Introduction

Orestias luteus along with 23 other Orestias sp. are endemic to Lake Titicaca and its tributaries, it is commonly known as “carachi amarillo” and is an important part of the economic activity of the inhabitants surrounding Lake Titicaca, generating direct and indirect jobs for the subsistence of rural families ²¹.

However, Orestias luteu biomass has decreased its catch volume from 21,920 to 18,724 t ¹¹; which is related to over fishing and massive mortality ². The latter due to pathological factors that could influence the biological behavior of the species; seriously impacting the economy and food security ¹⁷.

Current research is based on conservation and repopulation programs focused on food ^22,27, production and reproduction ¹⁷. The study of the health of these fish is being neglected, so much so that the only published works on Orestias luteus parasitosis are decades old ^8,26. Diplostomum sp are parasites whose metacercaria stage were recorded in more than 150 species of fish ⁹, mainly parasitizing the cerebrospinal fluid of the brain and eye cavities ⁴.

Heckmann ⁸ (1992) reported that infections of metacercariae of Diplostomum mordax in the cranial cavity of O. agassi, O. olivaceus, 0. luteus and Basilichthy sbonariensis 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.

Due to its low specificity Diplostomum sp affect both wild and cultivated fish species ¹⁰, causing negative impacts that are still unknown and neglected in the local aquaculture of Lake Titicaca. Lymneid snails and teleost fish are the intermediate hosts for Diplostomum sp. and piscivorous birds are the definitive hosts ¹⁸.

Diplostomum sp are prevalent, cosmopolitan parasites in the birds ^5,10. Severity of illness can be affected by the number of metacercariae present in the host ¹⁹, and severe illness leads to increased predation by birds ³⁰. The effects caused by the metacercaria in fish range from unnoticeable ¹⁶ to severe cases causing blindness, deformation, weight loss ⁵ and even death of the fish.

Parasitosis in fish causes economic and health losses due to the deterioration of meat ²³, ultimately impacting the food industry and those who depend on it for survival ²⁹. Therefore, the aim of this study is the ultra-structural characterization of meta-cercariae of Diplostomum sp in Orestias luteus of Lake Titicaca.

Material and methods

Fish collection and sample preparation: between February and May 2017, 35 Orestias luteus were collected from Peninsula of Capachica, Lake Titicaca - Puno, using chinchorros fishing gear ⁶. Fish were sacrificed by asphyxiation with Tricaine monosulfonate (Tricaine-s®) and then packed in an expanded polystyrene box with ice for shipment to Lima.

Necropsy and measurements: the necropsy and metacercaria collection of Diplostomum sp. was carried out in the Animal Parasitology Laboratory of the Faculty of Veterinary Medicine and Zootechnics of the Universidad Peruana Cayetano Heredia. At necropsy, the location of metacercariae in the fish was recorded and the frequency distribution and average parasitic intensity (total metacercariae / number of infected fish) of Diplostomum was determined ¹³.

Scanning electron microscopy: Scanning electron microscopy was performed in the specialized laboratory of the Faculty of Biological Sciences from the National University Greater than San Marcos, Lima - Peru.

The extracted metacercariae: were fixed in 4% glutaraldehyde and preserved in 70% ethyl alcohol. The samples were then dehydrated in 80%, 90% and 100% alcohol. The samples were desiccated to a critical point (Electron Microscopy Sciences, Model EMS 85), replacing water with carbon dioxide.

The metallization with gold was done using spray-metallizer coating equipment (Sputter Coater SPI, 11430E-AX), similar to the method used by ¹². Image registration was carried out using electronic microscope scanning with built-in camera (Fei Inspect S50).

Results

Of the 35 O. luteus analyzed, 17 (48.6%, IC 95%: 32.0-65.1) were infected by metacercariae of Diplostomum sp. In general, the mean parasitic intensity of infection was low 3.53 (1 to 5 metacercariae) in most fish; however, one fish had 23 parasites.

Metacercariae were found in the brain of the fish freely floating in the cerebrospinal fluid associated in the lobular in foldings, ventricular cavities and external meninges. Fish had parasites in the frontal lobes (58.8%), parietal lobes (11.8%), lateral lobes (17.6%), frontal and parietal lobes (5.9%) and in all three lobes (5.9%).

We did not observe a clear invasion of the cerebral parenchyma or severe inflammation in any fish. Also, the sampled fish did not show signs or symptoms of the disease (apparently healthy) and there was no evidence of metacercariae in other anatomical parts of the fish, as the target organ of the parasite is the brain.

Scanning electron microscopy analysis revealed that the parasites have papillae distributed irregularly on the surface of the body, both dorsal and ventral, and in greater numbers on the anterior end. The body is linguiform, 0.30 mm x 0.72 mm, slightly arched towards the ventral aspect.

The posterior border has a small conical prominence where the excretory pore is located, the anterior is slightly circular and a little ventral to this is the oral suction cup. The suction cup has a circular shape and its edges have roughness directed from the outside to the inside. On each side of the oral suction cup, a small pseudo-suction cup can be seen. The acetabulum has a transverse opening and the oval-shaped tribocytic organ is located posterior to it.

Discussion

The parasite observed in Orestias luteus from Lake Titicaca is likely Diplostomum mordax described by Tantaleán et al. (1975), Heckmann (1992) and Siegmund ²⁴ et al (1997) who described hemorrhage, cell necrosis and nerve fiber rupture associated with the parasite.

Diplostomum mordax differs from D. von Nord-man which parasitizes the brain and mesentery ¹⁰. D. mordax also differs from D. spathaceum and Diplostomum sp, which parasitize African Catfish and are normally located in the eyes ¹.

The frequency of parasities found in the present study was notably higher (48.6%) than the 15% and

Figures 1-6 Ultrastructural aspect of the metacercaria of Diplostomum sp. isolated from the brain of Orestias luteus from Lake Titicaca, Puno (Perú), revealed by electron microscopy. 1: whole body, dorsal surface with irregularly distributed papillae. 2: whole body, ventral surface, showing the oral suction cup (a), the acetabulum (b) and the tribocytic organ (c). 3 and 4: posterior region with small conical prominence where the excretory pore is located (d). 5 and 6: anterior region with oral suction (e) and pseudo-suction cup (f). 

11% prevalence observed by Heckmann (1992) and Sierralta ²⁵ et al (2020) in Lake Titicaca. But, slightly close than the prevalence of 60% and 7% to 100% reported in other fish species by Siegmund ²⁴ et al (1997) and Viozzi ²⁸ (2002), respectively.

The high prevalence may be due to the decreased water quality of Lake Titicaca ²⁰ and the increase in water temperature that occurs in the months of January, February, March and April. From January to April the average water temperature is 15.68° C ³, which would encourage the growth of snails of the genus Lymnaeidae ⁷. Also, Lyholt & Buchmann (1996) ¹⁵, showed that cercariae of Displostomun sphatacecum have higher infectivity at 15°C.

The mean intensity of Diplostomum sp is similar to what Siegmund et al (1997) reported in Chilean silver-side (Basilichthys australis), but lower than fish from tropical waters. Kembenya et al. (2012) ¹⁴, who identified Diplostomum sp in O. niloticus from three areas bordering the county of Uasin Gishu-Kenya; attributes the higher parasite intensities to a greater population of piscivorous birds and to fish farms with a higher density of fish compared to the natural environment.

Machado et al agrees but also mentions that the degree of barium infestation is higher in tropical waters and mentions that native fish have a lower degree of infestation compared to introduced fish. This indicates that the lesions produced by these parasites are not detrimental to the health of the fish unless the intensity increases ¹⁹.

The morpho anatomical details of the Diplostomum sp identified in this study coincide with that described by ¹⁰ with the exception of the acetabulum which the author describes as circular. Cavaleiro et al (2012) describe a tri lobed anterior end and the scanning electron microscopy described finger-like papillae, without apparent integument with a rounded base that ends in a kind of cilium.

Tantaleán et al (1975) describe a Diplostomum sp length between 0.860 to 0.890 mm and width from 0.470 to 0.490, which is slightly larger than that described in the present work. The size difference may have been caused by the procedures used for the measurement, since in the aforementioned study measurements were taken on flattened specimens for sheet preparation.

Additionally, mention that it is the first time that Diplostomum sp has been described at an ultra-structural level in the Peninsula of Capachica of Lake Titicaca, Puno. This supports the diagnosis of the disease in the various areas where Orestia luteus lives.

In the present study, the frequency of Diplostomum sp was 48.6% (IC 95%: 32.0 - 65.1) which is considered high. However the average parasitic intensity is low; suggesting that, at the moment, Diplostomum sp are not threatening the health of O. luteus. The location within the brain and the morpho anatomy of the parasite observed in this study coincide with Diplostomum mordax. Genotyping would be an essential tool to reach more specific conclusions.