Acta Herpetologica 10(1): 31-37, 2015 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-14988 Basal frequency of micronuclei and hematological parameters in the Side-necked Turtle, Phrynops hilarii (Duméril & Bibron, 1835) María A. Latorre1,2,*,#, Evelyn C. López González1,2,#, Pablo A. Siroski1,2,3, Gisela L. Poletta1,2,4 1 “Proyecto Yacaré” – Laboratorio de Zoología Aplicada: Anexo Vertebrados, Facultad de Humanidades y Ciencias, UNL/Ministerio de Aguas, Servicios Públicos y Medio Ambiente, Av. Aristóbulo del Valle 8700, Santa Fe, Argentina. *Corresponding author. E-mail: gul_16@hotmail.com 2 Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET- Av. Rivadavia 1917, CABA, Argentina 3 Instituto de Ciencias Veterinarias del Litoral, ICiVet Litoral-UNL-CONICET- R.P. Kreder 2805, Esperanza, Santa Fe, Argentina 4 Cát. Toxicol. y Bioq. Legal, FBCB-UNL, Ciudad Universitaria, Paraje El Pozo S/N, Santa Fe, Argentina # These authors contributed equally to this work Submitted on 2014, 15th September; revised on 2014, 22nd October; accepted on 2014, 27th October Editor: Giovanni Scillitani Abstract. The present study aimed to evaluate basal frequency of micronuclei (MN) and hematological values in adult Phrynops hilarii in order to propose this aquatic turtle, broadly distributed in our region, as a biological monitor for future studies of environmental pollution assessment. Thirty-two adult turtles from a semi-natural environment locat- ed at the Zoological Experimental Station (Santa Fe, Argentina) were used. Blood samples were taken and the follow- ing parameters were determined: basal frequency of MN (BFMN), total red blood cells (RBC) count, hematocrit (Ht), hemoglobin (Hb), total and differential white blood cells (WBC). The BFMN determined for the species was 3.56 ± 1.39, while hematological parameters showed the following reference values: 0.937  ±  0.12 x106 RBC/µl, 27062.50 ± 4565.43 WBC/mm3, hematocrit 18 ± 1.81% and Hb concentration 4.80 ± 0.45 g/dl. Differential WBC counts were: 76 ± 2.90% for lymphocytes, 20.12 ± 2.56% for heterophils, 1.5 ± 0.19% for monocytes, and 2.12 ± 0.61% for eosino- phils, while no basophils were observed in any of the samples analyzed. No differences were observed between males and females in any of the variables analyzed. Data provided in this work could be useful as reference values for future studies of natural regions where P. hilarii occurs, employing this species as a sentinel organism for genotoxic and immunotoxic evaluation of environmental pollutants. Keywords. Genotoxicity, immune system, hematological values, micronucleus test. INTRODUCTION Phrynops hilarii (Duméril and Bibron, 1835), com- monly called side-necked turtle, belongs to the Suborder Pleurodira, Family Chelidae. This species is distributed in the east of Uruguay, southern Brazil, almost all of Paraguay and Argentina (Cabrera, 1998; Van Dijk et al., 2012). In this country, it can be found in the provinces of Buenos Aires, Santa Fe, Entre Rios, Corrientes, Chaco, Misiones, Formosa, Córdoba, Tucuman and Mendoza (Richard, 1999). This species inhabits in streams and lagoons which in many cases constitute important areas of pollut- ants discharge, generally coming from agricultural and industrial activities. Evaluation of toxics effects pro- duced by chemicals on hematological, immunological and genetic parameters in wild species is of fundamen- tal importance for maintaining biodiversity, as they are 32 M.A. Latorre et alii good indicators of the health of populations in poten- tial exposure to different types of xenobiotics (Donald, 2004; Poletta et al., 2008). The immune system (IS) is an excellent indicator of the health of an organism (Burns et al., 1996). Its integri- ty is essential for the defense against infectious organisms and their toxic products and, therefore, for the survival of all individuals. The IS is also very sensitive to chemi- cals exposure and considering the fast response of some immune parameters, many of them are used as mark- ers of such exposure (Lafuente Giménez et al., 2001). In this sense, the exposure of wild or domestic animals to certain chemicals, whether acute or chronic, may affect the defense mechanisms. This information also allows the identification of associated diseases such as anemia, malnutrition, dehydration, inflammation, parasitemia, hematopoietic malignancies and disorders of hemostasis (Barboza et al., 2007). White blood cells (WBC) are the cellular compo- nents of the IS and are involved in a significant number of processes. Certain situations may cause an increase or decrease of the values of selected blood components; in turn, these are used for the interpretation of physiological phenomena or accurate diagnosis of diseases and nutri- tional status (González Fernández, 2003). In addition, traditional hematological parameters may provide infor- mation about the general stress, especially in birds and reptiles (Gross and Siegel, 1983; Morici et al., 1997; Lance and Elsey, 1999). As it has been mentioned above, many individual factors (age, sex, stress, nutritional condition, hormones concentrations, body hydration levels, etc.) as well as environmental factors (temperature, pressure, oxygen concentrations, etc.) may affect hematological val- ues and so they can be considered as biomarkers of expo- sure (Dessauer, 1970; Aguirre et al., 1995). On the other hand, biomarkers of genotoxicity reveal alterations induced by various agents on genetic material of organisms. In particular, the MN test (Schmid, 1975) detects the effects of agents which modify the structure and/ or segregation of chromosomes by the identification of acentric fragments and/ or lagging chromosomes that remain separated from the main nucleus of the daugh- ter cells during cell division. This technique allows the detection of early biological responses, before the damage is irreversible and causes imbalances in the health of an organism (Carballo and Mudry, 2006). Thus, the deter- mination of the frequency of MN (FMN) is used as an indicator of the effects of various agents on the genetic material and the “machinery” associated with cell divi- sion (Schmid, 1975). The basal frequency of micronuclei (BFMN) is the result of “normal” errors in the processes of replication and/ or cell division, which are not caused by the incidence of genotoxic agents. It is specific for each species and each cell population, and constitutes a reference value to determine the usefulness of a species as a sentinel or biological monitor. In reptiles, the MN test was applied to peripheral blood erythrocytes in dif- ferent species (Zuñiga González et al., 2000; Poletta et al., 2008; Schaumburg et al., 2012), being a useful tool to determine the genotoxic potential of chemical (Poletta et al., 2009; Poletta et al., 2011; López González et al., 2013) and physical agents (Schaumburg et al., 2010). Up to our knowledge, there is no report on the lit- erature about the application of the MN test in this spe- cies, while the studies on hematologic values in Phrynops hilarii are extremely scarce (Pitol et al., 2007). The aim of this study was to determine the BFMN and reference hematological values in adult P. hilarii, in order to propose this aquatic turtle of great importance in our region, as a biological monitor for future studies of environmental pollution assessment. MATERIALS AND METHODS Animals All animals in this study were treated in accordance with the Reference Ethical Framework for Biomedical Research: Ethical Principles for Research with Laboratory, Farm, and Wild Animals (National Scientific and Technical Research Council, 2005), using non-invasive techniques of blood collection and minimiz- ing stress and suffering by suitable management methods. Thirty-two adults Phrynops hilarii (from 0.61 to 5.46 kg, Fig.  1) from a semi-natural environment at Zoological Experi- mental Station (Santa Fe, Argentina), were bled (1 ml) immedi- ately from the external jugular vein (Rogers and Booth, 2004). All individuals were sexed, weighed and measured morphomet- rically. Micronucleus test (MN) The MN test was conducted according to the technique described by Poletta et al. (2008) for its application in periph- eral blood erythrocytes of another reptile species, with some modifications. Two smears were performed per animal, fixed with etha- nol and stained with 10% Giemsa solution previously centri- fuged and filtered. The baseline frequency of MN (BFMN: num- ber of cells with MN/1000 erythrocytes analyzed) was deter- mined under an optical microscope at 1000x. Hematological parameters For hematological study the following parameters were determined: RBC (red blood cells) and WBC (white blood cells) 33Micronuclei and hematological parameters in Phrynops hilarii counts, differential leukocyte count (DLC), Ht and Hb. The total leukocyte count was performed using a Neu- bauer chamber due to the presence of nucleated erythrocytes. In this species, like in all non-mammalian vertebrates, the use of automatic counter is impossible because the presence of nucle- ated erythrocytes gives wrong numbers. An aliquot of blood was diluted 1:200 with 0.6% NaCl (Lewis et al., 2008). The sam- ples were observed under an optical microscope at 400x. For the DLC two smears were made per animal on clean slides, fixed with ethanol for 10 min and stained with May Grünwald (50%)-Giemsa (10%) solutions. The samples were observed under an optical microscope at 1000x. Hb concentration was determined using an autoanalyzer. Statistical Analysis The statistical analysis was conducted with the software SPSS 17.0 for Windows. Mean and Standard Error (SE) of each variable analyzed were calculated from data of all animals. All variables were analyzed in normality by Kolmogorov-Smirnov test and homogeneity of variances by Levene test. We used the Mann Whitney U-test to compare the BFMN and monocytes between males and females, while the rest of the variables were analyzed trough the Student t-test. Linear regressions were car- ried out to analyze the relation between reference values and weight of the animals. A P value <  0.05 was considered statisti- cally significant. RESULTS Reference values of all variables analyzed and the particular values for males and females are shown in Table 1 (Fig. 2). Concerning hematological parameters no basophils were observed in any of the DLC analyzed and no differences were observed between males and females in any of the variables (P > 0.05, Table 1). In the case of the BFMN, females showed higher val- ues than males, but the difference was not statistically significant (P  >  0.05, Table 1). No relationship was found between BFMN and weight of the animals (P > 0.05, R2 = 0.001). DISCUSSION Currently, there is great interest in those studies that allow collecting information on the health status of wild populations. In the case of reptiles, hematological tech- niques have been evolving favorably (Wilmoth, 1994; Lowell, 1998), however, there are few hematology studies on the Testudines freshwater turtles (Metin et al., 2006; Pitol et al., 2007). Reference hematological values found for different species of turtles (Troiano et al., 1998; Montilla Fuen- mayor et al., 2006; Cabrera et al., 2011) differ from the values reported in our study, and this can be explained by the multiple factors that influence them, such as the spe- cies, the environment where they live and their geograph- ic distribution. In the case of RBC and WBC counts, we found higher values than those reported by other authors (Troiano et al., 1998; Montilla Fuenmayor et al., 2006; Cabrera et al., 2011; Table 2). It has to be noted that the conditions and habits of the turtles were different in the mentioned works and this clearly exert a particular influ- ence on certain hematological parameters. Hematocrit of Phrynops hilarii was similar to those found in other turtle species (Troiano et al., 1998; Mon- tilla Fuenmayor et al., 2006; Cabrera et al., 2011), while hemoglobin was lower than the values reported by Troi- ano et al. (1998) and Cabrera et al. (2011) (Table  2). Leu- kocytes populations showed different results. In the case of lymphocytes, our values were higher than those report- ed in similar species (Troiano et al., 1998, Montilla Fuen- Fig 1. Adult specimens of Phrynops hilarii (approximately 4-5 kg). Table 1. Reference values for all variables studied in P. hilarii, including males and females particular values (BFMN, baseline frequency of micronuclei; RBC, red blood cells; SE, standard error; WBC, white blood cells). Variables (mean ± SE) Males Females Total RBC (x106/µl) 0.937 ± 0.12 0.98 ± 0.23 0.91 ± 0.15 Total WBC (x103/µl) 27.06 ± 4.56 26 ± 2.75 27 ± 7.47 Hematocrit (%) 18 ± 1.81 20.17 ±0.35 16.73 ± 2.84 Hemoglobin (g/dl) 4.80 ± 0.45 5.17 ± 0.16 4.58 ± 0.73 Lymphocytes (%) 76 ± 2.90 77.0 ± 7.02 75.40 ± 2.94 Heterophils (%) 20.12 ± 2.56 18.33 ± 5.84 21.20 ± 2.71 Monocytes (%) 1.5 ± 0.19 1.33 ± 0.33 1.60 ± 0.24 Eosinophils (%) 2.12 ± 0.61 1.67 ± 0.33 2.4 ± 0.98 BFMN 3.56 ± 1.39 3.08 ± 2.39 4.27 ± 1.92 34 M.A. Latorre et alii mayor et al., 2006; Cabrera et al., 2011) but the opposite was detected for heterophils. With respect to monocytes, our values were lower than those found in Chelonia mydas (Montilla Fuenmayor et al., 2006) and Chelonoidis chilen- sis chilensis (Troiano et al., 1998), but higher than those of Geochelone denticulata (Cabrera et al., 2011). Unlike, eosinophils found in Phrynops hilarii were lower than those reported by Troiano et al. (1998) and Cabrera et al. (2011) in Chelonoidis chilensis chilensis and Geochelone denticulata, respectively, but higher than that of Chelonia mydas (Montilla Fuenmayor et al., 2006) (Table  2). Basophils were not observed in any of the ani- mals studied; this could be due to the small number of this cell type present in the circulation of healthy animals (Work et al., 1998). As we mentioned, the influence of factors such as age, sex, reproductive status, stress, temperature, time of year, capture techniques and methods of analysis could modify reference values substantially. Because of this, in order to be compared with those of other species, it is extremely important to describe in details most of the factors and conditions involved. Similar to our study, some authors found no signifi- cant differences in hematological values between females Fig 2. (1) Micronucleated erythrocyte, (2) lymphocyte and (3) eosinophil (arrows) of Phrynops hilarii stained with May Grunwald - Giemsa (1000x). Table 2. Hematological parameters reported by different authors in turtles (Hb, hemoglobin; Ht, hematocrit; RBC, red blood cells; WBC, white blood cells). Hematological Parameters ReferenceWBC (106/µl) RBC (103/ µl) Ht (%) Hb (g/dL) Lymphocytes (%) Heterophils (%) Monocytes (%) Eosinophils (%) Basophils (%) Phrynops hilarii 0.937 27.06 18 4.8 76 20.12 1.5 2.12 0 This study Chelonoidis chilensis chilensis 0.70 9.2 25.2 11 26 28 5 32 0 Troiano et al. (1998) Chelonia mydas 0.42 6.16 29.4 __ 14.7 82.9 1.97 0.47 ___ Montilla Fuenmayor et al., (2006) Geochelone denticulata 0.44 7.82 20.3 7.0 25.5 55.6 0.4 15.8 1.5 Cabrera et al. (2011) 35Micronuclei and hematological parameters in Phrynops hilarii and males in Caiman crocodilus (Rossini, 2004), while other authors reported such differences in other reptiles species (Frair, 1977; Wood and Ebanks, 1984; Hart et al., 1991). We only found a moderate positive correlation between heterophils and weight. Several studies in recent years have used the MN test as a biomarker of genotoxicity in various species of reptiles exposed to different chemicals (Poletta et al., 2009; 2011; Borrat et al., 2011; Capriglione et al., 2011; López González et al., 2013). Primarily, it is necessary to establish basal values of MN as it is species-specific and may vary with age, sex and health status of individuals. The results of our study have determined that BFMN of P. hilarii is 3.56  ±  1.39. This value is much higher than those found by our group in other endemic reptile spe- cies from Argentina. We found a BFMN of 0.87 ± 0.74 in the broad-snouted caiman (Caiman latirostris; Poletta et al., 2008) and of 0.95 ± 0.27 in the tegu lizard (Tupi- nambis merianae; Schaumburg et al., 2012). This finding could probably be attributed to factors intrinsic to the species, such as lifespan of circulating erythrocytes and removal time of senescent or damaged erythrocytes, or to phylogenetic distance (Caliani et al., 2014). Moreover, Zúñiga-González et al. (2000; 2001) reported BFMN in species of lizards, snakes, and croco- diles between 0.10 and 0.30/1000, while in species of tur- tles (Macroclemys temminckii; n = 1 and Kinosternon sub- rubrum; n = 2) the value reported was 0/1000. The low values obtained in the cited study could be due to the small number of individuals used, as no more than one or two animals were used per species. However, similar data were obtained by Strunjak-Perovic et al. (2010) for the snake Hierophis gemonensis (BFMN  = 0.30/1000; n = 10). Borrat et al. (2011) studied Chelonia mydas (marine green turtle) as a biological monitor to evaluate the lev- el of pollution in a contaminated area (n = 60). In this study, animals used as controls were rehabilitated speci- mens with a FMN of 9/1000 erythrocytes, while animals coming from two contaminated sites showed a FMN of 42 and 627/1000 erythrocytes, respectively. Even when the control FMN was much higher than the one found in our study, it is important to highlight that animals used were rehabilitated, so comparison is not appropriate. Finally, we found no relationship between BFMN and weight. These results agree with those reported in juve- nile Caiman latirostris (Poletta et al., 2008) and adults Tupinambis merianae (Schaumburg et al., 2012); new- born tegu lizards showed instead a weak negative correla- tion between the BFMN and weight, possibly indicating a relation between poor nutritional state and deficiencies in the mechanisms of protection and/or repair of genetic damage (Schaumburg et al., 2014). This study is the first report on the application of the MN test and determination of reference hematological values in Phrynops hilarii, considering individual varia- tion due to factors such as sex, age and health conditions of individuals. This represents an important contribution to the knowledge of the biological characteristics of this species and is the initial step to propose it as a sentinel organism for future studies on the biomonitoring of pol- lutants present in its natural habitat. 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Eremchenko (1949-2014) Leo J. Borkin1, Tatjana Dujsebayeva2, Roberto Sindaco3, Matthias Stöck4 Haplotype variation in founders of the Mauremys annamensis population kept in European Zoos Barbora Somerová1, Ivan Rehák2,*, Petr Velenský2, Klára Palupčíková1, Tomáš Protiva1, Daniel Frynta1 Reproductive ecology of Sichuan digging frogs (Microhylidae: Kaloula rugifera) Wei Chen1,*, Lina Ren2, Dujuan He2, Ying Wang2, David Pike3 Toxic effects of carbaryl on the histology of testes of Bufotes variabilis (Anura: Bufonidae) Özlem Çakici Basal frequency of micronuclei and hematological parameters in the Side-necked Turtle, Phrynops hilarii (Duméril & Bibron, 1835) María A. Latorre 1,2,*,#; Evelyn C. López González1,2, #; Pablo A. Siroski1,2,3; Gisela L. Poletta1,2,4 Into a box interiors: clutch size variation and resource allocation in the European pond turtle Marco. A.L. Zuffi1,*, Simonetta Citi2, Elena Foschi1, Francesca Marsiglia1, Eva Martelli1 Where to “Rock”? Choice of retreat sites by a gecko in a semi-arid habitat Andreia Penado1,2, Ricardo Rocha3,4,*, Marta Sampaio3, Vanessa Gil3, Bruno M. Carreira3, Rui Rebelo3 Age structure, growth and longevity in the common toad, Rhinella arenarum, from Argentina Clarisa de L. Bionda1,2,*, Silvia Kost 4, Nancy E. Salas1, Rafael C. Lajmanovich3, Ulrich Sinsch4, Adolfo L. Martino1 On a putative type specimen of Pleurodema bibroni Tschudi, 1838 from Chile (Anura: Leptodactylidae) Daiana Paola Ferraro Re-description of the external morphology of Phyllomedusa iheringii Boulenger, 1885 larvae (Anura: Hylidae), with comments on the external morphology of tadpoles of the P. burmeisteri group Samanta Iop¹, Victor Mendes Lipinski¹, Bruno Madalozzo¹, Franciele Pereira Maragno¹, Sonia Zanini Cechin¹, Tiago Gomes Dos Santos² Book Review: Harold Heatwole, John W. Wilkinson (Eds). Amphibians Biology. Volume 11 - Status of conservation and decline of Amphibians. Eastern Hemisphere. Part 4 . Southern Europe and Turkey Sebastiano Salvidio Book Review: Antonio Romano. Atlante degli anfibi del Parco Nazionale del Cilento Vallo di Diano e Alburni - Distribuzione, biologia, ecologia e conservazione Sebastiano Salvidio ACTA HERPETOLOGICA Journal of the Societas Herpetologica Italica