Sánchez, García, Vásquez, & Alcántara, 2011).Within this group of fishes, Corydoras splendens and Brochis multiradiatus are highly demanded, being exported for many aquarists of the Peruvian Amazon (Sánchez et al., 2011).
It is well known that exportation of ornamental fish may represent a gateway for both ecto and endoparasites to enter a new environment.Their introduction may have serious effects, especially when parasites come into contact with new hosts (Fujimoto, Barros, Marinho-Filho, Diniz, & Eiras, 2013).Several cases of introduced parasites, sometimes with devastating consequences, can be found in the literature (Moravec, Wolter, & Körting, 1999;Evans & Lester, 2001).
In the present study, metacercariae of Tylodelphys sp. of C. splendens and B. multiradiatus captured in the Peruvian Amazon are reported different organs.It is important to mention that parasites of these fish species have never previously been studied in this Region.

Material and methods
Adult specimens of B. multiradiatus (n = 20) and C. splendens (n = 20) were collected between July and August 2017 from local fishermen of the city of Iquitos, Peru.According to the fishermen, fish were captured in the River Tapiche.
Parasitological analysis was conducted in the Laboratorio de Hidrobiología of the Universidad Nacional de la Amazonía Peruana (UNAP) located in the city of Iquitos, Peru.In the laboratory, fishes were quickly immersed in a 75-mg clove oil•L −1 solution and euthanized (Conselho Nacional de Controle de Experimentação Animal [CONCEA], 2013).Posteriorly, fishes were measured and weighed.Looking for digeneas, internal organs were removed and examined under stereoscope.Metacercariae found were counted, fixed in hot water (65ºC) and conserved in 95% ethanol.Specimens were stained with Langeron´s alcoholic carmine, cleared in Eugenol and mounted in Canada balsam as permanent slides.Voucher specimens were deposited in the zoological collection of the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Brazil.
Taxonomical identification was according to Gibson, Jones, and Bray (2001).Illustrations were prepared with the aid of a microprojector.Parasites were photographed with a digital camera (Olympus Qcolor 5) and the images were used to obtain the measurements of the body and internal organs.Measurements are given in micrometers (μm), except the total body length that is given in millimeters (mm).Average measurements are followed by ranges in parentheses.The ecological terms in parasitology follow Bush, Lafferty, Lotz, and Shostak (1997).

Results
Tylodelphys sp.Higher parasitic indexes were reported for B. splendens (Table 1).Some parasites were found free, others in pairs and in groups varying in number, between 7-15 individuals inside of a circular membrane (Figure 2).

Discussion
Currently the genus Tylodelphys has 16 described species, distributed around the world.Of these, only 12 species were reported in the Americas, of which 7 were described only from metacercariae:  Hughes, 1929(Hughes, 1929;Lunaschi & Drago, 2004;Muzzal & Kilroy 2007;Drago & Lunaschi 2008).The other 5 species are T. adult, parasite of Podiceps major Boddaert, 1783 from Argentina; T. brevis, parasite of Mycteria americana Linnaeus, 1758, also from Argentina; T. elongata, parasite of Podiceps dominicus Linnaeus, 1766 from Cuba, Venezuela and Brazil; T. americana parasite of M. americana, and Podilymbus podiceps from Brazil, Venezuela and Mexico and Tylodelphys aztecae García-Varella, Sereno-Uribe, Pinacho-Pinacho, Hernández-Cruz, and de León, (2016).In the present study, the genus Tylodelphys is cited for the first time parasitizing a fish from Peru, expanding its geographic distribution to the Peruvian Amazon Region.Some helminths like digeneans infecting fish can induce cellular responses, (Richards, Hoole, Lewis, Ewens, & Arme, 1994a).Immature stages of parasites may be encapsulated with eosinophils, neutrophils and monocytes, there is, in addition, an increase in leucocyte numbers in lymphoid tissue due to infection (Richards, Hoole, Lewis, Evans, & Arme, 1994b).In the present study, some Tylodelphys individuals were found free and others were found in pairs or in multiple numbers inside of a membrane.Apparently, this can be a mechanism of immune response of fish against the infection of this parasite.
The differentiation of metacercariae, which are frequently encountered in fish, is especially problematic (Chappell, 1995;Niewiadomska, 1996;Niewiadomska & Laskowski, 2002).Identification of metacercariae is made more difficult by the lack of a key devoted to this stage in the life cycle.All present keys and monographs are based on adult stages (Niewiadomska, 2002).Additionally, identification of species of Tylodelphys is difficult due to striking morphological similarity, and especially at the metacercariae stage (Niewiadomska, 1996).In this study, it was not possible to identify the species, due to the stage of the parasite and due to the lack of taxonomic keys for this genus.This study represents a contribution to the study of this group of parasites.Parasitological studies on piscivorous birds that feed on callichthyids would be necessary to identify the parasite species.
The over dispersion of metacercariae in fish host populations has been attributed to heterogeneity in susceptibility, and uneven exposure of fish to infection that is a result of distribution of cercariae in the habitat caused by distribution of snail intermediate hosts (Bush, Fernandez, Esch, & Seed, 2001).According to Eiras (1994), cercariae penetrated the fishes through any part of their bodies.In the present study, the presence of Tylodelphys sp. in C. splendens and B. multiradiatus indicates the presence of snails and piscivorous birds, coexisting in the same habitat.In this way, it can be assumed that this parasite uses these fish as second intermediate hosts, being snails the first intermediate hosts and piscivorous birds the final ones.
Species of Callichthyidae are obligate airbreathers that use the intestine as an accessory respiratory organ: air is taken in through the mouth, passed through the gut and let out from the anus.(Gee & Graham, 1978).Kramer and Graham (1976) suggested synchronous airbreathing may reduce susceptibility to attack from aquatic and terrestrial predators and likened its effect to schooling which reduces the probability of contact between predator and prey.(Castelnau, 1855) and Brochis multiradiatus (Orcés V., 1960).AF = analyzed fish, PF = parasitized fish, P% = prevalence, N = number of parasites recorded, I = amplitude of intensity of infection, mI = mean intensity of infection, mA = mean abundance of infection.Parasites extract energy, in the form of nutrition, from their hosts.If hosts become starved for nutrients, their behavior might change.If energy drain impairs their physiology, they might become more sluggish or display lower physical performance.Alternatively, if a host is drained of energy, it might become more active and increase foraging rate.Either change in behavior could benefit the parasite responsible for the energy drain.(Lafferty & Shaw, 2013).Energy drain might also influence habitat selection.Taking risks to forage might place the host in situations where it is more likely to be eaten by a predator that acts as the final host for the parasite (Lafferty & Shaw, 2013).In the present study, the presence of Tylodelphys metacercariae in the intestine of C. splendens and B. multiradiatus may be a strategy to affect physiology of the fish or to alter their behavior.Probably the parasite not only extracts energy from the hosts, affecting their nutrition, but also affects the complementary respiratory function of the intestine, causing the rise of the fish to the surface to take oxygen, and thus, increasing the chances of being consumed by piscivorous birds.Additionally, energy taken by parasites might affect foraging decisions.A hungry host might be more likely to avoid shelter to increase foraging rates, and consequently, allowing the closing of the parasite's biological cycle.

Host
The finding of Tylodelphys sp. in C. splendens and B. multiradiatus could be very important, because they can harm the hosts' health.According to Eiras (1994), cercariae penetrated the fishes through any part of their bodies, and metacercariae of Tylodelphys sp. have similar behavior as Diplostomum spp., causing formation of cataracts and even death of the fish.

Conclusion
Considering possible nutritional and respiratory problems caused by the infection of Tylodelphys sp., prophylactic measures must be taken into consideration for exporters to avoid damages in the fish that can deteriorate their health (quality) and avoid the introduction of this parasite to other countries through ornamental fish exports.