Ciliated microeukaryotes ( Alveolata : Ciliophora ) of a lotic urban system located in Minas Gerais-Brazil

aLaboratório de Limnologia, Programa de Pós-graduação em Meio Ambiente e Recursos Hídricos, Instituto de Recursos Naturais, Universidade Federal de Itajubá – UNIFEI, Avenida BPS, 1303, Pinheirinho, CEP 37500-903, Itajubá, MG, Brasil bLaboratório de Protozoologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora – UFJF, Rua José Lourenço Kelmer, s/n, Martelos, CEP 36036-900, Juiz de Fora, MG, Brasil *e-mail: jessica_biol@outlook.com


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The phylum Ciliophora is organized in 14 classes with ~8,000 described species (Gao et al., 2016).Among unicellular microeukaryotes, ciliates are the most specialized, diversified and with the highest complexity in terms of cellular organization (Puytorac, 1994).They typically occupy basal trophic levels and display a wide geographical distribution occurring in almost all environments such as marine, fresh and brackish waters, and also in edaphic systems like soils, mosses and lichens (Lynn, 2008).
Their diversity in lotic systems have been extensively studied in the northern hemisphere because of their potential use as water quality bioindicators (Wiackowski, 1981;Primc, 1988;Groliére et al., 1990;Madoni, 2005).However, scarce information is available in literature about their diversity in aquatic ecosystems in Brazil, even though its privileged hydrological condition.The access to ciliate diversity and biogeography is a challenge tasks because they are diminute organisms, difficult to identify (Finlay and Fenchel, 1999), there is a lack of experienced specialists (Foissner, 2006) and a number of species stay encysted for most of their life cycles (Foissner, 2004).In Brazil, significant works were performed with this emphasis such as conducted in Rio Grande do Sul (Safi et al., 2014), Paraná (Pauleto et al., 2009;Buosi et al., 2011;Velho et al., 2005Velho et al., , 2013)), São Paulo (Bagantini et al., 2013;Regali-Seleghim et al., 2011), Pará (Castro et al., 2014), Minas Gerais (Dias et al., 2008(Dias et al., , 2010) ) and Rio de Janeiro (Paiva and Silva-Neto, 2004a, b) states.Inventory studies constitute the first step for development of applied biotechnological usage of ciliates.Regali-Seleghim et al. (2011) highlight the importance of more works surveying the diversity of ciliates in less studied regions of Brazil given their ecological importance.Moreover, establishment of in vitro cultures will contribute with information to biomonitoring programs (Madoni and Romeo, 2006;Shi et al., 2012) and for better evaluation of the biotechnological potential of these organisms (Mansano et al., 2016).This present work aimed to survey the diversity of the species of ciliated protists in a neotropical lotic urban system located in Southern region of Minas Gerais state, Brazil.
The samples were taken from José Pereira stream (45°27'31" and 45°20' 57W, 22°23'18" and 22°26'57"S) a highly impacted watercourse with in natura disposal of sewage (Thomaz da Silva, 2015) located in the municipality of Itajubá, Minas Gerais, Brazil.A Van Veen dredge was used to collect sediment monthly for over a year (October 2014 to October 2015).The sediment samples were readily transferred to 500 mL plastic containers and moved to the laboratory to be processed.Each sample were then divided (~20 mL) into three petri dishes and screened using glass micropipettes.Each Petri dish was analyzed in the day of collecting and weekly for up to 4 weeks.For in vitro cultures, ciliates were transferred to new Petri dishes filled with mineral water where rice grains with shells were added to served as carbon source for bacterial growth that would act to sustain the propagation of the tested ciliates.The ciliates were identified according to Foissner and Berger (1996).The photographic records of in vivo specimens were carried out with the aid of a camera attached to a microscope Olympus BX 51.The main features used in the identification of ciliates were: body shape, position and number of contractile vacuoles, oral and somatic ciliatures, position of macronucleus and shape of inclusions and color and the cytoplasm.Eventually, DAPI, a DNA specific staining method (Kapuscinski, 1995), protargol (Dieckmann, 1995) and dry silver nitrate (Klein, 1958) were used for species confirmation.
Among all these morphospecies, the species Euplotes aediculatus, Euplotes eurystomus, Spirostomum minus and Spirostomum teres, and Paramecium bursaria, Paramecium caudatum and Tetmemena pustulata were the ones that best adapted to the in vitro growth conditions (up to several months) using mineral water and rice grains and Cerophyl medium (Sonneborn, 1957), respectively (data not shown).
This study contributed to the understanding of the diversity of ciliated protists in Brazil, since this is the first work in the Southern region of Minas Gerais.Still, such studies can be useful to a better comprehension of the trophic relationships in aquatic environments, can support biomonitoring programs that assess the quality of water as well as the maintenance and conservation of the species with biotechnological potential (Madoni and Romeo, 2006;Regali-Seleghim et al., 2011;Gutièrrez et al., 2011).
In Brazil, the first work on ciliates from freshwater environments were carried out by Cunha in the early twentieth century (Cunha, 1913;Faria and Cunha, 1917;Cunha and Fonseca, 1918) with few recent studies on inventory of these micro-organisms in inland waters (Paiva and Silva-Neto, 2004a, b;Dias, 2007;Dias et al., 2008;Regali-Seleghim et al., 2011;Safi et al., 2014;Sartini, 2012;Mendonça, 2012;Castro et al., 2014;Kuhner et al., 2016).Cotterill et al. (2008) estimated that there are about 40,000 species of free-living ciliates, where only 4,500 species (~ 11%) have been described so far.Recent studies emphasize the need to increase sampling effort in South America for a better understanding of ciliates diversity in this region (Fenchel and Finlay, 2004;Foissner, 2006;Foissner and Hawksworth, 2009), as there are a large number of unexplored environments and potential implication to biomonitoring and conservation of these ecosystems (Mitchell and Meisterfeld, 2005;Cotterill et al., 2008).
The saprobic system for water quality evaluation, and more specifically organic pollution, developed by Kolkwitz andMarsson (1908, 1909), is widely used in biological classification of running water.The original list of indicator species, including ciliates, was revised and expanded (Foissner, 1988).Among the 48 morphospecies found in the stream José Pereira, 23 are included in the saprobic system and are considered biomarkers (Table 1), in which the vast majority were indicative of organically enriched environments (polluted or extremely polluted water), such as Loxodes striatus, Spirostomum teres, Paramecium caudatum, Euplotes aediculatus, Euplotes eurystomus, Tokophrya lemnarum, Cyclidium cf.glaucoma, Carchesium polypinum, Vorticella convallaria-complex, Spirostomum minus, Stentor polymorphus, Stentor roeselii, Aspidisca and Coleps hirtus.This observation is corroborated by a recent study (Thomaz da Silva, 2015) focusing in the quality of the water in this same sampling station, using physical and chemical parameters to classify this lotic system as Class III (highly polluted water) (Brasil, 2005), and highlighted the high levels of electrical conductivity, total coliforms, phosphorus, total nitrogen, ammonia and chlorophyll.
Moreover, we were able to stably maintain the in vitro growth of seven species of ciliates: Euplotes aediculatus, Euplotes eurystomus, Paramecium bursaria, Paramecium caudatum, Spirostomum minus, Spirostomum teres and Tetmemena pustulata.The ability to grow these organisms in vitro using cerophyl medium (Sonneborn, 1957) expands the possibilities of future applied studies such as acute trials (ecotoxicology), detection, characterization and isolation of secondary metabolites, characterization of molecules with antimicrobial activity, contributing to neotropical water monitoring programs (Madoni and Romeo, 2006;Petrelli et al., 2012;Mansano et al., 2016).This present study contributes to a better comprehension about the diversity of ciliated protists in limnic ecosystems in Brazil and emphasizes the importance of development Ciliated microeukaryotes of a lotic urban system Braz.J. Biol., 2019, vol. 79, no. 3, pp. 543-547 545/547 545  of new and efficient growth methods for in vitro culture of these microorganisms aiming future biotechnological end environmental monitoring studies.
in the same day of assay; 1 st = appeared in the first week of assay; 2 nd = in the second week; 3 rd = in the third week; 4 th = in the fourth; S = saprobicity(Foissner & Berger, 1996); p = polysaprobic; a = alpha-mesosaprobic; b = beta-mesosaprobic; **= not classified; + = occurred; -= absent.Dark names represent species that have been successfully in vitro cultivated in mineral water supplemented with crushed rice with shells; *First record in Minas Gerais state.