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Simple Summary: This commentary focuses on experimental techniques used by researchers to understand how the immune system of an animal can acclimate, or change, to function across different body temperatures. I categorized studies as using three different, broad techniques: (1) allowing animal body temperatures to change naturally; (2) manipulating body temperatures of live animals in a controlled environment; and (3) manipulating temperatures of immune components (plasma, cells, etc.) in laboratory assays. I reviewed how the current literature used these techniques, with the conclusion that combinations of the three different techniques increased a mechanistic understanding of acclimation. In particular, cell-based techniques seem under-utilized and can lead to a greater understanding of how an animal may be changing the numbers of cells in their bodies or changing the cells themselves to function across different temperatures. This short review focuses on current experimental designs to quantify immune acclimation in animals. Especially in the face of rapidly changing thermal regimes, thermal acclimation of immune function has the potential to impact host–pathogen relationships and the fitness of hosts. While much of the field of ecoimmunology has focused on vertebrates and insects, broad interest in how animals can acclimate to temperatures spans taxa. The literature shows a recent increase in thermal acclimation studies in the past six years. I categorized studies as focusing on (1) natural thermal variation in the environment (e.g., seasonal), (2) in vivo manipulation of animals in captive conditions, and (3) in vitro assays using biological samples taken from wild or captive animals. I detail the strengths and weaknesses of these approaches, with an emphasis on mechanisms of acclimation at different levels of organization (organismal and cellular). These two mechanisms are not mutually exclusive, and a greater combination of the three techniques listed above will increase our knowledge of the diversity of mechanisms used by animals to acclimate to changing thermal regimes. Finally, I suggest that functional assays of immune system cells (such as quantification of phagocytosis) are an accessible and non-taxa-specific way to tease apart the effects of animals upregulating quantities of immune effectors (cells) and changes in the function of immune effectors (cellular performance) due to structural changes in cells such as those of membranes and enzymes. [ABSTRACT FROM AUTHOR]

Mojave desert tortoises (Gopherus agassizii), a threatened species under the US Endangered Species Act, are long‐lived reptiles that experience a chronic respiratory disease. The virulence of primary etiologic agent, Mycoplasma agassizii, remains poorly understood, but it exhibits temporal and geographic variability in causing disease outbreaks in host tortoises. Multiple attempts to culture and characterize the diversity of M. agassizii have had minimal success, even though this opportunistic pathogen chronically persists in nearly every population of Mojave desert tortoises. The current geographic range and the molecular mechanisms of virulence of the type‐strain, PS6T, are unknown, and the bacterium is thought to have low‐to‐moderate virulence. We designed a quantitative polymerase chain reaction (qPCR) targeting three putative virulence genes annotated on the PS6T genome as exo‐α‐sialidases, enzymes which facilitate growth in many bacterial pathogens. We tested 140 M. agassizii‐positive DNA samples collected from 2010 to 2012 across the range of Mojave desert tortoises. We found evidence of multiple‐strain infections within hosts. We also found the prevalence of these sialidase‐encoding genes to be highest in tortoise populations surrounding southern Nevada, the area from which PS6T was originally isolated. We found a general pattern of loss or reduced presence of sialidase among strains, even within a single host. However, in samples that were positive for any of the putative sialidase genes, one particular gene (528), was positively associated with bacterial loads of M. agassizii and may act as a growth factor for the bacterium. Our results suggest three evolutionary patterns: (1) high levels of variation, possibly due to neutral changes and chronic persistence, (2) a trade‐off between moderate virulence and transmission, and (3) selection against virulence in environmental conditions known to be physiologically stressful to the host. Our approach of quantifying genetic variation via qPCR represents a useful model of studying host–pathogen dynamics. [ABSTRACT FROM AUTHOR]

Many measures of immune function used in ecoimmunological studies were optimized for endotherms and may not adequately characterize immunocompetence in ectothermic species. Lymphocytes are interpreted as functioning in adaptive immunity despite evidence that large proportions of these cells have innate immune functions, including phagocytosis, in ectotherms.We modified previously published protocols to remove many barriers that inhibited the wide application of assays to quantify phagocytic activity in lymphocytes. These barriers included the use of species‐specific reagents, high cost and incompatibility with field work. We statistically compared published methods to our simplified technique using blood samples collected from tortoises.We show the efficacy of the new technique and offer an outline for optimizing these methods for different species and to address a wide variety of ecoimmunological questions.A full understanding of immune function in ectothermic vertebrates is needed to comprehend complex epidemiological patterns. As our assay disentangles the relationship between effector cells (lymphocytes) and their function (phagocytosis), these methods provide a foundation for more thorough analyses of ecoimmunological patterns. [ABSTRACT FROM AUTHOR]

Mycoplasma agassizii is one of the known causative agents of upper respiratory tract disease (URTD) in Mojave desert tortoises (Gopherus agassizii) and in gopher tortoises (Gopherus polyphemus). We sequenced the genomes of M. agassizii strains PS6T (ATCC 700616) and 723 (ATCC 700617) isolated from the upper respiratory tract of a Mojave desert tortoise and a gopher tortoise, respectively, both with signs of URTD. The PS6T genome assembly was organized in eight scaffolds, had a total length of 1,274,972 bp, a G + C content of 28.43%, and contained 979 protein-coding genes, 13 pseudogenes and 35 RNA genes. The 723 genome assembly was organized in 40 scaffolds, had a total length of 1,211,209 bp, a G + C content of 28.34%, and contained 955 protein-coding genes, seven pseudogenes, and 35 RNA genes. Both genomes exhibit a very similar organization and very similar numbers of genes in each functional category. Pairs of orthologous genes encode proteins that are 93.57% identical on average. Homology searches identified a putative cytadhesin. These genomes will enable studies that will help understand the molecular bases of pathogenicity of this and other Mycoplasma species. [ABSTRACT FROM AUTHOR]

Mycoplasma testudineum is one of the pathogens that can cause upper respiratory tract disease in desert tortoises, Gopherus agassizii. We sequenced the genome of M. testudineum BH29T (ATCC 700618T = MCCM 03231T), isolated from the upper respiratory tract of a Mojave desert tortoise with upper respiratory tract disease. The sequenced draft genome, organized in 25 scaffolds, has a length of 960,895 bp and a G + C content of 27.54%. A total of 788 protein-coding sequences, six pseudogenes and 35 RNA genes were identified. The potential presence of cytadhesin-encoding genes is investigated. This genome will enable comparative genomic studies to help understand the molecular bases of the pathogenicity of this and other Mycoplasma species. [ABSTRACT FROM AUTHOR]