Infections of Hypostomus spp . by Trypanosoma spp . and leeches : a study of hematology and record of these hirudineans as potential vectors of these hemoflagellates

Among Kinetoplastida, the Trypanosoma is the genus with the highest occurrence infecting populations of marine fish and freshwater in the world, with high levels of prevalence, causing influences fish health and consequent economic losses, mainly for fish populations in situation stress. This study investigated infections of Hypostomus spp. by Trypanosoma spp. and leeches, as well as blood parameters of this host in the network of tributaries of the Tapajós River in the state of Pará, in the eastern Amazon region in Brazil. Of the 47 hosts examined, 89.4% were parasitized by Trypanosoma spp. and 55.4% also had leeches attached around the mouth. The intensity of Trypanosoma spp. increased with the size of the host, but the body conditions were not influenced by the parasitism. The number of red blood cells, and hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), total number of leukocytes and thrombocytes showed variations and negative correlation with the intensity of Trypanosoma spp. in the blood of the hosts. The results suggest that the leeches were vectors of Trypanosoma spp. in Hypostomus spp.


Introduction
The genus Hypostomus Lacepède, 1803 comprises small and large Loricariidae with a highly variable pattern of coloration, with or without spots.The abdomen may or may not be covered with scales; the caudal fin is forked, with the larger lobe on top; there are two or three pre-dorsal scales; five rows of scales on the caudal peduncle; and a caudal keel with or without lateral scales (ARMBRUSTER, 2004).However, there is still no consensus about the taxonomy of Loricariidae, indicating the need for more specific studies aimed at its accurate identification (ZAWADZKI et al., 2012).
Trypanosomatidae Doflein, 1901 (Kinetoplastida) species have a single nucleus, are elongated with a single flagellum or rounded with a very short flagellum, and are not free living.Many members of this family are heteroxenous, living one phase of life in the bloodstream or in a variety of tissues of different species of aquatic vertebrates (fish, amphibians and reptiles), and another phase in the intestine of bloodsucking invertebrates.Trypanosoma species (Gruby, 1843) are almost all heteroxenous and parasites of the blood of all classes of vertebrates, including marine and freshwater teleost and elasmobranch fish all over the world (WOO, 1998;EIRAS et al., 2008;ROBERTS & JANOVY, 2013;HAYES et al., 2014).Today, more than 200 species are known to parasite fish around the world (GUPTA & GUPTA, 2012).More than 60 of these Trypanosoma species have been recorded in fish in Brazilian hydrographic basins and approximately 18 species (almost one third) have been described in Loricariidae species (EIRAS et al., 2010).
Trypanosoma species are transmitted to fish through a blood-sucking host, usually a species of leech.However, isopod crustaceans can also be potential vectors of trypanosomiasis in fish.These hemoparasites may not be detrimental to the infected fish, but in some cases can cause severe alterations of blood.Some trypanosomatids are highly pathogenic and can cause the death of the host fish (ISLAM & WOO, 1991;WOO, 1998;AHMED et al., 2011;LEMOS et al., 2015;MAQBOOL & AHMED, 2016).However, the effects of these hemoflagellates on the host fish physiology are not well understood.In general, it is difficult to determine unequivocally, based solely on morphology, when a Trypanosoma species is undescribed.DNA sequence data can provide some insight into species designations (WOO, 1998;LEMOS et al., 2015), but there are few studies for these hemoflagellates in South America.Therefore, knowledge of these parasites hematozoa Neotropical fish remains limited.
The purpose of this study was to investigate infection by Trypanosoma spp.and leeches in Hypostomus spp., and the hematology of these hosts in the Tapajós River system in the state of Pará, Brazil.

Fish and collection site
Between September and October 2012, 47 species of Hypostomus spp.were collected, 17 from the Uruá Stream (S 0,4°31'58,7", W 56°18'2,2") and 30 from the mouth of the Jamaxinzinho River (S 0,4°53'58,0", W 56°27'00,3"), which are tributaries of the Tapajós River system in the state of Pará, in northern Brazil.The fish were collected during an inventory of the region's ichthyic and parasitic fauna to determine the components of the diversity of this biome prior to the construction of a complex of hydroelectric plants, in order to garner statistical data to underpin the assessment, prediction and mitigation of the consequences of the anthropogenic changes imposed on the fish in the rivers that run through these protected areas (Figure 1).

Collection and analysis of ectoparasites and hemoparasites
Leeches were collected from the oral region (Figure 2) of the specimens of Hypostomus spp., and then fixed in alcohol.To examine the hemoparasites, a blood sample was collected by cardiac puncture using 1.5 mL syringes containing sodium heparin (25,000 Ul/mL).The blood samples were collected at the collection site.Part of the blood (8 µL) was used to prepare blood smears with May-Grünwald-Giemsa stain (DACIE & LEWIS, 2007).These blood smears were used to quantify the Trypanosoma spp. in each host.The ecological terms used here were those recommended by Rohde et al. (1995) and Bush et al. (1997).
Extensions containing the blood parasites and leeches were deposited in the collection of the Continental Fish Hematology Laboratory -CEPTA/ICMBio in Pirassununga, state of São Paulo.All the Hypostomus sp.specimens were deposited in the fish collection of the Genetics Museum of UNESP at Botucatu, state of São Paulo.

Blood collection and analysis procedures
The remaining blood was used to determine the total number of erythrocytes and the hemoglobin and hematocrit concentration.This data was then used to calculate the mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).The blood smears were also used to determine the total white blood cells and thrombocytes (DACIE & LEWIS, 2007).
The body weight (g) and total length (cm) data were used to calculate the relative condition factor (Kn) of the host fish, which was then compared with the standard value (Kn = 1.00) by means of the Mann-Whitney (U) test.The Spearman correlation coefficient (rs) was used to verify possible correlations between the intensity of Trypanosoma spp.and the weight, length and blood parameters of the hosts (ZAR, 2010).In addition, weight and length data were used to calculate the length-to-weight ratio (W = aL b ) after logarithmic transformation of length and weight and subsequent two straight-line adjustments, thereby obtaining lny = lnA + Blnx (LE-CREN, 1951).

Results
The fish weighed 162.3 ± 95.6 g and were 25.1 ± 7.5 cm long.The pH of the Uruá Stream was 7.4 and its water temperature was 27.0 °C, while the pH at the mouth of the Jamaxinzinho River was 7.0 and its water temperature was 29.9 °C.
Were examined 47 fish of which 42 were infected with Trypanosoma spp. with their distribution in the area of collecting and presenting different types of Trypanosoma spp.(Table 1).
The shape of the Trypanosoma spp. was long and wide, with tapered ends and a highly prominent undulating membrane, with several folds.The kinetoplast was terminal or sub-terminal, round and somewhat stained, surrounded by a clear area.The nucleus, rounded to oval in shape and slightly stained, was located centrally and occupied the entire width of the cell, with few or no granulations.Its cytoplasm was highly granular and it had numerous vacuoles along the body.The flagellum was short, slightly stained and, in some cases, almost undetectable (Figure 3).
Apparently, one sees three distinct morphological types of Trypanosoma spp.Thus, we present a description of trypomastigotes in fish examined, highlighting the polymorphism with their respective measures (Table 2).
All the Hypostomus spp.specimens parasitized by leeches also presented Trypanosoma spp.infection.There was variation in the intensity of Trypanosoma spp.and leeches in the hosts examined (Table 3).The intensity of Trypanosoma spp. in the blood was positively correlated with the length (rs = 0.622, p = 0.0001) and weight (rs = 0.426, p = 0.003) of the hosts.
The equation for the weight-length relationship of Hypostomus spp.revealed a negative allometric relationship (Figure 4), indicating that there was a greater increase in body weight than in length.The Kn of hosts varied (Table 4), but did not differ (U = 940.0,p = 0.213) from the standard value (Kn = 1.00).

Discussion
Three morphotypes of Trypanosoma were found in Hypostomus spp., being two in hosts from Uruá Stream and one in hosts from Jamaxinzinho River.In addition, a high level of parasitism of Trypanosoma spp.and leeches in Hypostomus species from Tapajós River system was found.Fujimoto et al. (2013) reported low prevalence and high intensity of Trypanosoma spp.and leeches in Hypostomus species from the Guamá River.In marine and freshwater fish populations, Trypanosoma spp.maintain their life cycle by using hematophagous invertebrates as primary hosts, namely leech species, and subsequently fish populations (D' AGOSTO & SERRA-FREIRE, 1993;WOO, 1998;PÁDUA et al., 2011;HAYES et al., 2014;LEMOS et al., 2015).Trypanosoma spp.multiply in the digestive tract of leeches, with the non-infective forms migrating to the proboscis, where they transform into infective forms and are inoculated into fish when the leech feeds (WOO, 1998).In Brazil, it has been suggested that the main vector of these hemoflagellates in fish from the Loricariidae family is the Batracobdella gemmata leech (D' AGOSTO & SERRA-FREIRE, 1993).Therefore, in Hypostomus spp., the transmission to these hosts presumably occurs infected leeches feed on them.Moreover, the wide diversity of the leech fauna of the Neotropical region (SKET & TRONTELJ, 2008) leads to the assumption that the hemoflagellate fauna of this region is also diverse.
Species of Trypanosoma can cause anorexia in infected fish.This is most evident in cases of high parasitemia, although fish that survive the disease return to normal feeding.Anemia may alter the hosts' body conditions and the somatic indices of their liver, spleen and heart (WOO, 1998).However, the hosts' body     conditions in this study were not affected by parasitism, as indicated by the relative condition factor.In addition, stress conditions may influence the course of parasitemia in fish.At low and high temperatures, fish have decreased trypanosome levels in the blood (WOO, 1998;GUPTA & GUPTA, 2012).
Trypanosomes can cause anemia in infected fish (GUPTA & GUPTA, 2012;MAQBOOL & AHMED, 2016), whose erythrocytes frequently undergo alterations.As trypanosomes generally depend on the energy resources of the host fish, the impact of this parasitism in fish is considerable, ranging from physiological, metabolic, pathological and biochemical alterations to asymptomatic behavior.Woo (1998) stated that anemia caused by Trypanosoma spp.may be related to the inactivity of the host's hemopoietic system.The severity of this anemic process is directly tied to the intensity of hemoparasite load, and is partly caused by lytic factors and hemodilution.The lytic factor is secreted by living parasites and lyses red blood cells (RBC) in the absence of specific antibodies.It seems that the virulence factor that leads to anemia is a protease.However, Fujimoto et al. (2013) reported that the RBC parameters of Hypostomus sp., Ancistrus sp. and Rineloricaria lanceolata were not influenced by infection by Trypanosoma sp., but the RBC and hematocrit level in Lasiancistrus saetiger increased while hemoglobin concentration decreased.The RBC count, hematocrit and MCV of Hypostomus spp. in this study were similar to those reported by Fujimoto et al. (2013) for the same host infected by Trypanosoma spp., while the hemoglobin concentration, MCHC and MCH levels were lower.The hemoglobin concentration and RBC number showed an increase with the intensity of Trypanosoma spp. in blood.However, the hematocrit, MCV, MCH and MCHC of the hosts of this study presented negative correlation with the intensity of Trypanosoma spp. in blood, indicating that an increase in parasite number may lead fish to an anemiant process.
Hemostasis is a function of paramount importance when fish are responding to injuries, and piscine thrombocytes play a central role in this process.The number of thrombocytes can vary from 2,000-78,900 µL among healthy fish species due to intraspecific variations, which are attributed to biotic factors such as age, season and maturity, and abiotic factors such as water temperature, pH, dissolved oxygen content, sex, and maturity stage, as well as stress and diseases (TAVARES-DIAS & OLIVEIRA, 2009).However, the number of thrombocytes in Hypostomus spp.suggests that it was not influenced by the infection of Trypanosoma spp., since the host's hemopoietic system was not impaired.
Piscine leukocytes are involved in phagocytosis, immunoglobulin production, modulation of immune defense, inflammation process and defense against parasitic and bacterial infections and stress (DAVIS et al., 2008;RANZANI-PAIVA et al., 2013).Leukocytosis has been reported in Schizothorax plagiostomus infected with Trypanosoma spp.(MAQBOOL & AHMED, 2016), while lymphocytes decreased perceptibly in Hypostomus sp.(FUJIMOTO et al., 2013).In contrast, the total number of leukocytes in Hypostomus sp.appeared to have been unaffected by Trypanosoma spp.infection.
In summary, armored catfish species are usually infected by Trypanosoma spp., particularly the Hypostomus species.Hypostomus spp.sampling was carried out in dry season, when the water levels in lakes and streams decrease drastically, resulting in greater competition for resources such as food and shelter.Therefore, these factors combined with high temperatures may be related to the high infection levels that were found, representing a stress condition that reduces the immune status of the fish population and thus facilitating the survival of parasites.The morphometric features, alone, did not suffice to identify the species of Trypanosoma found.Thus, a review of the Trypanosoma species that infect fish species is needed, along with other factors such as host characteristics, isolation culture media, experimental infections and analysis of DNA sequences.Finally, this is first study of hematological parameters and infection by Trypanosoma in Hypostomus spp.parasitized by hirudineans in the Tapajós River system.

Figure 3 .
Figure 3. Forms of trypomastigotes viewed in light microscope.The blood smears of Hypostomus spp.were stained with Grünwald-Giemsa.
trypanosomes measured; FL: flagellum length; UM: Width of the undulating membrane; BL: body length; BW: body width; NL: nucleus length; NW: nucleus width; PD: distance from the front end of the nucleus; FA: distance from the posterior end nucleus; MK: midnucleus to kinetoplast and PK: posterior to kinetoplast.

Table 1 .
Morphotypes Trypanosoma number of infected fish and collection sites along the Tapajos River.
Infections of Hypostomus spp.by Trypanosoma spp.and leeches: study hematology these hemoflagellates

Table 2 .
Measures of morphometric characteristics with the values mean (minimum and maximum) expressed in µm of blood forms of Trypanosoma spp.

Table 3 .
Parasitological indices in Hypostomus spp.from Tapajós river system, state of Pará (Brazil).Count of Trypanosoma spp. in 8 µl of per host.