Recent studies have validated the use of biopsies as a minimally invasive way to identify pregnant females in several species of wild cetaceans: Balaenapteraacutorostrata , Delphinusdelphis , ...Lissodelphisborealis, and Lagenorhynchusobliquidens. These studies found that progesterone (P4) concentrations quantified from blubber attached to biopsy samples is diagnostic of pregnancy. Here we examine a broader group of cetacean species in efforts to investigate how progesterone levels vary between species with respect to pregnancy status. We compared P4 concentrations in blubber collected from fishery bycatch and beach-stranded specimens for 40 females of known reproductive condition from Delphinuscapensis (n = 18), Stenellaattenuata (n = 8), S. longirostris (n = 6), and Phocoenoidesdalli (n = 8). The P4 concentrations were different (t = -7.1, p = 1.79E-08) between pregnant and non-pregnant animals in all species, with the mean blubber P4 concentration for pregnant animals 164 times higher than that of non-pregnant animals. There was no overlap in concentration levels between sexually immature or non-pregnant sexually mature animals and pregnant animals. No significant differences (F = 0.354, p = 0.559) were found between mature non-pregnant and immature D. capensis and Pdalli, suggesting P4 level is not indicative of maturity state in female delphinoids. P4 concentrations in relation to reproductive state were remarkably similar across species. All samples were analyzed with two different enzyme immunoassay kits to gauge assay sensitivity to measure progesterone in small samples, such as biopsies. With the technique now validated for these cetacean species, blubber P4 is a reliable diagnostic of pregnancies across multiple species, and thus expands the utility of this method to study reproduction in free-ranging cetaceans using biopsies.
When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging ...cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measurements exhibited appropriate quality characteristics when analyzed via a bootstraped stepwise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 - 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75-150 mg; r(2) = 0.012, p = 0.78, slope = 0.022 ng(cortisol deviation)/ul(tissue extract added)). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality type (e.g., stranded or bycaught), 2) specimen condition (state of decomposition), 3) total body length, 4) sex, 5) sexual maturity state, 6) pregnancy status, 7) lactation state, and 8) adrenal mass, were assessed using a Bayesian generalized linear model averaging technique. Fatality type was the only factor correlated with blubber cortisol, and the magnitude of the effect size was substantial: beach-stranded individuals had on average 6.1-fold higher cortisol levels than those of bycaught individuals. Because of the difference in conditions surrounding these two fatality types, we interpret this relationship as evidence that blubber cortisol is indicative of stress response. We found no evidence of seasonal variation or a relationship between cortisol and the remaining cofactors.
•Induced stress activated the HPA axis with typical increases in stress hormones (e.g. cortisol).•Serum corticosteroids rapidly returned to baseline levels when dolphins were returned to the ...water.•Elevated cortisol was detectable in blubber as well as in feces.•These alternate matrices may be useful indicators of acute stress in dolphins.
Several hormones are potential indicators of stress in free-ranging animals and provide information on animal health in managed-care settings. In response to stress, glucocorticoids (GC, e.g. cortisol) first appear in circulation but are later incorporated into other tissues (e.g. adipose) or excreted in feces or urine. These alternative matrices can be sampled remotely, or by less invasive means, than required for blood collection and are especially valuable in highly mobile species, like marine mammals. We characterized the timing and magnitude of several hormones in response to a stressor in bottlenose dolphins (Tursiops truncatus) and the subsequent incorporation of cortisol into blubber, and its metabolites excreted in feces.
We evaluated the endocrine response to an acute stressor in bottlenose dolphins under managed care. We used a standardized stress protocol where dolphins voluntarily beached onto a padded platform and remained out of water for two hours; during the stress test blood samples were collected every 15 min and blubber biopsies were collected every hour (0, 60, and 120 min). Each subject was studied over five days: voluntary blood samples were collected on each of two days prior to the stress test; 1 and 2 h after the conclusion of the out-of-water stress test; and on the following two days after the stress test. Fecal samples were collected daily, each afternoon.
The acute stressor resulted in increases in circulating ACTH, cortisol, and aldosterone during the stress test, and each returned to baseline levels within 2 h of the dolphin’s return to water. Both cortisol and aldosterone concentrations were correlated with ACTH, suggesting both corticosteroids are at least partly regulated by ACTH. Thyroid hormone concentrations were generally unaffected by the acute stressor. Blubber cortisol increased during the stress test, and fecal GC excretion was elevated on the day of the stress test. We found that GCs in bottlenose dolphins can recover within hours of acute stress, and that cortisol release can be detected in alternate matrices within a few hours—within 2 h in blubber, and 3.5–5 h in fecal samples.
Antarctic humpback whales are recovering from near extirpation from commercial whaling. To understand the dynamics of this recovery and establish a baseline to monitor impacts of a rapidly changing ...environment, we investigated sex ratios and pregnancy rates of females within the Western Antarctic Peninsula (WAP) feeding population. DNA profiling of 577 tissue samples (2010–2016) identified 239 males and 268 females. Blubber progesterone levels indicated 63.5% of the females biopsied were pregnant. This proportion varied significantly across years, from 36% in 2010 to 86% in 2014. A comparison of samples collected in summer versus fall showed significant increases in the proportion of females present (50% to 59%) and pregnant (59% to 72%), consistent with demographic variation in migratory timing. We also found evidence of annual reproduction among females; 54.5% of females accompanied by a calf were pregnant. These high pregnancy rates are consistent with a population recovering from past exploitation, but appear inconsistent with recent estimates of WAP humpback population growth. Thus, our results will help to better understand population growth potential and set a current baseline from which to determine the impact of climate change and variability on fecundity and reproductive rates.
Determining sex ratios of endangered populations is important for wildlife management, particularly species subject to sex-specific threats or that exhibit temperature-dependent sex determination. ...Sea turtle sex is determined by incubation temperature and individuals lack external sex-based traits until sexual maturity. Previous research utilized serum/plasma testosterone radioimmunoassays (RIA) to determine sex in immature/juvenile sea turtles. However, there has been a growing application of enzyme-linked immunosorbent assay (ELISA) for wildlife endocrinology studies, but no study on sea turtles has compared the results of ELISA and RIA. This study provides the first sex ratio for a threatened East Pacific green sea turtle (Chelonia mydas) foraging aggregation, a critical step for future management of this species. Here, we validate a testosterone ELISA and compare results between RIA and ELISA of duplicate samples. The ELISA demonstrated excellent correspondence with the RIA for providing testosterone concentrations for sex determination. Neither assay proved reliable for predicting the sex of reproductively active females with increased testosterone production. We then applied ELISA to examine the sex ratio of 69 green turtles foraging in San Diego Bay, California. Of 45 immature turtles sampled, sex could not be determined for three turtles because testosterone concentrations fell between the ranges for either sex (females: 4.1-113.1 pg/mL, males: 198.4-2,613.0 pg/mL) and these turtles were not subsequently recaptured to enable sex determination; using a Bayesian model to predict probabilities of turtle sex we predicted all three 'unknowns' were female (> 0.86). Additionally, the model assigned all turtles with their correct sex (if determined at recapture) with 100% accuracy. Results indicated a female bias (2.83F:1M) among all turtles in the aggregation; when focusing only on putative immature turtles the sex ratio was 3.5F:1M. With appropriate validation, ELISA sexing could be applied to other sea turtle species, and serve as a crucial conservation tool.
While environmental pollutants have been associated with changes in endocrine health in cetaceans, efforts to link contaminant exposure with hormones have largely been limited to a list of known, ...targeted contaminants, overlooking minimally characterized or unknown compounds of emerging concern. To address this gap, we analyzed a suite of potential endocrine disrupting halogenated organic compounds (HOCs) in blubber from 16 male short-beaked common dolphins (Delphinus delphis) with known maturity status collected from fishery bycatch in the Southern California Bight. We employed a suspect screening mass spectrometry-based method to investigate a wide range of HOCs that were previously observed in cetaceans from the same region. Potential endocrine effects were assessed through the measurement of blubber testosterone. We detected 167 HOCs, including 81 with known anthropogenic sources, 49 of unknown origin, and 37 with known natural sources. The sum of 11 anthropogenic and 4 unknown HOC classes were negatively correlated with blubber testosterone. Evidence suggests that elevated anthropogenic HOC load contributes to impaired testosterone production in mature male D. delphis. The application of this integrative analytical approach to cetacean contaminant analysis allows for inference of the biological consequences of accumulation of HOCs and prioritization of compounds for future environmental toxicology research.
Blubber and serum testosterone levels were compared among 55 individual common bottlenose dolphins, Tursiops truncatus, in Sarasota Bay, FL during 2011-2019. A significant positive relationship ...between the matrices was found in male testosterone concentrations in 29 paired samples (r
= 0.932). Mature males (n = 17) had 300 times greater mean testosterone concentration in serum than immature males (n = 17). A comparison of blubber samples, including 12 females, 24 immature males, and 19 mature males, revealed significant differences in mean blubber testosterone values among all three demographics. Immature males had greater than 6 times the average blubber testosterone concentration of females and mature males had almost 100 times that of immature males. Estimated testis volume was highly correlated with blubber testosterone concentration and mature males had 60 times greater average testis volume than immature males. We observed seasonal variation in blubber testosterone in mature males, consistent with known reproductive patterns. These data suggest males can be distinguished from females and designated as mature or immature via blubber testosterone concentrations, an observation that validates dart biopsy sampling as a means of obtaining demographic data.
Cetaceans in the Southern California Bight (SCB) are exposed to high levels of halogenated organic contaminants (HOCs), which have previously been linked to impaired reproductive health and immune ...responses. We used a combination of molecular tools to examine the potential physiological impacts of HOC exposure in two bottlenose dolphin (Tursiops truncatus) ecotypes in the SCB. We quantified 25 HOCs in the blubber of 22 biopsies collected from males between 2012 and 2016. We then analyzed genome-wide gene expression in skin using RNA-sequencing and measured blubber testosterone to compare HOC exposure with cellular and endocrine biomarkers. We found high levels of HOCs in both ecotypes with significantly higher total polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), tris(4-chlorophenyl)methanol (TCPMOH), and chlordane-related compounds in the coastal ecotype versus the offshore ecotype. We found evidence of PBDE bioaccumulation in both ecotypes, however, the pattern of bioaccumulation or endocrine disruption for other HOCs was different between the ecotypes, suggesting potential endocrine disruption in the coastal ecotype. We also observed correlations between HOCs and gene coexpression networks enriched for xenobiotic metabolism, hormone metabolism, and immune response that could indicate cellular effects from HOC exposure. By integrating measurements of HOC load with both transcriptome profiling and endocrine biomarkers, our approach provides insight into HOC exposure and potential impacts on wild cetacean health in southern California.
As long-lived predators that integrate exposures across multiple trophic levels, cetaceans are recognized as sentinels for the health of marine ecosystems. Their utility as sentinels requires the ...establishment of baseline health parameters. Because cetaceans are protected, measurements obtained with minimal disruption to free ranging animals are highly desirable. In this study we investigated the utility of skin gene expression profiling to monitor health and contaminant exposure in common bottlenose dolphins (Tursiops truncatus). Remote integument biopsies were collected in the northern Gulf of Mexico prior to the Deepwater Horizon oil spill (May 2010) and during summer and winter for two years following oil contamination (2010-2011). A bottlenose dolphin microarray was used to characterize the skin transcriptomes of 94 individuals from three populations: Barataria Bay, Louisiana, Chandeleur Sound, Louisiana, and Mississippi Sound, Mississippi/Alabama. Skin transcriptomes did not differ significantly between populations. In contrast, season had a profound effect on gene expression, with nearly one-third of all genes on the array differing in expression between winter and the warmer seasons (moderated T-test; p<0.01, fold-change≥1.5). Persistent organic pollutants (POPs) in blubber changed concurrently, reaching >two-fold higher concentrations in summer compared to winter, due to a seasonal decrease in blubber thickness and loss of stored lipid. However, global gene expression did not correlate strongly with seasonally changing contaminant concentrations, most likely because the refractory, lipid-stored metabolites are not substrates for phase I or II xenobiotic detoxification pathways. Rather, processes related to cell proliferation, motility, and differentiation dominated the differences in expression in winter and the warmer seasons. More subtle differences were seen between spring and summer (1.5% of genes differentially expressed). However, two presumed oil-exposed animals from spring presented gene expression profiles more similar to the summer animals (presumed exposed) than to other spring animals. Seasonal effects have not previously been considered in studies assessing gene expression in cetaceans, but clearly must be taken into account when applying transcriptomic analyses to investigate their contaminant exposure or health status.
Timely detection and understanding of causes for population decline are essential for effective wildlife management and conservation. Assessing trends in population size has been the standard ...approach, but we propose that monitoring population health could prove more effective. We collated data from 7 bottlenose dolphin (Tursiops truncatus) populations in the southeastern United States to develop a method for estimating survival probability based on a suite of health measures identified by experts as indices for inflammatory, metabolic, pulmonary, and neuroendocrine systems. We used logistic regression to implement the veterinary expert system for outcome prediction (VESOP) within a Bayesian analysis framework. We fitted parameters with records from 5 of the sites that had a robust network of responders to marine mammal strandings and frequent photographic identification surveys that documented definitive survival outcomes. We also conducted capture–mark–recapture (CMR) analyses of photographic identification data to obtain separate estimates of population survival rates for comparison with VESOP survival estimates. The VESOP analyses showed that multiple measures of health, particularly markers of inflammation, were predictive of 1‐ and 2‐year individual survival. The highest mortality risk 1 year following health assessment related to low alkaline phosphatase (odds ratio OR = 10.2 95% CI: 3.41–26.8), whereas 2‐year mortality was most influenced by elevated globulin (OR = 9.60 95% CI: 3.88–22.4); both are markers of inflammation. The VESOP model predicted population‐level survival rates that correlated with estimated survival rates from CMR analyses for the same populations (1‐year Pearson's r = 0.99, p = 1.52 × 10–5; 2‐year r = 0.94, p = 0.001). Although our proposed approach will not detect acute mortality threats that are largely independent of animal health, such as harmful algal blooms, it can be used to detect chronic health conditions that increase mortality risk. Random sampling of the population is important and advancement in remote sampling methods could facilitate more random selection of subjects, obtainment of larger sample sizes, and extension of the approach to other wildlife species.
Un sistema basado en conocimiento experto para predecir la tasa de supervivencia a partir de datos de salud
Resumen
La detección y el entendimiento oportunos de la declinación poblacional son esenciales para que el manejo y la conservación de fauna tengan efectividad. La evaluación de las tendencias en el tamaño poblacional ha sido la estrategia estándar, pero proponemos que el monitoreo de la salud poblacional podría ser más efectivo. Recopilamos datos de siete poblaciones de delfines (Tursiops truncatus) en el sureste de Estados Unidos para desarrollar un método de estimación de la probabilidad de supervivencia con base en un conjunto de medidas sanitarias identificadas por expertos como índices para los sistemas inflamatorio, metabólico, pulmonar y neuroendocrino. Usamos la regresión logística para implementar el sistema de expertos veterinarios para la predicción de resultados (SEVPR) en un análisis bayesiano. Ajustamos los parámetros con los registros de cinco sitios que contaban con una buena red de respondientes a los varamientos de mamíferos marinos y censos de identificación fotográfica (foto‐ID) que documentaron los resultados de supervivencia definitivos. También realizamos análisis de marcaje‐recaptura (MR) en los datos de identificación fotográfica para obtener estimados separados de las tasas de supervivencia poblacional para compararlos con los estimados del SEVPR. Los análisis del SEVPR mostraron que varias medidas sanitarias, particularmente los marcadores de inflamación son buenos predictores de la supervivencia individual para uno y dos años. El riesgo de mortalidad más alto un año después de la valoración sanitaria se relacionó con una fosfatasa alcalina baja (cociente de probabilidades de 10.2 95% CI 3.41‐26.8), mientras que la mortalidad a los dos años estuvo más influenciada por una globulina elevada (9.60 95% CI 3.88‐22.4); ambas son marcadores de la inflamación. El modelo del SEVPR predijo las tasas de supervivencia a nivel poblacional en correlación con las tasas estimadas de supervivencia de los análisis de MR para las mismas poblaciones (Pearson de un año r = 0.99, p = 1.52e‐05; dos años r = 0.94, p = 0.001). Aunque nuestra propuesta no detecta las amenazas agudas de mortalidad que en su mayoría son independientes de la salud animal, como la proliferación de algas nocivas, puede usarse para detectar las condiciones crónicas de salud que incrementan el riesgo de mortalidad. Es importante el muestreo aleatorio de la población y los avances en los métodos de muestreo remoto podrían facilitar una selección más aleatoria de los sujetos, la obtención de muestras de mayor tamaño y la expansión de la estrategia a otras especies de fauna.
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