Is CpG Density the Link between Epigenetic Aging and Lifespan?

The genetic mechanisms contributing to lifespan variation remain unresolved. Based on recent conceptual advances in our understanding of epigenetic potential and the relocalization of chromatin modifiers (RCM), we hypothesize that increased CpG density is protective against age-related erosion of the epigenetic landscape and may explain interspecific variation in lifespan.

Maternal provisioning interacts with incubation temperature to affect hatchling mercury exposure in an oviparous reptile.

The thermal environment experienced by developing embryos can influence the utilization of maternally provisioned resources. Despite being particularly consequential for oviparous ectotherms, these dynamics are largely unexplored within ecotoxicological frameworks. Here, we test if incubation temperature interacts with maternally transferred mercury to affect subsequent body burdens and tissue distributions of mercury in hatchling American alligators (Alligator mississippiensis). Nine clutches of alligator eggs were collected from a mercury-contaminated reservoir and incubated at either female- or male-promoting temperatures. Total mercury (THg) concentration was measured in egg yolk collected during incubation and in a suite of tissues collected from hatchlings. THg concentrations in residual yolk and blood were higher in hatchlings incubated at cooler, female-promoting temperatures compared to the warmer, male-promoting temperatures. THg concentrations in most tissues were positively correlated with THg concentrations in blood and dermis, and egg yolk THg concentration was the best predictor of THg concentration in many resultant tissues. Our results highlight a hereto unknown role of the developmental environment in mediating tissue specific uptake of contaminants in an oviparous reptile.

Genome-wide DNA methylation patterns harbour signatures of hatchling sex and past incubation temperature in a species with environmental sex determination.

Conservation of thermally sensitive species depends on monitoring organismal and population-level responses to environmental change in real time. Epigenetic processes are increasingly recognized as key integrators of environmental conditions into developmentally plastic responses, and attendant epigenomic data sets hold potential for revealing cryptic phenotypes relevant to conservation efforts. Here, we demonstrate the utility of genome-wide DNA methylation (DNAm) patterns in the face of climate change for a group of especially vulnerable species, those with temperature-dependent sex determination (TSD). Due to their reliance on thermal cues during development to determine sexual fate, contemporary shifts in temperature are predicted to skew offspring sex ratios and ultimately destabilize sensitive populations. Using reduced-representation bisulphite sequencing, we profiled the DNA methylome in blood cells of hatchling American alligators (Alligator mississippiensis), a TSD species lacking reliable markers of sexual dimorphism in early life stages. We identified 120 sex-associated differentially methylated cytosines (DMCs; FDR < 0.1) in hatchlings incubated under a range of temperatures, as well as 707 unique temperature-associated DMCs. We further developed DNAm-based models capable of predicting hatchling sex with 100% accuracy (in 20 training samples and four test samples) and past incubation temperature with a mean absolute error of 1.2°C (in four test samples) based on the methylation status of 20 and 24 loci, respectively. Though largely independent of epigenomic patterning occurring in the embryonic gonad during TSD, DNAm patterns in blood cells may serve as nonlethal markers of hatchling sex and past incubation conditions in conservation applications. These findings also raise intriguing questions regarding tissue-specific epigenomic patterning in the context of developmental plasticity.

Intrinsic and extrinsic factors interact during development to influence telomere length in a long-lived reptile.

The mechanisms connecting environmental conditions to plasticity in biological aging trajectories are fundamental to understanding individual variation in functional traits and life history. Recent findings suggest that telomere biology is especially dynamic during early life stages and has long-term consequences for subsequent reproduction and survival. However, our current understanding is mostly derived from studies investigating ecological and anthropogenic factors separately, leaving the effects of complex environmental interactions unresolved. American alligators (Alligator mississippiensis) are long-lived apex predators that rely on incubation temperature during a discrete period during development and endocrine cues to determine sex, making them especially vulnerable to current climatic variability and exposure to anthropogenic contaminants interfering with hormone function. Here, we combine field studies with a factorial design to understand how the developmental environment, along with intrinsic biological variation contribute to persistent telomere variation. We found that exposure to a common endocrine disrupting contaminant, DDE, affects telomere length, but that the directionality is highly dependent upon incubation temperature. Variation in hatchling growth, underlies a strong clutch effect. We also assess concentrations of a panel of glucocorticoid hormones and find that contaminant exposure elicits an increase in circulating glucocorticoids. Consistent with emerging evidence linking stress and aging trajectories, GC levels also appear to trend with shorter telomere length. Thus, we add support for a mechanistic link between contaminants and glucocorticoid signalling, which interacts with ecological aspects of the developmental environment to alter telomere dynamics.

Spatial and temporal variation in nest temperatures forecasts sex ratio skews in a crocodilian with environmental sex determination.

Species displaying temperature-dependent sex determination (TSD) are especially vulnerable to the effects of a rapidly changing global climate due to their profound sensitivity to thermal cues during development. Predicting the consequences of climate change for these species, including skewed offspring sex ratios, depends on understanding how climatic factors interface with features of maternal nesting behaviour to shape the developmental environment. Here, we measure thermal profiles in 86 nests at two geographically distinct sites in the northern and southern regions of the American alligator's (Alligator mississippiensis) geographical range, and examine the influence of both climatic factors and maternally driven nest characteristics on nest temperature variation. Changes in daily maximum air temperatures drive annual trends in nest temperatures, while variation in individual nest temperatures is also related to local habitat factors and microclimate characteristics. Without any compensatory nesting behaviours, nest temperatures are projected to increase by 1.6-3.7°C by the year 2100, and these changes are predicted to have dramatic consequences for offspring sex ratios. Exact sex ratio outcomes vary widely depending on site and emission scenario as a function of the unique temperature-by-sex reaction norm exhibited by all crocodilians. By revealing the ecological drivers of nest temperature variation in the American alligator, this study provides important insights into the potential consequences of climate change for crocodilian species, many of which are already threatened by extinction.

Embryonic estrogen exposure recapitulates persistent ovarian transcriptional programs in a model of environmental endocrine disruption.

Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL, are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a nonaromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses; however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.

Assessing impacts of precocious steroid exposure on thyroid physiology and gene expression patterns in the American alligator (Alligator mississippiensis).

The thyroid gland is sensitive to steroid hormone signaling, and many thyroid disrupting contaminants also disrupt steroid hormone homeostasis, presenting the possibility that thyroid disruption may occur through altered steroid hormone signaling. To examine this possibility, we studied short-term and persistent impacts of embryonic sex steroid exposure on thyroid physiology in the American alligator. Alligators from a lake contaminated with endocrine disrupting contaminants (Lake Apopka, FL, USA) have been shown to display characteristics of thyroid and steroid hormone disruption. Previous studies suggest these alterations arise during development and raise the possibility that exposure to maternally deposited contaminants might underlie persistent organizational changes in both thyroidal and reproductive function. Thus, this population provides a system to investigate contaminant-mediated organizational thyroid disruption in an environmentally-relevant context. We assess the developmental expression of genetic pathways involved in thyroid hormone biosynthesis and find that expression of these genes increases prior to hatching. Further, we show that nuclear steroid hormone receptors are also expressed during this period, indicating the developing thyroid is potentially responsive to steroid hormone signaling. We then explore functional roles of steroid signaling during development on subsequent thyroid function in juvenile alligators. We exposed alligator eggs collected from both Lake Apopka and a reference site to 17ß-estradiol and a non-aromatizable androgen during embryonic development, and investigated effects of exposure on hatchling morphometrics and thyroidal gene expression profiles at 5 months of age. Steroid hormone treatment did not impact the timing of hatching or hatchling size. Furthermore, treatment with steroid hormones did not result in detectable impacts on thyroid transcriptional programs, suggesting that precocious or excess estrogen and androgen exposure does not influence immediate or long-term thyroidal physiology.

Analysis of PFAAs in American alligators part 1: Concentrations in alligators harvested for consumption during South Carolina public hunts.

Environmental contamination resulting from the production or release of harmful chemicals can lead to negative consequences for wildlife and human health. Perfluorinated alkyl acids (PFAAs) were historically produced as protective coatings for many household items and currently persist in the environment, wildlife, and humans. PFAAs have been linked to immune suppression, endocrine disruption, and developmental toxicity in wildlife and laboratory studies. This study examines the American alligator, Alligator mississippiensis, as an important indicator of ecosystem contamination and a potential pathway for PFAA exposure in humans. Alligator meat harvested in the 2015 South Carolina (SC) public hunt season and prepared for human consumption was collected and analyzed for PFAAs to determine meat concentrations and relationships with animal body size (total length), sex, and location of harvest. Of the 15 PFAAs analyzed, perfluorooctane sulfonate (PFOS) was found in all alligator meat samples and at the highest concentrations (median 6.73ng/g). No relationship was found between PFAA concentrations and total length or sex. Concentrations of one or all compounds varied significantly across sampling locations, with alligators harvested in the Middle Coastal hunt unit having the highest PFOS concentrations (median 16.0ng/g; p=0.0001). Alligators harvested specifically from Berkley County, SC (located in the Middle Coastal hunt unit) had the highest PFOS concentrations and the greatest number of PFAAs detected (p<0.0001). The site-specific nature of PFAA concentrations in alligator meat observed in this study suggests a source of PFAA contamination in Berkley County, SC.

Analysis of PFAAs in American alligators part 2: Potential dietary exposure of South Carolina hunters from recreationally harvested alligator meat.

Exposure to perfluorinated alkyl acids (PFAAs) has been linked to many harmful health effects including reproductive disorders, developmental delays, and altered liver and kidney function. Most human exposure to environmental contaminants, including PFAAs, occurs through consumption of contaminated food or drinking water. This study uses PFAA data from meat samples collected from recreationally harvested American alligators (Alligator mississippiensis) in South Carolina to assess potential dietary exposure of hunters and their families to PFAAs. Consumption patterns were investigated using intercept surveys of 23 hunters at a wild game meat processor. An exposure scenario using the average consumption frequency, portion size, and median perfluorooctane sulfonic acid (PFOS) concentration in alligator meat from all hunt units found the daily dietary exposure to be 2.11ng/kg body weight per day for an adult human. Dietary PFOS exposure scenarios based on location of harvest suggested the highest daily exposure occurs with alligator meat from the Middle Coastal hunt unit in South Carolina. Although no samples were found to exceed the recommended threshold for no consumption of PFOS found in Minnesota state guidelines, exposure to a mixture of PFAAs found in alligator meat and site-specific exposures based on harvest location should be considered in determining an appropriate guideline for vulnerable populations potentially exposed to PFAAs through consumption of wild alligator meat.

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation.

BACKGROUND: The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. RESULTS: Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. CONCLUSIONS: Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

In ovo treatment with an estrogen receptor alpha selective agonist causes precocious development of the female reproductive tract of the American alligator (Alligator mississippiensis).

The molecular signaling processes involved the differentiation of the Müllerian duct (MD) into the female reproductive tract, or oviduct, in non-mammalian vertebrates are not well understood. Studies in mammals and birds indicate that steroid hormones play a role in this process, as the embryonic MD has been shown to be vulnerable to exogenous estrogens and progestins and environmental endocrine disrupting contaminants. In a previous study, developmental treatment with an estrogen receptor α (ERα) agonist, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), induced significant enlargement of the MD in alligator embryos incubated at a male-producing temperature, which was not observed in embryos treated with an estrogen receptor ß (ERß) agonist, 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), or with 17ß-estradiol (E2). In order to understand the role of estrogen signaling in female alligator oviduct development, we incubated eggs at a female-producing temperature and treated them with E2 and these ER selective agonists, PPT and WAY 200070, just prior to the thermosensitive window of sex determination. At stage 27, one stage prior to hatching, PPT induced significant enlargement of the MD with precocious development of secretory glands and connective tissue differentiation similar to characteristics of mature adult oviduct. PPT treatment in ovo increased mRNA expression of ERß, progesterone receptor, androgen receptor and insulin-like growth factor 1 in MD at stage 27, while expression of ERα was decreased. Neither WAY 200070 nor E2 treatment induced these effects seen in PPT-treated MD. The results of this study provide insight into the critical factors for healthy reproductive system formation in this sentinel species, although further investigation is needed to determine whether the observed phenomena are directly due to selective stimulation of ERα or related to some other aspect of PPT treatment.

Epigenetic programming alterations in alligators from environmentally contaminated lakes.

Previous studies examining the reproductive health of alligators in Florida lakes indicate that a variety of developmental and health impacts can be attributed to a combination of environmental quality and exposures to environmental contaminants. The majority of these environmental contaminants have been shown to disrupt normal endocrine signaling. The potential that these environmental conditions and contaminants may influence epigenetic status and correlate to the health abnormalities was investigated in the current study. The red blood cell (RBC) (erythrocyte) in the alligator is nucleated so was used as an easily purified marker cell to investigate epigenetic programming. RBCs were collected from adult male alligators captured at three sites in Florida, each characterized by varying degrees of contamination. While Lake Woodruff (WO) has remained relatively pristine, Lake Apopka (AP) and Merritt Island (MI) convey exposures to different suites of contaminants. DNA was isolated and methylated DNA immunoprecipitation (MeDIP) was used to isolate methylated DNA that was then analyzed in a competitive hybridization using a genome-wide alligator tiling array for a MeDIP-Chip analysis. Pairwise comparisons of alligators from AP and MI to WO revealed alterations in the DNA methylome. The AP vs. WO comparison identified 85 differential DNA methylation regions (DMRs) with ⩾3 adjacent oligonucleotide tiling array probes and 15,451 DMRs with a single oligo probe analysis. The MI vs. WO comparison identified 75 DMRs with the ⩾3 oligo probe and 17,411 DMRs with the single oligo probe analysis. There was negligible overlap between the DMRs identified in AP vs. WO and MI vs. WO comparisons. In both comparisons DMRs were primarily associated with CpG deserts which are regions of low CpG density (1-2CpG/100bp). Although the alligator genome is not fully annotated, gene associations were identified and correlated to major gene class functional categories and pathways of endocrine relevance. Observations demonstrate that environmental quality may be associated with epigenetic programming and health status in the alligator. The epigenetic alterations may provide biomarkers to assess the environmental exposures and health impacts on these populations of alligators.

The influence of thermal signals during embryonic development on intrasexual and sexually dimorphic gene expression and circulating steroid hormones in American alligator hatchlings (Alligator mississippiensis).

Incubation temperatures experienced by developing embryos exert powerful influences over gonadal sex determination and differentiation in many species. However, the molecular mechanisms controlling these impacts remain largely unknown. We utilize the American alligator to investigate the sensitivity of the reproductive system to thermal signals experienced during development and ask specifically whether individuals of the same sex, yet derived from different incubation temperatures display persistent variation in the expression patterns of sex biased transcripts and plasma sex hormones. Our analysis focuses on assessments of circulating sex steroids and transcript abundance in brain and gonad, two tissues that display sexually dimorphic gene expression and directly contribute to diverse sexually dimorphic phenotypes. Whereas our results identify sexually dimorphic patterns for several target gonadal genes in postnatal alligators, sex linked variation in circulating 17ß-estradiol, testosterone, and expression of two brain transcripts (aromatase and gonadotropin releasing hormone) was not observed. Regarding intrasexual variation, we found that AMH transcript abundance in hatchling testes is positively correlated with temperatures experienced during sexual differentiation. We also describe highly variable patterns of gene expression and circulating hormones within each sex that are not explained by the intensity of embryonic incubation temperatures. The magnitude of sexually dimorphic gene expression, however, is directly associated with temperature for SOX9 and AMH, two transcripts with upstream roles in Sertoli cell differentiation. Collectively, our findings regarding temperature linked variation provide new insights regarding the connections between embryonic environment and persistent impacts on sexual differentiation in a reptile species that displays temperature dependent sex determination.

Incubation history prior to the canonical thermosensitive period determines sex in the American alligator.

Despite the widespread occurrence of environmental sex determination (ESD) among vertebrates, our knowledge of the temporal dynamics by which environmental factors act on this process remains limited. In many reptiles, incubation temperature determines sex during a discrete developmental window just prior to and coincident with the differentiation of the gonads. Yet, there is substantial variation in sex ratios among different clutches of eggs incubated at identical temperatures during this period. Here, we test the hypothesis that temperatures experienced prior to the reported thermosensitive period for alligators (Alligator mississippiensis) can impact how the sex determination system responds to thermal cues later in development. Temperature shift experiments on eggs collected from the field within 24  h of oviposition were employed to decouple various maternal influences from thermal effects, and results demonstrate a previously undefined window of thermosensitivity occurring by stage 15 of embryonic development, six stages earlier than previously reported. We also examine the intrasexual expression of several male- and female-biased genes and show that while male-biased genes display no intrasexual differences, ovarian CYP19A1 (aromatase) transcript abundance differs by approximately twofold depending on thermal exposures experienced at early stages of embryonic development. These findings expand our understanding of the ESD in the alligator and provide the rationale for reevaluation of the temporal dynamics of sex determination in other crocodilians.

Differential incubation temperatures result in dimorphic DNA methylation patterning of the SOX9 and aromatase promoters in gonads of alligator (Alligator mississippiensis) embryos.

Environmental factors are known to influence sex determination in many nonmammalian vertebrates. In all crocodilians studied thus far, temperature is the only known determinant of sex. However, the molecular mechanisms mediating the effect of temperature on sex determination are not known. Aromatase (CYP19A1) and SOX9 play critical roles in vertebrate sex determination and gonadogenesis. Here, we used a variety of techniques to investigate the potential roles of DNA methylation patterning on CYP19A1 and SOX9 expression in the American alligator, an organism that relies on temperature-dependent sex determination. Our findings reveal that developing gonads derived from embryos incubated at a male-producing temperature (MPT) show elevated CYP19A1 promoter methylation and decreased levels of gene expression relative to incubation at a female-producing temperature (FPT). The converse was observed at the SOX9 locus, with increased promoter methylation and decreased expression occurring in embryonic gonads resulting from incubation at FPT relative to that of MPT. We also examined the gonadal expression of the three primary, catalytically active DNA methyltransferase enzymes and show that they are present during critical stages of gonadal development. Together, these data strongly suggest that DNA methylation patterning is a central component in coordinating the genetic cascade responsible for sexual differentiation. In addition, these data raise the possibility that DNA methylation could act as a key mediator integrating temperature into a molecular trigger that determines sex in the alligator.

Influence of tissue, age, and environmental quality on DNA methylation in Alligator mississippiensis.

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography-tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2'-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

Epigenetic drift underlies epigenetic clock signals, but displays distinct responses to lifespan interventions, development, and cellular dedifferentiation.

Changes in DNA methylation with age are observed across the tree of life. The stereotypical nature of these changes can be modeled to produce epigenetic clocks capable of predicting chronological age with unprecedented accuracy. Despite the predictive ability of epigenetic clocks and their utility as biomarkers in clinical applications, the underlying processes that produce clock signals are not fully resolved, which limits their interpretability. Here, we develop a computational approach to spatially resolve the within read variability or "disorder" in DNA methylation patterns and test if age-associated changes in DNA methylation disorder underlie signals comprising epigenetic clocks. We find that epigenetic clock loci are enriched in regions that both accumulate and lose disorder with age, suggesting a link between DNA methylation disorder and epigenetic clocks. We then develop epigenetic clocks that are based on regional disorder of DNA methylation patterns and compare their performance to other epigenetic clocks by investigating the influences of development, lifespan interventions, and cellular dedifferentiation. We identify common responses as well as critical differences between canonical epigenetic clocks and those based on regional disorder, demonstrating a fundamental decoupling of epigenetic aging processes. Collectively, we identify key linkages between epigenetic disorder and epigenetic clocks and demonstrate the multifaceted nature of epigenetic aging in which stochastic processes occurring at non-random loci produce predictable outcomes.

Plasmid size determines adsorption to clay and breakthrough in a saturated sand column.

Horizontal gene transfer (HGT) is a major factor in the spread of antibiotic resistant genes (ARG). Transformation, one mode of HGT, involves the acquisition and expression of extracellular DNA (eDNA). eDNA in soils is degraded rapidly by extracellular nucleases. However, if bound to a clay particle, eDNA can persist for long periods of time without losing its transformation ability. To better understand the mechanism of eDNA persistence in soil, this experiment assessed the effects of 1) clay mineralogy, 2) mixed salt solution, 3) plasmid size on DNA adsorption to clay and 4) breakthrough behavior of three differently sized plasmids in an environmentally relevant solution. Batch test methods were used to determine adsorption trends of three differently sized DNA plasmids, pUC19, pBR322, and pTYB21, to several pure clay minerals, goethite (α-FeOOH), illite, and kaolinite, and one environmental soil sample. Results show not all sorbents have equal adsorption capacity based on surface area with adsorption capacities decreasing from goethite > illite = kaolinite > bulk soil, and low ionic strength solutions will likely not significantly alter sorption trends. Additionally, plasmid DNA size (i.e., length) was shown to be a significant predictor of adsorption efficiency and that size affects DNA breakthrough, with breakthroughs occurring later with larger plasmids. Given that DNA persistence is linked to its adsorption to soil constituents and breakthrough, eDNA size is likely an important contributor to the spread of ARG within natural microbial communities.

Association of size, climatic factors, and mercury body burdens with movement behavior in American alligators.

Animal movement behavior provides insight into organismal and ecological function. These functions are often disturbed by anthropogenic influences, such as urbanization and habitat fragmentation, yet the effects of long-term exposures to environmental contaminants on movement have yet to be examined. The long lifespans and broad diets of crocodilians often lead to bioaccumulation of persistent contaminants and confer a marked vulnerability to consequent physiological effects. In this study, we investigate the relationships between blood concentrations of mercury (Hg), a widespread contaminant with well characterized neurotoxicity, and movement patterns in free living, naturally exposed American alligators (Alligator mississippiensis). We sampled adult male alligators from two former nuclear cooling reservoirs with different Hg contamination histories and placed GPS transmitters on a subset of individuals from each reservoir (13 total). Data collected over the ensuing two years were analyzed using a linear mixed effects framework combined with AICc model selection to resolve the relationships linking seasonal alligator movement (daily activity (s) and daily distance (m)) and home range to climate conditions, individual traits, and blood Hg concentrations (mg/kg; wet weight). We found that climate conditions, alligator size (snout-vent-length), and blood Hg concentrations all influence alligator daily activity but do not contribute to alligator daily movement (distance). Furthermore, we found that blood Hg concentrations were strongly correlated with seasonal home range size where individuals with elevated Hg had larger home ranges in spring, fall, and winter. These findings provide insight into how climate, anthropogenic contaminants, and individual traits relate to alligator movement patterns across seasons.

The influence of incubation temperature on offspring traits varies across northern and southern populations of the American alligator (Alligator mississippiensis).

Maternal provisioning and the developmental environment are fundamental determinants of offspring traits, particularly in oviparous species. However, the extent to which embryonic responses to these factors differ across populations to drive phenotypic variation is not well understood. Here, we examine the contributions of maternal provisioning and incubation temperature to hatchling morphological and metabolic traits across four populations of the American alligator (Alligator mississippiensis), encompassing a large portion of the species' latitudinal range. Our results show that whereas the influence of egg mass is generally consistent across populations, responses to incubation temperature show population-level variation in several traits, including mass, head length, head width, and residual yolk mass. Additionally, the influence of incubation temperature on developmental rate is greater at northern populations, while the allocation of maternal resources toward fat body mass is greater at southern populations. Overall, our results suggest that responses to incubation temperature, relative to maternal provisioning, are a larger source of interpopulation phenotypic variation and may contribute to the local adaptation of populations.