Microparticles as a biomarker of decompression sickness

Recent studies of stranded marine mammals indicate that exposure to underwater military sonar may induce symptoms consistent with decompression sickness (DCS).

Without a measurable indicator or biomarker, DCS has been difficult to accurately diagnose in marine mammals. As part of the dolphin health assessment project, our team of researchers at Woods Hole Oceanographic Institution, Texas A&M University – Corpus Christi, and University of Maryland is investigating the use of Microparticles (MPs) as a tool to diagnose DCS in marine mammals.

MPs (measured as number/μl plasma) are fragments of various cells, such as platelets that circulate in blood.  Studies of MPs in humans and mice have shown that with increased decompression stress, circulating MPs also increase. The origin and cause of elevated MPs is not clear; however, tissue injury as a result of gas bubble formation from decompression stress is likely a factor.  With support from the many supporters of the Sarasota Dolphin Research Program and the Office of Naval Research, we are collecting and analyzing blood samples from captive Steller sea lions, wild-caught-and-released dolphins, and stranded dolphins in order to verify the use of MPs as a biomarker for DCS.

As part of the Sarasota Dolphin Research Program, we have collected blood samples from healthy dolphins in Sarasota Bay.  In 2012 and 2013, we analyzed blood samples from nearly 40 dolphins to determine baseline MPs values.  Our next step is to collect and analyze blood samples from single and mass-stranded dolphins in Cape Cod in early 2014 to compare against the baseline MPs levels from Sarasota dolphins.  We will also perform statistical analyses of all data by utilizing associated morphometric data, environmental variables, and health assessment parameters collected during the sampling events.

Preliminary results from a related study in Steller sea lions support the use of MPs as a biomarker for decompression stress.  We measured MPs in Steller sea lions before and after experimental dive trials.  Compared to Pre-dive MPs, Post-dive MPs increased on average by 303% at 3 hours and 458% at 24 hours.  This Steller sea lion study provides preliminary verification that MPs may potentially be used as biomarker for DCS in marine mammals.  Although data collection and analyses are ongoing, confirmation of MPs as a diagnostic tool could have significant implications for marine mammal conservation by improving management decisions during a stranding event.

 This article was published on 15 in the January 2014 Nicks n Notches.

Evaluating stress hormones in bottlenose dolphins

As inhabitants of a constantly changing coastal environment, estuarine bottlenose dolphins are exposed to a variety of internal and external stressors. Their stress response is modulated by various hormones, several of which are secreted from the adrenal and thyroid glands.

The objective of this study was to use data from long-term capture-release projects conducted in Sarasota Bay to evaluate influential factors (for example, demographic, seasonal, temporal, physical, and sampling) related to individual variation in circulating concentrations of adrenal and thyroid hormones commonly considered to be involved in the marine mammalian stress response (cortisol, aldosterone, T3, TT4, and FT4).

Previous studies have implicated the influence of these variables on the secretion of these hormones following exposure to various stressors; however most of these studies relied upon short-term data or single-sampling events.  Our study, funded by the Office of Naval Research, used hormone concentrations measured in blood samples collected during capture-release events during 2000-2012.   In total, 185 observations representing 121 individuals (61 males, 60 females) were used for statistical analyses to examine associations between stress hormone measurements and factors such as sex, age, sexual maturity, time of day, length, mass, and elapsed sampling time (the time between capture and blood collection).  Statistical analyses were used to determine that elapsed time and mass were significant influences on cortisol levels, while sex, age, and sexual maturity were associated with thyroid hormone concentrations.

While it is helpful to understand demographic and environmental influences on endocrine hormone concentrations among individuals and across populations, the impact of various biological and artificial stressors cannot be evaluated without a comparison to baseline “normal” values.

Reference intervals provide an opportunity to evaluate an individual’s health status among populations for which baseline data are not available, as they represent a range of expected values for a normal, healthy population.  Results of the analyses to determine influential factors were used to stratify reference intervals for each of the stress hormones.  For example, since elapsed sampling time was significantly associated with cortisol levels, separate reference intervals were calculated for animals sampled in less than 30 minutes following capture and greater than 30 minutes post-capture.  Similarly, reference intervals for T3 were stratified based on sexual maturity, where separate reference intervals were calculated for immature and mature dolphins. Bottlenose dolphin capture-release health assessments have increased in frequency over the past decade due to questions regarding health effects from harmful algal bloom exposure, PCB contamination, and in response to environmental disasters such as the Deepwater Horizon Oil Spill.  In many cases, baseline health data have not been available for these study populations.  Reference intervals, based on unbiased statistical analyses and long-term data, provide an opportunity to quantitatively and objectively compare and evaluate health parameters of individuals for which historical data are unavailable.

This article was published on page 15 in the January 2014 Nicks n Notches.

Genetic susceptibility to red tides

Recent advances in molecular technologies have made it possible to study genetic variation across whole genomes of bottlenose dolphins and other species in the natural environment.

These powerful techniques greatly benefit studies of genetic adaptation, where researchers search for genes linked to higher fitness and health.  In Sarasota Bay, these techniques have greatly enhanced my study of a genetic basis for resistance to harmful algal blooms (HABs) in bottlenose dolphins.

Bottlenose dolphins in central-west Florida, including Sarasota Bay, are exposed to HABs of the toxic dinoflagellate Karenia brevis almost every year.  These HABs, also known as red tides, produce neurotoxins that can be lethal to bottlenose dolphins and other marine animals, such as fish, manatees, sea birds, and sea turtles, but the dolphins in central-west Florida appear to be relatively resistant to the toxin.  I compared bottlenose dolphins that died due to red tide exposure to dolphins that survived, from both central-west Florida and the Florida Panhandle.  Dolphins in the Panhandle appear to be relatively susceptible to red tides and have experienced several large-scale unusual mortality events associated with K. brevis blooms.

My comparisons of live and dead dolphins from apparently resistant and susceptible populations involved thousands of genetic markers from across the bottlenose dolphin genome.  These genetic data are generated using a technique called restriction-associated DNA sequencing, which produces short sequences of DNA representing a subset of the genome.  I found that some variation in these DNA sequences correlates with survival in bottlenose dolphins from both central-west Florida and the Panhandle. These survival-associated DNA sequences may be linked to several genes potentially related to red tide resistance, including genes involved in the immune system, the response to stress, and the generation of neurons.  Overall, this finding of genetic variation associated with survival suggests a genetic basis for resistance to red tides in bottlenose dolphins, and we have identified potential candidate genes that may encode this resistance.

This research was supported by the Duke University Marine Lab, the American Fisheries Society, and the PADI Foundation.  Samples were provided by the NOAA Fisheries SEFSC DNA Archives and the Sarasota Dolphin Research Program.


This article was published on pages 14-15 in the January 2014 Nicks n Notches.


Bottlenose dolphins: A biosensor to detect ocean health risks

The environmental quality of marine ecosystems is often assessed by the variety of species present and their relative abundance, the levels of pollutants, and the frequency and severity of harmful algal blooms.

As top level predators, bottlenose dolphins are particularly sensitive to chemical and biological toxins that accumulate and biomagnify in the marine food chain.

We are currently investigating the potential of screening for multiple contaminant and/or algal toxin exposure through their associated immunological and/or endocrine perturbations using gene expression profile analysis. Here we present the evidence of how strongly chemical contaminants impact dolphin gene expression.

Gene expression analysis at a global scale, which is where we look at the behavior of most genes in the entire genome, can be employed using specific tools called microarrays.  A dolphin microarray representing 24,418 gene sequences was developed and used to analyze blood samples collected from dolphins sampled during capture-release health assessments from four geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island and Brunswick, GA). Organochlorine pesticide and polychlorinated biphenyl congener concentrations were determined in blubber biopsy samples from the same animals.

Sets of samples with the highest and the lowest measured values of contaminants in their blubber were used as a guide to tell us how the animals with the highest exposure to these contaminants compared to the animals with the lowest level of exposure.  By looking to see which genes were different between these two sets of animals, we can identify genes that are increasing or decreasing as a result of the exposure extremes. The resultant gene set could be a signature of contaminant exposure.

In order to test this hypothesis we next investigated the blood transcriptomes (the type and level of genes expressed at any given time) of the remaining dolphin samples using machine-learning approaches, artificial systems that can learn from data. Using the derived gene set to train the system, the algorithms worked very well at distinguishing and classifying dolphins according to the contaminant load accumulated in their blubber.

The transcriptomic data analysis will be a first step towards identification of markers and patterns indicative of exposure to chemical contaminants as well as marine toxins and will promote an understanding of toxic mechanisms and/or pathways that are currently not well understood in marine mammals.

This study was supported by the NOAA Oceans and Human Health Initiative, Dolphin Quest, Georgia Aquarium, Disney’s Animal Programs, Morris Animal Foundation’s Betty White Wildlife Rapid Response Fund, and NOAA Fisheries Service.

This article was published on page 14 in the January 2014 Nicks n Notches.

A visual body condition index for bottlenose dolphins

Bottlenose dolphin health assessments are very useful for investigating the potential impacts of natural and anthropogenic factors on dolphin populations, but they are expensive and logistically complex.

In response to the need to develop a simple and cost-effective means of initially assessing the condition of individuals within a population, I conducted my master’s research to create a visual body condition index for bottlenose dolphins, using photographs.

To develop the index, I investigated the post-nuchal depression (PND), a concavity behind the blowhole.  PNDs can be recognized in lateral photos and are commonly considered by dolphin researchers to be indicative of poor body condition.  Although PNDs have been used as a sign of emaciation for bottlenose dolphins, very few studies have related this trait to quantifiable body condition metrics.  The two objectives of my study were (1) to create a technique to objectively and systematically identify PNDs in photos and (2) to determine if PNDs were indicative of poor body condition by comparing length-weight measurements and body mass index (BMI) values of animals with and without this trait.

For this study, I used records from the Stranding Investigations Program (SIP) at Mote Marine Laboratory because they included both photos and measurements of dolphins that died from a variety of causes, with and without a relationship to body condition.  I investigated data from 2000-2012, controlling for carcass decomposition and tissue loss, pregnancy, neonatal development, geographical variation in size, and ecotype.  I also accounted for photo quality by considering the angle a photo was taken, focus/clarity, contrast, body position, and the amount of dorsal surface included in the photo.  For the first objective, I developed a technique that involved drawing a straight, horizontal line across the post-nuchal region of a dolphin in a digital photo.  If a space was visible between the line and the dolphin’s dorsal surface in this region, it was considered to have a PND.  For each dolphin, I tested four different line drawing approaches to determine if the amount of dorsal surface that was considered affected the PND outcome.  For the second objective, I compared the body condition of PND and non-PND animals for each line drawing method using length-weight data and BMI values.

The results showed that the amount of dorsal surface considered for PND analysis can affect the PND outcome.  I recommend including as much dorsal surface in a photo as possible as far caudally as the anterior insertion of the dorsal fin to increase the accuracy of assessing PND presence.  In addition, dolphins with PNDs generally had length-weight values that were lower and had lower BMI values than individuals without PNDs.  The PND index appears to be a viable tool for assessing bottlenose dolphin body condition and can readily be applied to photos of both free-swimming and stranded dolphins.

This project was made possible by the support from an anonymous donation to the Chicago Zoological Society and by the University of Florida.  I would like to thank all of the SDRP and SIP for collecting the decades of health assessment and stranding data that were vital to my research.  I also thank Gretchen Lovewell, the SIP manager, for her generosity in giving her time to help me collect and organize my stranding sample.  I thank the SDRP staff for all of their help whenever I have needed it and for having taught me so much both as an intern and graduate student.  Finally, I thank my committee, Drs. Randall Wells, Bill Pine, and D. Ann Pabst for their guidance and support.

This article was published on pages 13-14 in the January 2014 Nicks n Notches.