SALAMANDRA 55(3) 160–172 15 AugustGabriela 2019 Ramírez-HernándezISSN 0036–3375 et al. Effects of habitat disturbance on parasite infection and stress of the endangered Mexican stream salamander Ambystoma ordinarium Gabriela Ramírez-Hernández1, Ireri Suazo-Ortuño1, Javier Alvarado-Díaz1, Luis H. Escalera-Vázquez2, Yurixhi Maldonado-López3 & David Tafolla-Venegas4 1) Instituto de Investigaciones sobre los Recursos Naturales, Universidad Michoacana de San Nicolás de Hidalgo, Avenida Juanito Itzícuaro SN, Nueva Esperanza, 58330 Morelia, Michoacán, México 2) Laboratorio de Biología Acuática, Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo, edificio R, planta baja, Ciudad Universitaria, Morelia, Michoacán, México 3) CONACyT-Instituto de Investigaciones sobre los Recursos Naturales, Universidad Michoacana de San Nicolás de Hidalgo, Avenida Juanito Itzícuaro SN, Nueva Esperanza, 58330 Morelia, Michoacán, México 4) Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo. Edificio R, planta baja, Ciudad Universitaria, 58000, Morelia Michoacán, México Corresponding author: Ireri Suazo-Ortuño, e-mail: [email protected] Manuscript received: 1 May 2018 Accepted: 3 April 2019 by Edgar Lehr Abstract. Amphibians are one of the most vulnerable groups of tetrapods with 41% of species considered threatened with extinction. In amphibians, an increase in stress level is an early physiological response to factors associated with habitat degradation. In vertebrates, stress increases the susceptibility to parasite infections and is associated with changes in the number of blood cells, and therefore the proportion of the leukocytes, neutrophils to lymphocytes (N/L ratio) is used as a proxy measure of stress hormones. In this study, we used such leukocyte profiles to analyze stress levels and evaluate para- site load to elucidate the health condition of the endangered Mexican stream salamander, Ambystoma ordinarium. The habitat quality of streams inhabited by A. ordinarium was evaluated by Rapid Bioassessment Protocols (RBPs). We selected three streams with the highest RBPs scores as the undisturbed streams and three with the lowest scores as the disturbed streams. In each stream, we determined leukocyte profile and searched for ecto/endoparasites on sampled individuals. We report for the first time the leukocyte profile of A. ordinarium. A significantly higher N/L ratio was found in salamanders from disturbed vs. undisturbed streams. We also found a higher parasitic infection in salamanders from disturbed streams. Our results suggest that low habitat quality can increase stress levels and susceptibility to parasitic infections, thereby com- promising the long-term persistence of populations of this species in disturbed habitats. Key words. Amphibia, Caudata, Ambystomatidae, health, leukocytes, habitat quality, neutrophils/lymphocytes ratio, am- phibian. Introduction cal response in amphibians (Carey & Bryant 1995, Mar- co 2002, Johnstone et al. 2012). This response is regulated At present, amphibians are considered one of the most by the hypothalamic-pituitary-interrenal axis that actives vulnerable groups of tetrapods to anthropogenic activi- the corticosterone production (Romero 2004). As a con- ties (e.g. Green 2003, Stuart et al. 2004, Baillie et al. sequence, overproduction of reactive oxygen species oc- 2010, Hoffmann et al. 2010, Pimm et al. 2014). According cur in multiple tissues, affecting functions of the organism to IUCN Red List, 41% of amphibian species are threatened such as reproduction, behavior, and growth (Denver et al. with extinction, the highest percentages reported for ter- 2002, Kindermann et al. 2013). restrial vertebrates, followed by 25% of mammals and 13% Many amphibian species that are subjected to chronic of birds. Many threats have been associated to amphibian stress, show detrimental effects such as suppression of im- decline and extinction, such as emergent diseases and cli- mune activity, which might result in an increase of sus- mate change, however habitat change associated with an- ceptibility to parasite and bacterial infections, enhancing thropogenic activities has been identified as the principal the risk of death or decreasing individuals’ performance factor (e.g. Suazo-Ortuño et al. 2008, Becker & Zamu- (Martin et al. 2005, Kiank et al. 2006). This stress re- dio 2011, Hof et al. 2011). Environmental stress associated sponse is associated with an increase in glucocorticoid hor- to anthropogenic disturbances, cause an early physiologi- mones and alterations in the number of leukocytes (Davis © 2019 Deutsche Gesellschaft für Herpetologie und Terrarienkunde e.V. (DGHT), Mannheim, Germany Available160 at http://www.salamandra-journal.com Habitat quality and parasite infection in Ambystoma ordinarium & Maerz 2008a). Specifically, in amphibian species, such Volcanic Belt. This species is restricted to the north-east- as Ambystoma talpoideum, Notophthalmus viridescens and ern parts of the State of Michoacán and western parts of Rana catesbeiana, a rise in glucocorticoids results in an the State of Mexico, at altitudes between 2,200–2,850 m, increase in the number of neutrophils (phagocytic leuko- and inhabits mountain streams in moist pine and fir forests cytes that proliferate in response to infections and stress) (Anderson & Worthington 1971, Weisrock et al. 2006, and a decline in the number of lymphocytes (leukocytes Alvarado-Díaz et al. 2013, Ruiz-Martínez et al. 2014). involved in the modulation of the immune system) in the Ambystoma ordinarium often become the apex predator circulating blood (e.g. Bennett et al. 1972, Dhabhar et in the absence of carnivorous fishes, playing an important al. 1996, Davis et al. 2008, Davis & Maerz 2010). In am- role in trophic cascades and energy flow in these mountain phibians, the ratio of neutrophils to lymphocytes (N/L) has ecosystems (Shaffer 1989). Climatic variables is highly been used as an indirect measure of hormones associat- associated with geographic distribution of A. ordinarium. ed to stress (Davis & Maerz 2008a, b, 2009, Davis et al. Mainly, precipitation and temperature show high correla- 2008) and infection levels (Davis et al. 2004, 1010). It has tion with movement, migration and reproductive activity been suggested that levels in amphibians of N/L close to of Ambystoma species (e.g. Palis 1997). Most suitable habi- 0.30, are typical of unstressed individuals, while an aver- tats are streams with high temporal variation in air temper- age ratio of highly stressed populations is closer or greater ature and precipitation (Escalera-Vázquez et al. 2018). It to 1.0 (Davis & Durso 2009, Davis & Maerz 2011). Addi- has also been reported that the breeding season of A. ordi­ tionally, a high number of eosinophils is associated to the narium takes place between the early rainy season and late defense against metazoan parasites (Kiesecker 2002) and winter, with the highest abundance of juveniles and adults bacterial infections increases the number of circulating from March to May (Anderson & Worthington 1971). monocytes (Turner 1988, Davis et al. 2004). Studies using leukocyte profiles have been frequently used as an indirect measure of health and stress in amphibians, mainly in the Study area United States (e.g. Ussing & Rosenkilde 1995, Davis & Maerz 2008a, b, 2009, 2010, Davis & Durso 2009). We collected Ambystoma ordinarium individuals in six Mexico harbors a high number of microendemic am- mountain streams located in the central part of the trans- phibian species, as a result of the interaction between the Mexican Volcanic Belt in the State of Michoacán within complex topography and climate variety, that creates a an altitudinal range of 2,090 to 2,715 m from the locality very diverse mosaic of environmental and micro-environ- of Cruz de Plato, municipality of Tacámbaro (19°22’07’’ N, mental conditions, where rivers and streams are present 101°22’54.8’’ W) in the west to Carindapaz, municipality (Ochoa-Ochoa & Flores-Villela 2006). Although it of Indaparapeo (19°41’07.5 ’’N, 100°54’28.8’’ W) in the east has been widely recognized that neotropical montane am- (Fig. 1). Riparian vegetation was dominated by Agnus acu­ phibian species associated with streams are at high risk of minate, Fraxinus uhdei, Ilex tolucana and Salix bonpladia­ decline (Beebee & Griffiths 2005), studies that evaluate na, and Pinus, Quercus and Pinus/Quercus forests domi- the physiological status of amphibians in degraded habi- nated the upslope adjacent vegetation. Disturbances of tats in these areas in Mexico are scarce. Only one study on anthropogenic origin include stream bank erosion from Mexican ambystomatids (Ambystoma rivulare) includes trampling by cattle and people, selective logging of ripar- information on blood cell profiles in relation to individu- ian vegetation, dam construction and conversion of up- als’ health (Barriga-Vallejo et al. 2015). slope areas to agricultural activities (Ruiz-Martínez et al. In the present study, we used leukocyte profile and ecto- 2014). endoparasite load to estimate the health condition of popu- lations of the Mexican stream salamander Ambystoma ordi­ narium, cataloged as endangered in the IUCN Red List of Selection of streams according to habitat conservation Endangered Species (IUCN 2015) and under special protec- condition tion (Pr) by Mexican Law (DOF 2010). We compared leu- kocyte profile and parasitic load between populations in- Eleven streams located within the historical distribution habiting