Herpetological Conservation and Biology 10(2):592–601. Submitted: 29 May 2014; Accepted: 6 March 2015; Published: 31 August 2015. ASSESSING POPULATION HEALTH OF THE TOLUCA AXOLOTL AMBYSTOMA RIVULARE (TAYLOR, 1940) FROM MÉXICO, USING LEUKOCYTE PROFILES CARLOS BARRIGA-VALLEJO1,2, OSWALDO HERNÁNDEZ-GALLEGOS2, IONE HUNT VON HERBING3, ANA ESTHELA LÓPEZ-MORENO2, MARÍA DE LOURDES RUIZ-GÓMEZ2, GISELA GRANADOS-GONZALEZ2, MÓNICA VANESA GARDUÑO-PAZ2, JOSÉ FERNANDO MÉNDEZ- SÁNCHEZ2, JAVIER BANDA-LEAL4, AND ANDREW K. DAVIS5,6 1Laboratorio de Ecofisiología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Apartado Postal - 513, C.P. 66450, Nuevo León, México 2Facultad de Ciencias, Universidad Autónoma del Estado de México, Instituto Literario 100, Toluca Centro, C.P. 50000, México 3Department of Biological Sciences, University of North Texas, Denton, Texas 76201 4Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Laboratorio de Herpetología, Apartado Postal # 513, San Nicolás de los Garza, Nuevo León, C.P. C.P. 66450, México 5Odum School of Ecology, University of Georgia, Athens, Georgia, USA 30602 6Corresponding author, e-mail: [email protected] Abstract.—World-wide declines of amphibians have heightened the need for information relating to their health status and immune function under natural conditions. Evaluation of differential white blood cell (leukocyte) counts from thin blood smears is one way to gain this information, and this approach is increasingly being used by herpetologists to gauge the integrity of amphibian populations. This approach is especially useful in natural settings because amphibian leucocyte profiles can vary depending on biological and physiological processes, including those caused by environmental factors. In this study we examined, for the first time, the leukocyte profile of the Toluca Axolotl (Ambystoma rivulare) in Central México, for the purposes of evaluating population health. This species is poorly studied with respect to its life history and biology. We collected 163 individuals over a 6 mo time span from a population in the municipality of Amanalco de Becerra, in the state of México. From microscopic evaluation of blood smears from each individual, we determined the average leukocyte profile of this population to be 77.1% lymphocytes, 7.3% neutrophils, 12.1% eosinophils 2.3% basophils, and 1.2% monocytes. These values are similar to that reported for other members of this genus, so these counts could be considered normal. Moreover, the average ratio of neutrophils to lymphocytes (N/L ratio), which reflects stress levels in vertebrates, appeared especially low in this population compared to other Ambystomatid salamanders. Relative numbers of all cell types varied by month and there were also gender differences in the density of certain cells. Key Words.—amphibians; conservation; N/L ratios; physiology INTRODUCTION Counts of the number of circulating immune cells (white blood cells, or leukocytes) are especially useful In many zoological disciplines, including herpetology, for gauging the immune status of animals, and are researchers are often tasked with conducting assessments increasingly being used by researchers in a variety of of animal populations that are at risk or declining. While conservation and/or research settings (Rohr et al. 2008; assessment of population size or animal abundance can Shutler and Marcogliese 2011; Peterson et al. 2013; Das be useful for this task, a secondary approach is to and Mahapatra 2014). In these situations the numbers of conduct non-invasive hematological assessments of each of the five white cell types (the leukocyte profile) animals in the population, which can provide are typically ascertained from microscopic examination information on the health state of animals. In all of thin blood films. Each cell type performs a different vertebrates, blood is responsible for important bodily function in the innate immune system. Neutrophils are functions like immune response, nutrient transport, the first phagocytic leukocytes that act and proliferate in transport of gases and metabolic wastes (Solis et al. the circulation in response to infections, inflammations, 2007; Álvarez-Mendoza et al. 2011). Thus, any and stress (Jain 1993; Thrall et al. 2006; Davis et al. alteration of these parameters can indicate potential 2008). Lymphocytes are involved in several health problems in the animal, and by extension, immunological functions such as the production of problems with the surrounding environment. Copyright © 2015.Carlos Barriga-Vallejo 592 All Rights Reserved. Barriga-Vallejo et al.—Leucocyte profiles of Ambystoma rivulare. FIGURE 1. Toluca Axolotl (Ambystoma rivulare) from the study site in Mexico. (Photographed by Oswaldo Hernández-Gallegos). immunoglobulin and modulation of the immune defense. distributed from southern Canada and Alaska into the Eosinophils act in inflammatory processes and are Trans-Mexicana volcanic belt. Nineteen species of the associated with defense against metazoan parasites. genus Ambystoma are only found in Mexico, and most of Monocytes are associated with defense against infections these are poorly studied with respect to their life history, and bacteria. Finally, the exact role of basophils is not biology, and physiology (Bille 2009; Parra-Olea et al. yet clarified, but they are known to be implicated in the 2012). inflammation process (Jain 1993; Thrall et al. 2006). The Toluca Axolotl, Ambystoma rivulare (Fig. 1), is Counts of specific leukocyte types present in distributed in the states of México, Michoacán, and circulation can also provide information concerning the Guerrero (in the country of México), and it is a species overall stress levels of vertebrate animals (reviewed in defined by the IUCN Red List as Data-Deficient (Shaffer Davis et al. 2008). This stems from the unique et al. 2008). It is considered microendemic because it is physiological effect of elevated stress hormones on only found above 2,800 m elevation, mainly in the lotic certain subsets of the leukocyte population. Specifically, ecosystems within Pine, Pine-Oyamel, and Pine-Oak increases in stress hormones cause neutrophils to forests (Huacuz 2001; Casas-Andreu et al. 2004). In a increase in circulation while lymphocytes tend to recent report by the Secretaria del Medio Ambiente y decline, leading to a rise in the ratio of these cells Recusos Naturales of México (Secretary of Environment (neutrophil-to-lymphocyte, or N/L ratio). While there and Natural Resources), its status was listed as are aspects of this relationship that remain unclear, this threatened (SEMARNAT 2010). In this study we metric has been demonstrated to depict elevated stress conducted a hematologic characterization (i.e., leukocyte levels in a variety of animals, including amphibians, assessment) of a population of Ambystoma rivulare under laboratory and field conditions (Davis and Maerz occurring in the Los Hoyos stream, within the Corral de 2008b, 2009; Davis and Maerz 2010; Davis and Maerz Piedra watershed, which is located in the municipality of 2011; Shutler and Marcogliese 2011), making it a useful Amanalco de Becerra in the state of México. This study tool for population health assessments. represents the first description of leukocyte profiles of Health assessment of amphibian populations are this species, and these data should provide useful becoming increasingly important because of global-scale information for evaluating the health status of this declines in their populations due largely to diseases and population. habitat degradation (Stuart et al. 2004). In fact some species are declining even before they can be fully MATERIALS AND METHODS studied. Many species of salamanders within the Ambystomatidae family are examples of this. This Study site.—The study was conducted in the Los family has two genera and 31 species, and these are Hoyos stream, which is located in the basin of the 593 Herpetological Conservation and Biology FIGURE 2. Map of the study site for the Toluca Axolotl (Ambystoma rivulare) where we collected salamanders. The study site was within the "Los Hoyos" stream, which is located in the Corral de Piedra Ranch property, Amanalco Becerra, State of Mexico. Balsas, within the Corral de Piedra watershed (Fig. 2). sampling for all individuals. The average time from This watershed is west of the state of México in the capture to blood sampling of the salamanders for the municipality of Amanalco de Becerra between project was 55 min. We released all salamanders at the coordinates 19°10´29´´N and 99°53´27´´W. This area site of capture. represents the highest point of the natural Los Hoyos stream (Salinas 1999). At this site the average annual Blood smear analysis.—In the lab, we examined all temperature is 13.4° C and the mean annual precipitation blood films with a light microscope to obtain a is 1,200 mm. The site where we collected most of the differential leukocyte count for each salamander. We salamanders was in the Los Encinos trout-farm drainage identified leukocytes based on morphological description system (Fig. 2). given in other studies (e.g., Friedmann 1970; Davis and Durso 2009; Davis and Maerz 2010; Das and Mahapatra Capturing salamanders.—We sampled between the 2014). Counting was performed by a single person to months of October 2011 and May 2012, capturing avoid observer variation, using 400 × magnification, and between 20 and 30 individuals per month (except April). the slide was moved in