Bioaccumulation and metabolic impact of environmental PFAS residue on wild-caught urban wetland tiger snakes (Notechis scutatus)

PFAS contamination of urban waters is widespread but understanding the biological impact of its accumulation is limited to humans and common ecotoxicological model organisms. Here, we combine PFAS exposure and bioaccumulation patterns with whole organism responses and omics-based ecosurveillance methods to investigate the potential impacts of PFAS on a top predator of wetlands, the tiger snake (Notechis scutatus). Tiger snakes (18 male and 17 female) were collected from four wetlands with varying PFAS chemical profiles and concentrations in Perth, Western Australia. Tiger snake livers were tested for 28 known PFAS compounds, and Σ28PFAS in liver tissues ranged between 322 ± 193 μg/kg at the most contaminated site to 1.31 ± 0.86 μg/kg at the least contaminated site. The dominant PFAS compound detected in liver tissues was PFOS. Lower body condition was associated with higher liver PFAS, and male snakes showed signs of high bioaccumulation whereas females showed signs of maternal offloading. Biochemical profiles of snake muscle, fat (adipose tissue), and gonads were analysed using a combination of liquid chromatography triple quadrupole (QqQ) and quadrupole time-of-flight (QToF) mass spectrometry methodologies. Elevated PFAS was associated with enriched energy production and maintenance pathways in the muscle, and had weak associations with energy-related lipids in the fat tissue, and lipids associated with cellular genesis and spermatogenesis in the gonads. These findings demonstrate the bioavailability of urban wetland PFAS in higher-order reptilian predators and suggest a negative impact on snake health and metabolic processes. This research expands on omics-based ecosurveillance tools for informing mechanistic toxicology and contributes to our understanding of the impact of PFAS residue on wildlife health to improve risk management and regulation

A novel MRA-based framework for the detection of changes in cerebrovascular blood pressure.

Background: High blood pressure (HBP) affects 75 million adults and is the primary or contributing cause of mortality in 410,000 adults each year in the United States. Chronic HBP leads to cerebrovascular changes and is a significant contributor for strokes, dementia, and cognitive impairment. Non-invasive measurement of changes in cerebral vasculature and blood pressure (BP) may enable physicians to optimally treat HBP patients. This manuscript describes a method to non-invasively quantify changes in cerebral vasculature and BP using Magnetic Resonance Angiography (MRA) imaging. Methods: MRA images and BP measurements were obtained from patients (n=15, M=8, F=7, Age= 49.2 ± 7.3 years) over a span of 700 days. A novel segmentation algorithm was developed to identify brain vasculature from surrounding tissue. The data was processed to calculate the vascular probability distribution function (PDF); a measure of the vascular diameters in the brain. The initial (day 0) PDF and final (day 700) PDF were used to correlate the changes in cerebral vasculature and BP. Correlation was determined by a mixed effects linear model analysis. Results: The segmentation algorithm had a 99.9% specificity and 99.7% sensitivity in identifying and delineating cerebral vasculature. The PDFs had a statistically significant correlation to BP changes below the circle of Willis (p-value = 0.0007), but not significant (p-value = 0.53) above the circle of Willis, due to smaller blood vessels. Conclusion: Changes in cerebral vasculature and pressure can be non-invasively obtained through MRA image analysis, which may be a useful tool for clinicians to optimize medical management of HBP

For the record

This section lists discussion papers DP2009/11-16 issued in December 2009, and news releases made by the Bank in the period October-December 2009.

An Analysis of Prey Resistance and Long-Term Temporal Changes in Venom Composition Within Rattlesnake Populations

Venoms are complex mixtures of toxic constituents used by venomous snakes to incapacitate prey, to defend against threats, and to aid in pre-digestion of prey items. Snake venoms vary based on a number of characteristics, including but not limited to individual identity, species, geographic range, and ontogeny. While much is understood about snake venom variability, relatively little is known as to how venom changes or is stable through time. This doctoral dissertation focuses on elucidating the nature and mechanism of compositional change in the venoms of two rattlesnake species, the Northern Pacific Rattlesnake (Crotalus oreganus oreganus) and the Desert Massasauga (Sistrurus tergeminus edwardsii). Investigating compositional change in snake venom, this dissertation has three major objectives: O1 To verify the quality of long-term stored samples for use in comparative analyses. O2 To compare the structure and function of snake venoms collected from the same geographic location at two, distant time points. O3 To detect and describe venom resistance in a rodent prey species of the rattlesnake S. t. edwardsii from Lincoln County, Colorado. Tracking changes in populations over time requires the use of long-term stored samples, so the second chapter discusses the viability of using stored samples, evaluating whether degradation is a limiting factor. Venom samples collected ~35 years ago were examined to detect changes in protein content, identity and enzyme activity. Enzyme iv assay data collected at the time of venom sample extraction were compared to data collected using the same samples and assay procedure in the present. SDS polyacrylamide gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC) were used to detect features indicating degradation, such as lateral shifts in the position of stereotypical RP-HPLC peaks or the appearance of extra, unexpected SDS-PAGE bands that may result from proteolysis. Qualitative analyses did not reveal significant effects of degradation, but two enzyme activities were significantly lower for stored samples. While proteomic information was retained, some enzyme activity was lost after long-term storage, indicating that stored snake venom samples are viable sources of biological information, but require careful scrutiny of which feature are reliably retained over time. Special attention should be paid to the effects of degradation from storage in venom toxin quantity and quality. The third chapter explores whether venom composition change is detectable after ~35 years in three populations of C. o. oreganus. Comparing samples from three populations in California collected ~35 years apart, changes in presence/absence of toxins by SDS-PAGE band analysis, in relative abundance of toxins by RP-HPLC peak area analysis, and in specific activities of six enzymes were evaluated. Evidence in the literature suggested that changes are possible, but it was hypothesized that there would be no major differences in venom composition between the two time points. Multivariate analyses revealed no significant changes in SDS-PAGE band presence/absence over ~35 years for any population. There was some evidence that enzyme activities differed significantly in one of three populations; however, activity values varied similarly between age group and geographic location, producing low support for ecologically v relevant differences between groups. There was a strong signal for separation of samples by their collection date in the HPLC peak area analysis; more recently collected samples all contained significantly more of a specific L-amino acid oxidase and snake venom metalloprotease compared to long-term stored samples. Multivariate approaches documented variation within and between samples; however, this variation appeared to be shared among samples, regardless of the date they were collected or their collection location. While rapid compositional change is hypothetically possible within such a short time period, the venoms of these populations have not diverged dramatically over the past four decades. The fourth chapter addresses the hypothesis that diet plays a major role in driving snake venom evolution. Previous work in Lincoln County, Colorado, with S. t. edwardsii indicated that the venom of this rattlesnake species might differentially affect possible mammalian prey species. Initial assays evaluated whether some prey species are more or less resistant to the venom of S. t. edwardsii from Lincoln County, Colorado. It was determined that Peromyscus maniculatus resistance was restricted to S. t. edwardsii venom and did not confer resistance to a second sympatric rattlesnake, the Prairie Rattlesnake (Crotalus viridis viridis). Additionally, resistance was only found in P. maniculatus captured in Lincoln Co., indicating that this is a local adaptation. Finally, standard protein chemistry methods were used to attempt to isolate and describe a resistance molecule. Previous studies have described serum inhibitor molecules in other systems; however, mouse serum was not able to neutralize venom enzyme activity, and mass spectrometry identified a candidate resistance molecule as mouse serum albumin. vi The final chapter summarizes the findings of the preceding chapters, and this dissertation presents a new trajectory in the study of natural toxins. The use of long-term stored snake venom samples is validated and comparing these samples against those collected from extant members of the same populations confirms that the continuum of a population’s venom variation remains stable over several generations. The mechanism of this stability is not yet known, but there is increasing evidence that diet plays a role in genetic fixation of the types of toxins and combinations of toxins found in a given venom

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa

DETECTION AND OCCURRENCE OF NERODIA HARTERI (SERPENTES: COLUBRIDAE) IN AN UPPER PORTION OF THE BRAZOS RIVER WATERSHED

Semiaquatic snakes are disproportionately impacted by habitat alteration adjacent to wetlands because of their reliance on both terrestrial and aquatic habitats. Brazos River Watersnakes, Nerodia harteri, are endemic natricines with one of the most restricted geographic ranges in North America. I quantified detection and site occupancy probabilities of N. harteri and its sympatric congeners (N. erythrogaster and N. rhombifer), as well as microhabitat selection within the Nerodia community. Within occupied transects, search effort was positively correlated with detecting N. harteri, whereas environmental and/or habitat variables were better predictors for detecting the sympatric congeners. Microhabitat selection between the three Nerodia species varied, with N. harteri selecting for riffle presence and increased canopy cover. The focal species was 97% less likely to be found in habitat characterized by bank vegetation containing saltcedar (Tamarix spp.). The results indicate the sympatric Nerodia spp. are partitioning their habitat to some degree, likely related to foraging activities. To better understand demographic trends of Nerodia harteri, long-term monitoring is needed, and habitat management — such as the preservation of riffle habitat — might be necessary to minimize the risk of continued population declines

Phenotypic Plasticity of Rattlesnake Trophic Morphology

The trophic morphology of gape-limited predators constrains the shape and size of prey items they can ingest. Trophic morphology consists of any morphological feature that is involved in the handling and ingestion of food. Diet has a profound effect on the morphology of many gape-limited predators. Identifying how prey type and resource level affect the morphology of different populations is an essential step in understanding the mechanisms contributing to patterns of morphological diversity. Species interactions (Chapter 1) induce plasticity in morphology that can lead to increased fitness, morphological divergence, and eventually speciation. In Chapter 2, a laboratory study tested the effects of defensive strikes on snake trophic morphology. In conjunction with morphological measurements, the metabolic costs of replacing venom were quantified. Control and milked snakes had baseline metabolic rate and morphology quantified before treatment manipulation. Milked snakes showed no significant difference in metabolic rate after expending venom than. Trophic morphology was not significantly different after snakes struck defensively. Venom expenditure does not impose significant increases to metabolic rate or changes in trophic morphology. In Chapter 3, I quantified the effects of starvation on trophic morphology and its allometric relationship with body size. Starvation periods of short (100 days) and long (200 days) intervals were compared to control snakes. Shorter periods of starvation had no effect on morphology or its relationship with body size, but longer periods resulted in a significant shift in the relationship between head and body size. Results showed extended periods of starvation can significantly alter the allometry of trophic morphology in snakes

2007 Annual Research Symposium Abstract Book

2007 annual volume of abstracts for science research projects conducted by students at Trinity College