Direct and Latent Effects of Pathogen Exposure Across Native and Invasive Amphibian Life Stages.

Emerging infectious diseases are one of the multiple factors contributing to the current "biodiversity crisis". As part of the worldwide biodiversity crisis, amphibian populations are declining globally. Chytridiomycosis, an emerging infectious disease, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is a major cause of amphibian population declines. This fungus primarily affects keratinized structures in larval, juvenile, and adult amphibians as well as heart function. However, we know little about how Bd can impact embryos as well as potential latent effects of Bd exposure over ontogeny. Using two different Bd strains and multiple exposure times, we examined the effects of Bd exposure in Pacific chorus frog (Pseudacris regilla), Western toad (Anaxyrus boreas) and American bullfrog (Lithobates catesbeianus) life stages. Using a factorial experimental design, embryos of these three species were exposed to Bd at early and late embryonic stages, with some individuals re-exposed after hatching. Embryonic Bd exposure resulted in differential survival as a function of host species, Bd strain and timing of exposure. P. regilla experienced embryonic mortality when exposed during later developmental stages to one Bd strain. There were no differences across the treatments in embryonic mortality of A. boreas and embryonic mortality of L. catesbeianus occurred in all Bd exposure treatments. We detected latent effects in A. boreas and L. catesbeianus larvae, as mortality increased when individuals had been exposed to any of the Bd strains during the embryonic stage. We also detected direct effects on larval mortality in all three anuran species as a function of Bd strain, and when individuals were double exposed (late in the embryonic stage and again as larvae). Our results suggest that exposure to Bd can directly affect embryo survival and has direct and latent effects on larvae survival of both native and invasive species. However, these impacts were highly context dependent, with timing of exposure and Bd strain influencing the severity of the effects.

Estimating the genetic diversity of Pacific salmon and trout using multigene eDNA metabarcoding.

Genetic diversity underpins species conservation and management goals, and ultimately determines a species' ability to adapt. Using freshwater environmental DNA (eDNA) samples, we examined mitochondrial genetic diversity using multigene metabarcode sequence data from four Oncorhynchus species across 16 sites in Oregon and northern California. Our multigene metabarcode panel included targets commonly used in population genetic NADH dehydrogenase 2 (ND2), phylogenetic cytochrome c oxidase subunit 1 (COI) and eDNA (12S ribosomal DNA) screening. The ND2 locus showed the greatest within-species haplotype diversity for all species, followed by COI and then 12S rDNA for all species except Oncorhynchus kisutch. Sequences recovered for O. clarkii clarkii were either identical to, or one mutation different from, previously characterized haplotypes (95.3% and 4.5% of reads, respectively). The greatest diversity in O. c. clarkii was among coastal watersheds, and subsets of this diversity were shared with fish in inland watersheds. However, coastal streams and the Umpqua River watershed appear to harbour unique haplotypes. Sequences from O. mykiss revealed a disjunction between the Willamette watershed and southern watersheds suggesting divergent histories. We also identified similarities between populations in the northern Deschutes and southern Klamath watersheds, consistent with previously hypothesized connections between the two via inland basins. Oncorhynchus kisutch was only identified in coastal streams and the Klamath River watershed, with most diversity concentrated in the coastal Coquille watershed. Oncorhynchus tshawytscha was only observed at one site, but contained multiple haplotypes at each locus. The characterization of genetic diversity at multiple loci expands the knowledge gained from eDNA sampling and provides crucial information for conservation actions and genetic management.

Reproductive characteristics of American bullfrogs (Lithobates catesbeianus) in their invasive range of the Pacific Northwest, USA.

Invasive species pose a major threat to global biodiversity. The effects of invasive species can be strongly influenced and potentially mediated by their reproductive characteristics, such as fecundity, egg production, and duration and number of reproductive events. Selection for smaller body size at first reproduction can also play a role in their establishment, facilitating colonization and spread. The American bullfrog, native to the eastern U.S. (Lithobates catesbeianus), is a species that has invaded more than 40 countries across 4 continents. This species has become especially prevalent in the western United States since its introduction in the early 1900s. This study characterized reproductive characteristics of bullfrogs with emphasis on the minimum size at which males and females reach sexual maturity in the Willamette Valley, Oregon, USA invasion range. We collected and dissected 121 individuals in 2013 and 2017, quantifying characteristics of sexual maturity including snout-vent length, total length, sex, tympanum diameter, presence of distended oviducts or eggs for females, and testes length and sperm activity in males. Our results showed that the minimum reproductive size of both males and females was smaller relative to bullfrogs in their native range as well as in populations across their invasive range. Reduction in size at reproductive maturity is likely impacting the invasive success of American bullfrogs and this study gives us insight on management actions to control the invasion. Applying this insight, managers can adjust their definition of reproductively active adults, increasing the target population of culling and other control methods.

Evaluating adaptive, carry-over, and plastic antipredator responses across a temporal gradient in Pacific chorus frogs.

The development of antipredator traits is dependent on the frequency and intensity of predator exposure over evolutionary and ecological time. We hypothesized that prey species would respond with increasing accuracy when exposed to predators across generational, ontogenetic, and immediate time scales. We assessed larval Pacific chorus frog (PSRE; Pseudacris regilla) individuals that varied in population sympatry, embryonic conditioning, and immediate exposure to stocked populations of rainbow trout (Oncorhynchus mykiss). Using PSRE populations from sites with and without resident rainbow trout, we conditioned embryos to trout odor, PSRE alarm cues, trout odor in combination with alarm cues, or control water. After being hatched and reared in control water, individuals were exposed to the four predator cue treatments using a fully factorial design. Tadpoles from populations with resident rainbow trout did not behave or develop differently than tadpoles originating from fishless sites. However, we found evidence that PSRE reduced predation risk with a combination of carry-over effect (i.e., transfer of information across life history stages) and within-life stage phenotypically plastic mechanisms. We found both developmental and behavioral carry-over effects: tadpoles conditioned with trout odor as embryos grew more slowly and took refuge more often than control animals. Within-life-stage behavioral plasticity was observed in tadpoles from all treatment groups, responding to predator cues with increased refuge use. Potentially additive effects of predator exposure on prey response should be considered when predicting the ability of prey to recognize novel threats.

The integrative effects of behavior and morphology on amphibian movement.

Animal movement and dispersal are key factors in population dynamics and support complex ecosystem processes like cross-boundary subsidies. Juvenile dispersal is an important mechanism for many species and often involves navigation in unfamiliar habitats. For species that metamorphose, such as amphibians, this transition from aquatic to terrestrial environments involves the growth and use of new morphological traits (e.g., legs). These traits strongly impact the fundamental ability of an organism to move in novel landscapes, but innate behaviors can regulate choices that result in the realized movements expressed. By assessing the integrative role of morphology and behavior, we can improve our understanding of juvenile movement, particularly in understudied organisms like amphibians. We assessed the roles of morphological (snout-vent length and relative leg length) and performance (maximal jump distance) traits in shaping the free movement paths, measured through fluorescent powder tracking, in three anuran species, Pacific treefrog (Hyliola regilla), Western toad (Anaxyrus boreas), and Cascades frog (Rana cascadae). We standardized the measurement of these traits to compare the relative role of species' innate differences versus physical traits in shaping movement. Innate differences, captured by species identity, were the most significant factor influencing movement paths via total movement distance and path sinuosity. Relative leg length was an important contributor but significantly interacted with species identity. Maximal jump performance, which was significantly predicted by morphological traits, was not an important factor in movement behavior relative to species identity. The importance of species identity and associated behavioral differences in realized movement provide evidence for inherent species differences being central to the dispersal and movement of these species. This behavior may stem from niche partitioning of these sympatric species, yet it also calls into question assumptions generalizing anuran movement behavior. These species-level effects are important in framing differences as past research is applied in management planning.

Warming-induced shifts in amphibian phenology and behavior lead to altered predator-prey dynamics.

Climate change-induced phenological variation in amphibians can disrupt time-sensitive processes such as breeding, hatching, and metamorphosis, and can consequently alter size-dependent interactions such as predation. Temperature can further alter size-dependent, predator-prey relationships through changes in species' behavior. We thus hypothesized that phenological shifts due to climate warming would alter the predator-prey dynamic in a larval amphibian community through changes in body size and behavior of both the predator and prey. We utilized an amphibian predator-prey system common to the montane wetlands of the U.S. Pacific Northwest: the long-toed salamander (Ambystoma macrodactylum) and its anuran prey, the Pacific chorus frog (Pseudacris regilla). We conducted predation trials to test if changes in predator phenology and environmental temperature influence predation success. We simulated predator phenological shifts using different size classes of the long-toed salamander representing an earlier onset of breeding while using spring temperatures corresponding to early and mid-season larval rearing conditions. Our results indicated that the predator-prey dynamic was highly dependent upon predator phenology and temperature, and both acted synergistically. Increased size asymmetry resulted in higher tadpole predation rates and tadpole tail damage. Both predators and prey altered activity and locomotor performance in warmer treatments. Consequently, behavioral modifications resulted in decreased survival rates of tadpoles in the presence of large salamander larvae. If predators shift to breed disproportionately earlier than prey due to climate warming, this has the potential to negatively impact tadpole populations in high-elevation amphibian assemblages through changes in predation rates mediated by behavior.

Opioid distribution trends (2006-2017) in the US Territories.

BACKGROUND: The US mainland is experiencing an epidemic of opioid overdoses. Unfortunately, the US Territories (Guam, Puerto Rico, and the Virgin Islands) have often been overlooked in opioid pharmacoepidemiology research. This study examined common prescription opioids over the last decade. METHODS: The United States Drug Enforcement Administration's Automation of Reports and Consolidated Orders System (ARCOS) was used to report on ten medical opioids: buprenorphine, codeine, fentanyl, hydrocodone, hydromorphone, meperidine, methadone, morphine, oxycodone, and oxymorphone, by weight from 2006 to 2017. Florida and Hawaii were selected as comparison areas. RESULTS: Puerto Rico had the greatest Territorial oral morphine mg equivalent (MME) per capita (421.5) which was significantly higher (p < .005) than the Virgin Islands (139.2) and Guam (118.9) but significantly lower than that of Hawaii (794.6) or Florida (1,509.8). Methadone was the largest opioid by MMEs in 2017 in most municipalities, accounting for 41.1% of the total in the Virgin Islands, 37.9% in Florida, 36.6% in Hawaii but 80.8% in Puerto Rico. Puerto Rico and Florida showed pronounced differences in the distribution patterns by pharmacies, hospitals, and narcotic treatment programs for opioids. CONCLUSIONS: Continued monitoring of the US Territories is needed to provide a balance between appropriate access to these important agents for cancer related and acute pain while also minimizing diversion and avoiding the opioid epidemic which has adversely impacted the US mainland.

Embryonic learning and developmental carry-over effects in an invasive anuran.

Carry-over effects influence trait responses in later life stages as a result of early experience with environmental cues. Predation risk is an influential stressor and selection exists for early recognition of threats. In particular, invasive species may benefit from carry-over effects by preemptively recognizing and responding to novel predators via latent developmental changes and embryonic learning. In a factorial experiment, we conditioned invasive American bullfrog embryos (Lithobates catesbeianus) to the odor of a novel fish predator, largemouth bass (Micropterus salmoides) alone or in combination with injured conspecific cues. We quantified developmental carryover in the larval life stage and found that individuals conditioned to the highest risk (fish and injured conspecific cues) grew into longer bodied larvae relative to larvae from lower risk treatments. We also assessed embryonic learning, a behavioral carry-over effect, and found an interaction between embryonic conditioning and larval exposure. Behavioral responses were only found in scenarios when predation risk varied in intensity across life history stages, thus requiring a more flexible antipredator strategy. This indicates a potential trade-off between the two strategies in larval growth and development rates, and time until metamorphosis. Our results suggest that early predator exposure and carry-over effects have significant impacts on life history trajectories for American bullfrogs. This research contributes to our understanding of a potentially important invasion mechanism in an anuran species of conservation concern.

Calling at the highway: The spatiotemporal constraint of road noise on Pacific chorus frog communication.

Loss of acoustic habitat due to anthropogenic noise is a key environmental stressor for vocal amphibian species, a taxonomic group that is experiencing global population declines. The Pacific chorus frog (Pseudacris regilla) is the most common vocal species of the Pacific Northwest and can occupy human-dominated habitat types, including agricultural and urban wetlands. This species is exposed to anthropogenic noise, which can interfere with vocalizations during the breeding season. We hypothesized that Pacific chorus frogs would alter the spatial and temporal structure of their breeding vocalizations in response to road noise, a widespread anthropogenic stressor. We compared Pacific chorus frog call structure and ambient road noise levels along a gradient of road noise exposures in the Willamette Valley, Oregon, USA. We used both passive acoustic monitoring and directional recordings to determine source level (i.e., amplitude or volume), dominant frequency (i.e., pitch), call duration, and call rate of individual frogs and to quantify ambient road noise levels. Pacific chorus frogs were unable to change their vocalizations to compensate for road noise. A model of the active space and time ("spatiotemporal communication") over which a Pacific chorus frog vocalization could be heard revealed that in high-noise habitats, spatiotemporal communication was drastically reduced for an individual. This may have implications for the reproductive success of this species, which relies on specific call repertoires to portray relative fitness and attract mates. Using the acoustic call parameters defined by this study (frequency, source level, call rate, and call duration), we developed a simplified model of acoustic communication space-time for this species. This model can be used in combination with models that determine the insertion loss for various acoustic barriers to define the impact of anthropogenic noise on the radius of communication in threatened species. Additionally, this model can be applied to other vocal taxonomic groups provided the necessary acoustic parameters are determined, including the frequency parameters and perception thresholds. Reduction in acoustic habitat by anthropogenic noise may emerge as a compounding environmental stressor for an already sensitive taxonomic group.

Evaluating Multi-Level Models to Test Occupancy State Responses of Plethodontid Salamanders.

Plethodontid salamanders are diverse and widely distributed taxa and play critical roles in ecosystem processes. Due to salamander use of structurally complex habitats, and because only a portion of a population is available for sampling, evaluation of sampling designs and estimators is critical to provide strong inference about Plethodontid ecology and responses to conservation and management activities. We conducted a simulation study to evaluate the effectiveness of multi-scale and hierarchical single-scale occupancy models in the context of a Before-After Control-Impact (BACI) experimental design with multiple levels of sampling. Also, we fit the hierarchical single-scale model to empirical data collected for Oregon slender and Ensatina salamanders across two years on 66 forest stands in the Cascade Range, Oregon, USA. All models were fit within a Bayesian framework. Estimator precision in both models improved with increasing numbers of primary and secondary sampling units, underscoring the potential gains accrued when adding secondary sampling units. Both models showed evidence of estimator bias at low detection probabilities and low sample sizes; this problem was particularly acute for the multi-scale model. Our results suggested that sufficient sample sizes at both the primary and secondary sampling levels could ameliorate this issue. Empirical data indicated Oregon slender salamander occupancy was associated strongly with the amount of coarse woody debris (posterior mean = 0.74; SD = 0.24); Ensatina occupancy was not associated with amount of coarse woody debris (posterior mean = -0.01; SD = 0.29). Our simulation results indicate that either model is suitable for use in an experimental study of Plethodontid salamanders provided that sample sizes are sufficiently large. However, hierarchical single-scale and multi-scale models describe different processes and estimate different parameters. As a result, we recommend careful consideration of study questions and objectives prior to sampling data and fitting models.

Elevational differences in trait response to UV-B radiation by long-toed salamander populations.

Amphibian species capable of optimizing trait response to environmental stressors may develop complex strategies for defending against rapid environmental change. Trait responses may differ between populations, particularly if stressor strength varies across spatial or temporal gradients. Ultraviolet-B (UV-B) radiation is one such stressor that poses a significant threat to amphibian species. We examined the ability of long-toed salamanders (Ambystoma macrodactylum) at high- and low-elevation breeding sites to cooperatively employ behavioral and physiological trait responses to mediate UV-B damage. We performed a microhabitat survey to examine differences in oviposition behavior and UV-B conditions among breeding populations at high- (n = 3; >1,500 m) and low-elevation (n = 3; <100 m) sites. We found significant differences in oviposition behavior across populations, with females at high-elevation sites selecting oviposition substrates in UV-B protected microhabitats. We also collected eggs (n = 633) from each of the breeding sites for analysis of photolyase activity, a photoreactivating enzyme that repairs UV-B damage to the DNA, using a photoproduct immunoassay. Our results revealed no significant differences in photolyase activity between long-toed salamander populations at high and low elevations. For high-elevation salamander populations, relatively low physiological repair capabilities in embryos appear to be buffered by extensive behavioral modifications to reduce UV-B exposure and standardize developmental temperatures. This study provides valuable insight into environmental stress responses via the assessment of multiple traits in allowing sensitive species to persist in rapidly changing landscapes.

A meta-analysis of the effects of pesticides and fertilizers on survival and growth of amphibians.

The input of agrochemicals has contributed to alteration of community composition in managed and associated natural systems, including amphibian biodiversity. Pesticides and fertilizers negatively affect many amphibian species and can cause mortality and sublethal effects, such as reduced growth and increased susceptibility to disease. However, the effect of pesticides and fertilizers varies among amphibian species. We used meta-analytic techniques to quantify the lethal and sublethal effects of pesticides and fertilizers on amphibians in an effort to review the published work to date and produce generalized conclusions. We found that pesticides and fertilizers had a negative effect on survival of -0.9027 and growth of -0.0737 across all reported amphibian species. We also observed differences between chemical classes in their impact on amphibians: inorganic fertilizers, organophosphates, chloropyridinyl, phosphonoglycines, carbamates, and triazines negatively affected amphibian survival, while organophosphates and phosphonoglycines negatively affected amphibian growth. Our results suggest that pesticides and fertilizers are an important stressor for amphibians in agriculturally dominated systems. Furthermore, certain chemical classes are more likely to harm amphibians. Best management practices in agroecosystems should incorporate amphibian species-specific response to agrochemicals as well as life stage dependent susceptibility to best conserve amphibian biodiversity in these landscapes.

Exploring factors influencing patient request for epidural analgesia on admission to labor and delivery in a predominantly Latino population.

Ethnic disparities in labor pain management exist. Our purpose is to identify patients' attitudes and beliefs about epidural analgesia in order to develop a culturally competent educational intervention. A prospective observational study was conducted in patients admitted for vaginal delivery between July 1st-31st, 2009. Inclusion criteria were: singleton, term, cephalic, normal fetal heart tracing and no contraindications for epidural. Patients were surveyed regarding their wishes for analgesia, and their reasons for declining epidural. The obstetrics physician performed pain management counseling as is usually done. Patients were asked again about their choice for analgesia. Likert scale questionnaires were used. Wilcoxon signed ranked test was used for categorical variables. Logistic regression was performed to look for predictors of epidural request. Fifty patients were interviewed. Average age was (27.9 ± 6.7), gestational age (39.3 ± 1.3), and a median parity of 2 (range 0-6). 72% declined epidural upon admission, and 61% after counseling (P = 0.14). Most common reasons for declined epidural were 'women should cope with labor pain' (57%), 'fear of back pain' (54%) and 'family/friends advise against epidural' (36%). Acculturation was assessed by years living in the US (10 ± 6.3), preferred language (Spanish 80%) and ethnic self-identification (Hispanic 98%). 38% were high school graduates. In multivariate logistic regression, graduation from high school was the only variable associated to request for epidural in labor (OR 4.94, 95% CI 1.6-15.1). Educational level is associated to requesting an epidural in labor. Knowledge of patients' fears and expectations is essential to develop adequate counseling interventions.

Color change and color-dependent behavior in response to predation risk in the salamander sister species Ambystoma barbouri and Ambystoma texanum.

Although many organisms show multiple types of trait responses to predation risk (e.g., shifts in behavior, morphology, color, chemistry or life history), relatively few studies have examined how prey integrate these multiple responses. We studied the joint expression of color and behavioral responses to predation risk in two sister species of salamander larvae that live in habitats with different selection pressures. We examined responses to predation risk in three situations that differed in availability of refuge and substrate color heterogeneity, and thus availability of behavioral options for reducing risk. Relative to Ambystoma texanum, A. barbouri larvae were darker in color and showed a greater range of color change. With no variation in background color or refuge available, both species exhibited color change to better match the available background. The degree of color change showed by both species, however, did not depend on predation risk. Given the option to choose between light and dark substrates, A. texanum exhibited behavioral background matching (i.e., they preferred substrates that matched their own body color), while A. barbouri's substrate preferences did not depend on their initial body color. Instead, A. barbouri responded to risk by showing a strong preference for dark substrates, followed by a change to a darker body color. With refuge available, A. texanum's refuge use was color-dependent; larvae that were well camouflaged spent less time in refuge. In contrast, A. barbouri showed strong refuge use in response to risk, regardless of their body color. Overall, these results reflect how conflicting selection pressures (predation risk, habitat ephemerality, risk of UV damage) and species differences in mean color and ability to change color can govern the interplay of complementary and compensatory behavioral and color responses to predation risk.