Integrating Gene Transcription-Based Biomarkers to Understand Desert Tortoise and Ecosystem Health

Abstract

Tortoises are susceptible to a wide variety of environmental stressors, and the influence of human disturbances on health and survival of tortoises is difficult to detect. As an addition to current diagnostic methods for desert tortoises, we have developed the first leukocyte gene transcription biomarker panel for the desert tortoise (Gopherus agassizii), enhancing the ability to identify specific environmental conditions potentially linked to declining animal health. Blood leukocyte transcript profiles have the potential to identify physiologically stressed animals in lieu of clinical signs. For desert tortoises, the gene transcript profile included a combination of immune or detoxification response genes with the potential to be modified by biological or physical injury and consequently provide information on the type and magnitude of stressors present in the animal's habitat. Blood from 64 wild adult tortoises at three sites in Clark County, NV, and San Bernardino, CA, and from 19 captive tortoises in Clark County, NV, was collected and evaluated for genes indicative of physiological status. Statistical analysis using a priori groupings indicated significant differences among groups for several genes, while multidimensional scaling and cluster analyses of transcription C T values indicated strong differentiation of a large cluster and multiple outlying individual tortoises or small clusters in multidimensional space. These analyses highlight the effectiveness of the gene panel at detecting environmental perturbations as well as providing guidance in determining the health of the desert tortoise.

Chronic disease in the Mojave desert tortoise: Host physiology and recrudescence obscure patterns of pathogen transmission

“…Active management of Mojave desert tortoise populations is becoming increasingly widespread, as it is for other species of reptiles and amphibians that are facing population declines (e.g., Germano, Ewen, Mushinsky, McCoy, & Ortiz-Catedral, 2014). As long-lived animals, adapted to survive extreme variations in their environment, common biological markers of health-such as glucocorticoid levels and gene expression-have proven to be difficult to interpret (Bowen et al, 2015;Drake et al, 2012;O'Connor, Grumbles, George, Zimmerman, & Spotila, 1994). The prevalence of recrudescent, chronic diseases themselves may serve as an integrative biological marker of health in long-lived species and which could prove useful in managing populations in changing environments.…”

Complex immune responses and molecular reactions to pathogens and disease in a desert reptile (Gopherus agassizii)

“…RNA extractions and cDNA synthesis from blood were performed as described by Bowen et al () on each sample collected for gene transcription. PCR primers developed for G. agassizii were used to amplify 11 genes of interest and one ribosomal housekeeping gene within each sample (see Bowen et al, and Drake et al, ). Gene transcription cycle threshold values ( C T ) were measured for the housekeeping gene (18S) and the genes of interest: AHR ‐Arylhydrocarbon Receptor, ATF , CaM ‐Calmodulin, CD9 , CL ‐Cathepsin L, HSP70 ‐Heat Shock Protein 70, Lep ‐Leptin, Mx1 , MyD88 ‐Myeloid Differentiation Factor 88, SAA ‐Serum Amyloid A, and SOD ‐Superoxide dismutase from each sample in duplicate using quantitative PCR (Supporting Information Table ).…”

“…RNA extractions and cDNA synthesis from blood were performed as described by Bowen et al (2015) on each sample collected for gene transcription. PCR primers developed for G. agassizii were used to amplify 11 genes of interest and one ribosomal housekeeping gene within each sample (see Bowen et al, 2015 andDrake et al, 2016). Gene transcription cycle threshold values (C T ) were We analyzed the qPCR gene transcript data using normalized C T values.…”

“…However, given the limited understanding of immunity in reptiles (Zimmerman, Vogel, et al, ) and the additional time and resources needed to elicit an induced response (Sandmeier, Weitzman et al, ), a broader approach may be more appropriate to assess their responses to infection or other stressors. Recent studies using gene transcription profiling have advanced the evaluation of immune function in reptiles (Bowen et al, ; Drake et al, ; Krivoruchko & Storey, ) and other ectotherms (Connon et al, ). Gene transcription works by targeting specific genes that respond to intrinsic (e.g.…”

“…with traditional assays (Brown et al, 2002;Wendland et al, 2007) in captive and wild Mojave desert tortoises over time. We investigated the presence and timing of immune responses after exposure to Myag and whether transcript profiles for genes involved in immune responses to pathogenic microbes (SAA, ATF, CD9, MX1, MyD88;Zhou, Guo, & Dai, 2008;Zhou, Wang, Feng, Guo, & Dai, 2011;Kibenge, Munir, & Kibenge, 2005;Tumpey et al, 2007;Li et al, 2011) and genes often correlated with malnutrition (Lep; Otero et al, 2005) and cellular stress (SOD; Walsh, Leggett, Carter, & Colle, 2010) would be higher in tortoises exposed to Myag and suspected of having bacteria-related infection and disease (Bowen et al, 2015;Drake et al, 2017). Our research asks how molecular biomarkers can improve and expedite diagnosis of immune responses and diseases in tortoises, and advance reptile ecoimmunology.…”