Assessing spatial distribution, genetic variants, and virulence of pathogen Mycoplasma agassizii in threatened Mojave desert tortoises.

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.

Potential Facilitation Between a Commensal and a Pathogenic Microbe in a Wildlife Disease.

We assessed the potential for microbial interactions influencing a well-documented host-pathogen system. Mycoplasma agassizii is the known etiological agent of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii), but disease in wild animals is extremely heterogeneous. For example, a much larger proportion of animals harbor M. agassizii than those that develop disease. With the availability of a new quantitative PCR assay for a microbe that had previously been implicated in disease, Pasteurella testudinis, we tested 389 previously collected samples of nasal microbes from tortoise populations across the Mojave desert. We showed that P. testudinis is a common commensal microbe. However, we did find that its presence was associated with higher levels of M. agassizii among the tortoises positive for this pathogen. The best predictor of P. testudinis prevalence in tortoise populations was average size of tortoises, suggesting that older populations have higher levels of P. testudinis. The prevalence of co-infection in populations was associated with the prevalence of URTD, providing additional evidence for an indirect interaction between the two microbes and inflammatory disease. We showed that URTD, like many chronic, polymicrobial diseases involving mucosal surfaces, shows patterns of a polymicrobial etiology.

Physiological control of water exchange in anurans.

Research on water exchange in frogs has historically assumed that blood osmotic potential drives water exchange between a frog and its environment, but here we show that the "seat patch" (the primary site of water exchange in many anurans), or other sites of cutaneous water uptake, act as an anatomic "compartment" with a water potential controlled separately from water potential of the blood, and the water potential of that compartment can be the driver of water exchange between the animal and its environment. We studied six frog species (Xenopus laevis, Rana pipiens, R. catesbeiana, Bufo boreas, Pseudacris cadaverina, and P. regilla) differing in ecological relationships to environmental water. We inferred the water potentials of seat patches from water exchanges by frogs in sucrose solutions ranging in water potential from 0 to 1000-kPa. Terrestrial and arboreal species had seat patch water potentials that were more negative than the water potentials of more aquatic species, and their seat patch water potentials were similar to the water potential of their blood, but the water potentials of venters of the more aquatic species were different from (and less negative than) the water potentials of their blood. These findings indicate that there are physiological mechanisms among frog species that can be used to control water potential at the sites of cutaneous water uptake, and that some frogs may be able to adjust the hydric conductance of their skin when they are absorbing water from very dilute solutions. Largely unexplored mechanisms involving aquaporins are likely responsible for adjustments in hydric conductance, which in turn, allow control of water potential at sites of cutaneous water uptake among species differing in ecological habit and the observed disequilibrium between sites of cutaneous water uptake and blood water potential in more aquatic species.

Mycoplasma agassizii, an opportunistic pathogen of tortoises, shows very little genetic variation across the Mojave and Sonoran Deserts.

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises.

Detecting trends in body size: empirical and statistical requirements for intraspecific analyses.

Attributing biological explanations to observed ecogeographical and ecological patterns require eliminating potential statistical and sampling artifacts as alternative explanations of the observed patterns. Here, we assess the role of sample size, statistical power, and geographic inclusivity on the general validity and statistical significance of relationships between body size and latitude for 3 well-studied species of turtles. We extend those analyses to emphasize the importance of using statistically robust data in determining macroecological patterns. We examined intraspecific trends in body size with latitude in Chelydra serpentina, Chrysemys picta, and Trachemys scripta using Pearson's correlations, diagnostic tests for influential points, and resampling. Existing data were insufficient to ascertain a latitudinal trend in body size for C. serpentina or T. scripta. There was a significant relationship for C. picta, however, resampling analyses show that, on average, 16 of the 23 available independent populations were needed to demonstrate a significant relationship and that at least 20 of 23 populations were required to obtain a statistically powerful correlation between body size and latitude. Furthermore, restricting the latitudes of populations resampled shows that body size trends of C. picta were largely due to leveraging effects of populations at the edge of the species range. Our results suggest that broad inferences regarding ecological trends in body size should be made with caution until underlying (intraspecific) patterns in body size can be statistically and conclusively demonstrated.

Host species, pathogens and disease associated with divergent nasal microbial communities in tortoises.

Diverse bacterial communities are found on every surface of macro-organisms, and they play important roles in maintaining normal physiological functions in their hosts. While the study of microbiomes has expanded with the influx of data enabled by recent technological advances, microbiome research in reptiles lags behind other organisms. We sequenced the nasal microbiomes in a sample of four North American tortoise species, and we found differing community compositions among tortoise species and sampling sites, with higher richness and diversity in Texas and Sonoran desert tortoises. Using these data, we investigated the prevalence and operational taxonomic unit (OTU) diversity of the potential pathogen Pasteurella testudinis and found it to be common, abundant and highly diverse. However, the presence of this bacterium was not associated with differences in bacterial community composition within host species. We also found that the presence of nasal discharge from tortoises at the time of sampling was associated with a decline in diversity and a change in microbiome composition, which we posit is due to the harsh epithelial environment associated with immune responses. Repeated sampling across seasons, and at different points of pathogen colonization, should contribute to our understanding of the causes and consequences of different bacterial communities in these long-lived hosts.

High quality draft genome sequences of Mycoplasma agassizii strains PS6T and 723 isolated from Gopherus tortoises with upper respiratory tract disease.

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.

High quality draft genome sequence of Mycoplasma testudineum strain BH29T, isolated from the respiratory tract of a desert tortoise.

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.

Co-infection does not predict disease signs in Gopherus tortoises.

In disease ecology, the host immune system interacts with environmental conditions and pathogen properties to affect the impact of disease on the host. Within the host, pathogens may interact to facilitate or inhibit each other's growth, and pathogens interact with different hosts differently. We investigated co-infection of two Mycoplasma and the association of infection with clinical signs of upper respiratory tract disease in four congeneric tortoise host species (Gopherus) in the United States to detect differences in infection risk and disease dynamics in these hosts. Mojave Desert tortoises had greater prevalence of Mycoplasma agassizii than Texas tortoises and gopher tortoises, while there were no differences in Mycoplasma testudineum prevalence among host species. In some host species, the presence of each pathogen influenced the infection intensity of the other; hence, these two mycoplasmas interact differently within different hosts, and our results may indicate facilitation of these bacteria. Neither infection nor co-infection was associated with clinical signs of disease, which tend to fluctuate across time. From M. agassizii DNA sequences, we detected no meaningful differentiation of haplotypes among hosts. Experimental inoculation studies and recurrent resampling of wild individuals could help to decipher the underlying mechanisms of disease dynamics in this system.

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

A seminatural, factorial-design experiment was used to quantify dynamics of the pathogen Mycoplasma agassizii and upper respiratory tract disease in the Mojave desert tortoise (Gopherus agassizii) over 2 years. Groups of initially healthy animals were separated into serologically positive (seropositive), seronegative, and artificially infected groups and paired into 23 pens. We found no evidence of long-term immune protection to M. agassizii or of immunological memory. Initially seronegative, healthy tortoises experienced an equal amount of disease when paired with other seronegative groups as when paired with seropositive and artificially infected groups-suggesting that recrudescence is as significant as transmission in introducing disease in individuals in this host-pathogen system. Artificially infected groups of tortoises showed reduced levels of morbidity when paired with initially seronegative animals-suggesting either a dilution effect or a strong effect of pathogen load in this system. Physiological dynamics within the host appear to be instrumental in producing morbidity, recrudescence, and infectiousness, and thus of population-level dynamics. We suggest new avenues for studying diseases in long-lived ectothermic vertebrates and a shift in modeling such diseases.

COMPARISON OF CURRENT METHODS FOR THE DETECTION OF CHRONIC MYCOPLASMAL URTD IN WILD POPULATIONS OF THE MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII).

Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.

Mycoplasmal upper respiratory tract disease across the range of the threatened Mojave Desert tortoise: associations with thermal regime and natural antibodies.

Most research of upper respiratory tract disease (mycoplasmal URTD) in the threatened Mojave Desert tortoise (Gopherus agassizii) has worked under the hypothesis that the pathogen, Mycoplasma agassizii, has a relatively consistent and predictable effect on tortoise populations across their natural range. In contrast, we hypothesized that multiple factors influence the prevalence of disease and analyzed biological and environmental variables that vary significantly across the Mojave Desert. We used multiple regression models to analyze associations between mycoplasmal URTD and the genetic structure of 24 tortoise populations, levels of natural antibody (NAb) to M. agassizii in tortoises (one component of the innate immune system), precipitation, and colder thermal regimes. We detected a significant, positive association between mean levels of NAb and seroprevalence to M. agassizii. We hypothesized that NAbs may provide tolerance to mycoplasmal infections and that more tolerant populations may act as host reservoirs of disease. We also detected significant associations between colder winters and mycoplasmal URTD, suggesting that colder winters may depress tortoise immune resistance against M. agassizii or enhance conditions for the growth of M. agassizii.

A trade-off between natural and acquired antibody production in a reptile: implications for long-term resistance to disease.

Vertebrate immune systems are understood to be complex and dynamic, with trade-offs among different physiological components (e.g., innate and adaptive immunity) within individuals and among taxonomic lineages. Desert tortoises (Gopherus agassizii) immunised with ovalbumin (OVA) showed a clear trade-off between levels of natural antibodies (NAbs; innate immune function) and the production of acquired antibodies (adaptive immune function). Once initiated, acquired antibody responses included a long-term elevation in antibodies persisting for more than one year. The occurrence of either (a) high levels of NAbs or (b) long-term elevations of acquired antibodies in individual tortoises suggests that long-term humoral resistance to pathogens may be especially important in this species, as well as in other vertebrates with slow metabolic rates, concomitantly slow primary adaptive immune responses, and long life-spans.

Not just small, wet, and cold: effects of body size and skin resistance on thermoregulation and arboreality of frogs.

We used simulations from a biophysical model that integrates interlinked exchanges of energy and water between frogs and their environments to address questions about the limits to thermoregulation and about adaptations for arboreality. Body size and cutaneous resistance (Rc) both significantly affected body temperature (Tb) and the time to desiccate to 70% of standard mass (an ecologically relevant metric of desiccation). Cutaneous resistances < 25 s/cm allow basking frogs to elevate their Tb several degrees above ambient, but Rc above 25 had little additional effect on Tb. Small frogs (<10 g) are able to elevate their Tb above ambient while basking, even with small Rc. Large frogs must have greater skin resistances to be able to elevate body temperatures above ambient, yet large frogs take longer to desiccate to 70% of their standard mass. Frogs can avoid rapid desiccation with high Rc, a large body size, or some combination of these traits. Our literature survey indicates that frogs with a combination of Rc and body size that would result in long times to desiccate to 70% of standard mass tend to be arboreal, suggesting that those species may be selectively favored in a niche that often requires frogs to be away from water sources for extended periods of time.

Short-term soil inorganic N pulse after experimental fire alters invasive and native annual plant production in a Mojave Desert shrubland.

Post-fire changes in desert vegetation patterns are known, but the mechanisms are poorly understood. Theory suggests that pulse dynamics of resource availability confer advantages to invasive annual species, and that pulse timing can influence survival and competition among species. Precipitation patterns in the American Southwest are predicted to shift toward a drier climate, potentially altering post-fire resource availability and consequent vegetation dynamics. We quantified post-fire inorganic N dynamics and determined how annual plants respond to soil inorganic nitrogen variability following experimental fires in a Mojave Desert shrub community. Soil inorganic N, soil net N mineralization, and production of annual plants were measured beneath shrubs and in interspaces during 6 months following fire. Soil inorganic N pools in burned plots were up to 1 g m(-2) greater than unburned plots for several weeks and increased under shrubs (0.5-1.0 g m(-2)) more than interspaces (0.1-0.2 g m(-2)). Soil NO(3) (-)-N (nitrate-N) increased more and persisted longer than soil NH(4) (+)-N (ammonium-N). Laboratory incubations simulating low soil moisture conditions, and consistent with field moisture during the study, suggest that soil net ammonification and net nitrification were low and mostly unaffected by shrub canopy or burning. After late season rains, and where soil inorganic N pools were elevated after fire, productivity of the predominant invasive Schismus spp. increased and native annuals declined. Results suggest that increased N availability following wildfire can favor invasive annuals over natives. Whether the short-term success of invasive species following fire will direct long-term species composition changes remains to be seen, yet predicted changes in precipitation variability will likely interact with N cycling to affect invasive annual plant dominance following wildfire.

Clutch size determination in shorebirds: revisiting incubation limitation in the pied avocet (Recurvirostra avosetta).

1. Traits strongly related to fitness, such as offspring number, are expected to show intraspecific variation among individuals. However, offspring number is invariant in several reptiles, birds, and mammals. Most shorebirds (210+ species), for example, have an invariant clutch size of four eggs, which is unexpected in such an ecologically, behaviourally and socially diverse group. 2. The incubation-limitation hypothesis (ILH) suggests that shorebird clutch size is limited by the inability of adults to incubate clutches larger than four eggs. Several recent studies reported no overall costs of incubating experimentally enlarged clutches and concluded no support for the traditional ILH. However, most studies have not measured all potential costs, and none has quantified costs beyond egg hatching. We conducted a clutch-enlargement experiment and measured potential costs both during incubation and chick rearing in pied avocets (Recurvirostra avosetta L.). 3. Hatching was more asynchronous and egg hatchability was marginally lower in enlarged clutches than in controls. Nonetheless, more young hatched from enlarged clutches (mean: 4.2 +/- 0.17 SE) than from controls (3.4 +/- 0.09), and the two groups did not differ in incubation period, complete or partial clutch failure, or hatchling body size, apparently refuting the ILH. 4. However, pairs incubating enlarged clutches occupied poorer feeding territories during chick rearing, experienced higher chick mortality, and eventually raised fewer young to independence (mean adjusted for season: 0.7 +/- 0.16 SE juveniles) than did control pairs (1.2 +/- 0.13). Chick survival was primarily associated with prey availability, and predation risks were not higher in larger broods. 5. Our results provide evidence that incubating unusually large clutches can affect post-hatching performance and lead to lower annual reproductive success in shorebirds. This study, therefore, supports the ILH and points to the importance of monitoring reproductive success beyond the hatching of the chicks.

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii).

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.