Trypanosomatids of four Loricariidae fish species in western Amazon rivers

A presença de tripanossomatídeos em peixes é bem conhecida e pode ocorrer em muitas espécies de peixes em todo o mundo. Nesse sentido, este estudo tem como objetivo relatar a ocorrência de Trypanosoma em quatro espécies da família Loricariidae na Amazônia ocidental, bem como a morfologia desses hemoparasitas. Os peixes foram coletados em três sub-bacias dos estados do Acre e Amazonas, Brasil. Amostras de sangue de peixes foram coletadas por punção cardíaca, com esfregaços feitos para quantificação, medidas morfométricas e morfotipagem dos tripanossomas encontrados. Estimamos os dados de prevalência e densidade de parasitismo. As variáveis morfométricas de Trypanosoma sp. foram submetidos à análise de variância. Encontramos novos relatos de distribuição e ocorrência de Trypanosoma em espécies de Loricariidae para a Amazônia ocidental. Os peixes examinados mostraram infecção por mais de um Trypanosoma o morfotipo A foi identificado infectando Loricariichthys anus, Sturisoma robustum e Loricaria cataphracta, o morfotipo B ocorreu em todas as espécies de peixes, e os morfotipos C, D e E ocorreram apenas em Loricaria sp. Houve diferença significativa entre os morfotipos de acordo


INTRODUCTION
The fish fauna of the Loricariidae family (Actinopterygii: Siluriformes) is one of the most diverse within the order Siluriformes (NELSON, 1994), playing an important role in aquatic ecosystems (BUCKUP, 1999). Besides, they have a high export potential in the aquarium market, which increases the scientific and economic level of these organisms (SANTOS et al., 2005).
Loricariids are commonly defined as detritivorous species with a tendency to algivory. They are scrapers with benthic and semi-sedentary habits (DELARIVA e AGOSTINHO, 2001), which can, according to Froés et al. (1979), make them potential fish hosts for leeches (Hirudinea). These invertebrates may develop Trypanosoma infection and transmit these hemoparasites to their host fish (JONES e WOO 1991).
Trypanosomatidae (Kinetoplastida) have a single nucleus, are elongated with a flagellum or rounded with a very short flagellum, and do not live freely. Many members of this family are heteroxenous, living a phase of life in the bloodstream or many tissues of different aquatic vertebrates (fish, amphibians, and reptiles), and another phase in the intestine of invertebrates, such as Hirudinea (CORRÊA et al., 2016).
These hemoparasites may not be harmful to infected fish, however in some cases, depending on the degree of infection, they can cause vital changes in the blood and some organs, causing anemia and anorexia (AHMED et al., 2011;LEMOS et al., 2015; MAQBOOL e AHMED 2016).
The presence of these trypanosomatids in fish is well known in the literature, occurring in more than 200 species of teleosts and elasmobranchs in marine and freshwater environments worldwide (EIRAS et al. 2012;GUPTA e GUPTA, 2012). More than 35 species of Trypanosoma have been described only in fish of the Loricariidae family (EIRAS et al. 2012). In the Amazon, few studies have been carried out on the distribution and occurrence of Trypanosoma in fish (FUJIMOTO et al., 2013;CORRÊA et al., 2016;NEVES et al., 2018;De SOUZA e CORRÊA, 2019), which may indicate a knowledge gap about these hemoparasites. This gap is mainly due to the region's high fish species diversity (REIS et al., 2016). Thus, identifying new Trypanosoma hosts is crucial since this information can help to solve significant problems regarding the study of pathogens caused by these parasites, in addition to contributing to the phylogeny of the trypanosomatid group (LOM, 1979).
In this sense, the present study aims to report the occurrence of Trypanosoma sp.
in four species of the Loricariidae family in western Amazonia and to evaluate the morphology of these hemoparasites in individuals of the same and different species of fish.

Study area
The fish were collected (authorization from the Brazilian Institute of the   m long and 3.0 m high, with meshes sizes of 1.5 cm, 2.5 cm, 3.5 cm, and 5.5 cm between opposing nodes. We used two nets, each with 12 mm mesh, 2 m high, and 12 m open. The nets were launched ten times at each collection point. We also used beach trawls of 9 m long and 2.4 m high, with a 13 mm mesh. The fish collected were sent to the Aquatic Ecology Laboratory of the Federal University of Acre (UFAC), where they were identified, measured, and weighed.
The specimens collected were anesthetized using menthol in a similar way to benzocaine. Menthol has many characteristics that qualify it as a suitable anesthetic for fish due to its efficacy and a sufficient safety margin for fish and the operator in the concentration used (GOMES et al., 2001). Then, to assess the presence of hemoparasites, blood samples were collected by cardiac puncture, using a hypodermic syringe containing an anticoagulant (5% EDTA), with duplicate blood smears made per fish sample. The blood smears were stained using Quick Panoptic/LABORCLIN® and examined by optical microscopy with 400x and 1000x magnification in the Microscopy Laboratory I at the Federal University of Acre (UFAC), Cruzeiro do Sul Campus, Acre, Brazil.
We selected ten Trypanosoma specimens of each morphotype for the morphometric evaluation. The parasites found were photographed using a Leica DM 500 optical microscope with an ICC50 HD coupled camera. The photos were used to determine the morphometric characteristics of the trypanosomatids using the ImageJ software, and the cytomorphometric measurements of the trypanosomatids were performed according to Borges (2016) (Figure 2).

Data analysis
The parameters of prevalence were calculated according to Bush et al. (1997). We used the direct method, adapted from De Carli (2001), to estimate the infection intensity (expressed in parasites/mL). We recorded and calculated all parasites found in 100 microscopic fields using 1000x magnification. It is estimated that 100 microscopic fields are equivalent to 0.2 μL of blood. Thus, the intensity of infection = (number of parasites × 5) × 1,000 = (parasites/mL) (De SOUZA e CORRÊA, 2019).
The morphometric variables of Trypanosoma spp. underwent homoscedasticity (Levene) and normality (Shapiro-Wilk) tests. Thus, we used an analysis of variance (ANOVA) followed by Tukey's post-hoc test to verify whether there was a difference in morphometric parameters among the morphotypes of Trypanosoma sp. Analyses were performed using the R software version 3.6.1.

RESULTS
Most studies on trypanosomes of Loricariidae in the Amazon have hitherto been concentrated in the eastern Amazon, where Hypostomus was the most studied genus (Table 1).
The present study shows new reports of Trypanosoma distribution and occurrence in four Loricariidae fish species for the western Amazon.
The fish species examined showed infection by more than one morphotype of Trypanosoma. Morphotype A has been identified infecting Loricariichthys anus, Sturisoma cf. robustum, and Loricaria cataphracta, morphotype B occurred in all fish species, and morphotypes C, D, and E occurred only in Loricaria sp. (Tables 2 and 3).  Of the four species examined, Sturisoma cf. robustum and Loricaria sp. were the most parasitized species and with the highest prevalence of parasites (P = 100% and P = 100%, respectively). However, Loricaria sp. had a higher number of parasites (n = 146) and intensity of parasite infection with 6 x 10⁴ parasites/mL, whereas Sturisoma cf.
We selected ten individuals from each Trypanosoma morphotype for morphometric characterization (Table 3). We found a significant difference among the morphotypes (TL, BL, NL, F, BW, NK, NA, and PN), according to the morphometric parameters (Table 3). Therefore, morphotypes A and E, LT, BL, and NA were significantly different from the other morphotypes (p<0.005). Morphotype E also showed a significant difference between NK and PN and the other morphotypes (p<0.005). Morphotype B was different, mainly in the BW data, whereas morphotype D was different in PN and morphotype C, NA (p<0.005) (Figure 2).

DISCUSSION
The present study demonstrated that research on Trypanosoma in Loricariidae is concentrated in the eastern Amazon. Here, we reported for the first time the occurrence of Trypanosoma ssp. to the western Amazon, expanding the distribution of these hemoparasites. We also reported trypanosome infection in Loricaria sp., Loricariichthys anus, Sturisoma robustum, and Loricaria cataphracta. Some studies reported trypanosomatids in Loricaria and Loricariichthys species (EIRAS et al., 1998;FROES et al., 1978); however, it is the first report for these hemoparasites in Loricaria cataphracta and Sturisoma cf. robustum. Many fish species may have infections by hemoparasites of the genus Trypanosoma. Loricariidae has the highest number of species with reports of infection (EIRAS et al., 2010(EIRAS et al., , 2012MOLINA et al., 2016), where the genus Hypostomus is considered the most common host (RIBEIRO et al. 1990).
Loricaria sp. showed a higher prevalence and parasitemia of Trypanosoma sp., in addition to indicating a higher variety of morphotypes. Trypanosoma infection in Loricaria is well documented (FONSECA e VAZ 1929;FONSECA e VAZ, 1928;LOPES et al., 1991;EIRAS et al., 2012) and may be associated with the species' habits, which are usually semi-sedentary. They are found on the margins or backwater regions, which may allow a higher infection by ectoparasites, such as leeches, which transmit protozoa (FROÉS et al., 1978). Trypanosoma infections can cause anorexia in infected fish, pronounced in cases of high parasitemia, although fish that survive the disease return to their regular diet (CORRÊA et al., 2016). Anemia can change the body conditions of hosts and somatic indexes of their livers, spleens, and hearts (ARDELLI e WOO, 1998).
The present study indicated many morphological forms of Trypanosoma in the blood of individuals of the same species and among species of Loricariidae. However, we cannot infer that these are different hemoparasite species since trypanosomes can show pleomorphism, which hinders the identification of these individuals (FRÓES et al., 1978).
Thus, due to the plasticity of these hemoparasites, molecular tools are crucial for accurate identification (FUJIMOTO et al., 2013). However, the morphological identification of Trypanosoma in fish is essential, since these data can characterize how the infection settles in the blood system. For example, according to some authors (LOM, 1979;LETCH, 1980), during the onset of infection, trypanosomes can show short and thin forms, and as the infection matures, the trypanosomes become long and wide. That is similar to some morphotypes in the same specimen of Loricaria sp. found in the present study. However, morphological studies associated with morphometry are helpful tools to elucidate the diversity of these flagellates, offering a low-cost methodology that can demonstrate the differences between the numerous specimens observed in the hosts' bloodstream (SOUZA e CORRÊA, 2019).
The aspects that expressed, in the present study, the high morphological variation of the blood trypomastigotes of Loricariidae were the changes in the body total length and width (thin or wide forms), the number of undulations in the wavy membrane, the presence and number of cytoplasmic granules, the distance of the kinetoplast to the posterior part, and the free flagellum length. Characteristics that may indicate variation in the development stage of these hemoparasites (LOM, 1979) and confirm the hypothesis that the morphological parameters of the bloodstream trypomastigote forms of fish are weak factors for the differentiation of parasite species (FIGUEROA et al., 1999;GU et al., 2007).
In this regard, studying Trypanosoma spp. in fish helps to clarify the morphological variation, distribution, and diversity of hosts, aiming to provide information about possible infections of neotropical fish farming and increase the knowledge about the diversity of these hemoparasites.