The learning curve never stops as the GRANITE project begins its seventh field season

Clara Bird, PhD Student, OSU Department of Fisheries, Wildlife, and Conservation Sciences, Geospatial Ecology of Marine Megafauna Lab

When I thought about what doing fieldwork would be like, before having done it myself, I imagined that it would be a challenging, but rewarding and fun experience (which it is). However, I underestimated both ends of the spectrum. I simultaneously did not expect just how hard it would be and could not imagine the thrill of working so close to whales in a beautiful place. One part that I really did not consider was the pre-season phase. Before we actually get out on the boats, we spend months preparing for the work. This prep work involves buying gear, revising and developing protocols, hiring new people, equipment maintenance and testing, and training new skills. Regardless of how many successful seasons came before a project, there are always new tasks and challenges in the preparation phase.

For example, as the GEMM Lab GRANITE project team geared up for its seventh field season, we had a few new components to prepare for. Just to remind you, the GRANITE (Gray whale Response to Ambient Noise Informed by Technology and Ecology) project’s field season typically takes place from June to mid-October of each year. Throughout this time period the field team goes out on a small RHIB (rigid hull inflatable boat), whenever the weather is good enough, to collect photo-ID data, fecal samples, and drone imagery of the Pacific Coast Feeding Group (PCFG) gray whales foraging near Newport, OR, USA. We use the data to assess the health, ecology and population dynamics of these whales, with our ultimate goal being to understand the effect of ambient noise on the population. As previous blogs have described, a typical field day involves long hours on the water looking for whales and collecting data. This year, one of our exciting new updates is that we are going out on two boats for the first part of the field season and starting our season 10 days early (our first day was May 20th). These updates are happening because a National Science Foundation funded seismic survey is being conducted within our study area starting in June. The aim of this survey is to assess geophysical structures but provides us with an opportunity to assess the effect of seismic noise on our study group by collecting data before, during, and after the survey. So, we started our season early in order to capture the “before seismic survey” data and we are using a two-boat approach to maximize our data collection ability.

While this is a cool opportunistic project, implementing the two-boat approach came with a new set of challenges. We had to find a second boat to use, buy a new set of gear for the second boat, figure out the best way to set up our gear on a boat we had not used before, and update our data processing protocols to include data collected from two boats on the same day. Using two boats also means that everyone on the core field team works every day. This core team includes Leigh (lab director/fearless leader), Todd (research assistant), Lisa (PhD student), Ale (new post-doc), and me (Clara, PhD student). Leigh and Todd are our experts in boat driving and working with whales, Todd is our experienced drone pilot, I am our newly certified drone pilot, and Lisa, Ale, and myself are boat drivers. Something I am particularly excited about this season is that Lisa, Ale, and I all have at least one field season under our belts, which means that we get to become more involved in the process. We are learning how to trailer and drive the boats, fly the drones, and handling more of the post-field work data processing. We are becoming more involved in every step of a field day from start to finish, and while it means taking on more responsibility, it feels really exciting. Throughout most of graduate school, we grow as researchers as we develop our analytical and writing skills. But it’s just as valuable to build our skillset for field work. The ocean conditions were not ideal on the first day of the field season, so we spent our first day practicing our field skills.

For our “dry run” of a field day, we went through the process of a typical day, which mostly involved a lot of learning from Leigh and Todd. Lisa practiced her trailering and launching of the boat (figure 1), Ale and Lisa practiced driving the boat, and I practiced flying the drone (figure 2). Even though we never left the bay or saw any whales, I thoroughly enjoyed our dry run. It was useful to run through our routine, without rushing, to get all the kinks out, and it also felt wonderful to be learning in a supportive environment. Practicing new skills is stressful to say the least, especially when there is expensive equipment involved, and no one wants to mess up when they’re being watched. But our group was full of support and appreciation for the challenges of learning. We cheered for successful boat launchings and dockings, and drone landings. I left that day feeling good about practicing and improving my drone piloting skills, full of gratitude for our team and excited for the season ahead.

Figure 1. Lisa (driving the truck) launching the boat.
Figure 2. Clara (seated, wearing a black jacket) landing the drone in Ale’s hands.

All the diligent prep work paid off on Saturday with a great first day (figure 3). We conducted five GoPro drops (figure 4), collected seven fecal samples from four different whales (figure 5), and flew four drone flights over three individuals including our star from last season, Sole. Combined, we collected two trifectas (photo-ID images, fecal samples, and drone footage)! Our goal is to get as many trifectas as possible because we use them to study the relationship between the drone data (body condition and behavior) and the fecal sample data (hormones). We were all exhausted after 10 hours on the water, but we were all very excited to kick-start our field season with a great day.

Figure 3. Lisa on the bow pulpit during our first sighting of the day.
Figure 4. Lisa doing a GoPro drop, she’s lowering the GoPro into the water using the line in her hands.
Figure 5. Clara and Ale collecting a fecal sample.

On Sunday, just one boat went out to collect more data from Sole after a rainy morning and I successfully flew over her from launching to landing! We have a long season ahead, but I am excited to learn and see what data we collect. Stay tuned for more updates from team GRANITE as our season progresses!

Are bacteria important? What do we get by analyzing microbiomes?

By Leila Lemos, PhD candidate, Fisheries and Wildlife Department, OSU

As previously mentioned in one of Florence’s blog posts, the GEMM Lab holds monthly lab meetings, where we share updates about our research and discuss articles and advances in our field, among other activities.

In a past lab meeting we were asked to bring an article to discuss that had inspired us in the past to conduct research in the marine field or in our current position. I brought to the meeting a literature review regarding methodologies to overcome the challenges of studying conservation physiology in large whales [1]. This article discusses different non-invasive or minimally invasive matrices (e.g., feces, blow, skin/blubber) that can be gathered from whales, and what types of analyses could be carried out, as well as their pros and cons.

One of the possible analyses that can be performed with fecal samples that was discussed in the article is the gut microflora (i.e., bacterial gut community) via genetic analysis. Since my PhD project analyzes fecal samples to determine/quantify stress responses in gray whales, we have since discussed the possibility of integrating this extra parameter to our analysis.

But… what is the importance of analyzing the gut microflora of a whale? What is the relationship between microflora and stress responses? Should we really use our limited sample size, time and money to work on this extra analysis? In order to be able to answer all of these questions, I began reading some articles of the field to better understand its importance and what kind of research questions this analysis can answer.

The gut of a mammal comprises a natural habitat for a large and dynamic community of bacteria [2] that is first developed in early life. Colonization of facultative bacteria (i.e., aerobic bacteria) begins at birth [3], and later, anaerobic bacteria also colonizes the gut. In humans, at the age of 1 year old, the microbiome should have a stable adult-like signature (Fig. 1).

Figure 01: Development of the microbiome in early life.
Source: [3]
 

The gut bacterial community is important for the physiology and pathology of its host and plays an important role in mammal digestion and health [2], responsible for many metabolic activities, including:

  • fermentation of non-digestible dietary residue and endogenous mucus [2];
  • recovery of energy [2];
  • recovery of absorbable nutrients [2];
  • cellulose digestion [4];
  • vitamin K synthesis [4];
  • important trophic effects on intestinal epithelia (cell proliferation and differentiation) [2];
  • angiogenesis promotion [4];
  • enteric nerve function [4];
  • immune structure [2];
  • immune function [2];
  • protection of the colonized host against invasion by alien microbes (barrier effect) [2];

Despite all the benefits, the bacterial community might also be potentially harmful when changes in the community composition (i.e., dysbiosis) occur due to the use of antibiotics, illness, stress, aging, lifestyle, bad dietary habits [4], and prolonged food and water deprivation [5]. Thus, potential pathological disorders might emerge when the microbiome community changes, such as allergy, obesity, diabetes, autism, multisystem organ failure, gastrointestinal and prostate cancers, inflammatory bowel diseases (IBD), and cardiovascular diseases [2, 4].

Changes in gut bacterial composition may also alter the brain-gut axis and the central nervous system (CNS) signaling [3]. More specifically, the core pathway affected is the hypothalamic-pituitary-adrenal (HPA) axis, which is activated by physical/psychological stressors. According to a previous study [6], the microbial community in the gut is critical for the development of an appropriate stress response. In addition, the microbial colonization in early life should occur within a certain time window, otherwise an abnormal development of the HPA axis might happen.

However, the gut microbiome can not only affect the HPA axis, but the opposite can also occur [3]. Signaling molecules released by the axis can alter the gastrointestinal (GIT) environment (i.e., motility, secretion, and permeability) [7]. Stress responses, as well as diseases, may also alter the gut permeability, causing the bacteria to cross the epithelial barrier (reducing the overall numbers of bacteria in the gut), activating immune responses that also alter the composition of the bacterial community in the gut [8, 9].

Figure 02: Communication between the brain, gut and microbiome in a healthily and in a stressed or diseased (mucosal inflammation) mammal.
Source: [3]
 

Thus, when thinking about whales, monitoring of the gut microflora might allow us to detect changes caused by factors such as aging, illness, prolonged food deprivation, and stressful events [2, 5]. However, since these are two-way factors, it is important to find an association between bacterial composition alterations and stressful events, such as the presence of predators (e.g., killer whales), illness (e.g., bad body condition), prolonged food deprivation (e.g., low prey availability and high competition), noise (e.g., noisy vessel traffic, fisheries opening and seismic surveys), and stressful reproductive status (e.g., pregnancy and lactating period). Examination of possible shifts in the gut microflora may be able to detect and be linked to many of these events, and also forecast possible chronic events within the population. In addition, the bacterial community monitoring study could aid in validating the hormone data (i.e., cortisol) we have been working with.

Therefore, the main research questions that arise in this context that can aid in elucidating the stress physiology in gray whales are:

  1. What is the microflora community content in guts of gray whales along the Oregon coast?
  2. Is it possible to detect shifts in the gut microflora from our gray fecal samples over time?
  3. How do gut microflora and cortisol levels correlate?
  4. Am I able to correlate shifts in gut microflora with any of the stressful events listed above?

We can answer so many other questions by analyzing the microbiome of baleen whales. Microbiomes are mainly correlated with host diet [10], so the composition of a microbiome can be associated with specific diets and functional gut capacity, and consequently, be linked to other animal populations, which helps to decode evolutionary questions. Results of a previous study on baleen whale microbiomes [10] point out that whales harbor unique gut microbiomes that are actually similar to those of terrestrial herbivores. Baleen whales and terrestrial herbivores have a shared physical structure of the GIT tract itself (i.e., multichambered foregut) and a shared hole for fermentative metabolisms. The multichambered foregut of baleen whales fosters the maintenance of the gut microbiome that is capable of extracting relatively unavailable nutrients from zooplankton (i.e., chitin, “sea cellulose”).

Figure 03: The similarities between whale and other terrestrial herbivore gut microbiomes: sea and land ruminants.
Source: [11]
 

Thus, the importance of studying the gut microbiome of a baleen whale is clear. Monitoring of the bacterial community and possible shifts can help us elucidate many questions regarding diet, overall health, stress physiology and evolution. Thinking about my PhD project, it may also help in validating our cortisol level results. I am confident that a microbiome analysis would significantly enhance my studies on the health and ecology of gray whales.

 

References

  1. Hunt, K.E., et al., Overcoming the challenges of studying conservation physiology in large whales: a review of available methods.Conservation Physiology, 2013. 1: p. 1-24.
  2. Guarner, F. and J.-R. Malagelada, Gut flora in health and disease.The Lancet, 2003. 360: p. 512–519.
  3. Grenham, S., et al., Brain–gut–microbe communication in health and disease.Frontiers in Physiology, 2011. 2: p. 1-15.
  4. Zhang, Y., et al., Impacts of Gut Bacteria on Human Health and Diseases.International Journal of Molecular Sciences, 2015. 16: p. 7493-7519.
  5. Bailey, M.T., et al., Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium.Infection and Immunity, 2010. 78: p. 1509–1519.
  6. Sudo, N., et al., Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice.The Journal of Physiology, 2004. 558: p. 263–275.
  7. Rhee, S.H., C. Pothoulakis, and E.A. Mayer, Principles and clinical implications of the brain–gut–enteric microbiota axis Nature Reviews Gastroenterology & Hepatology, 2009. 6: p. 306–314.
  8. Kiliaan, A.J., et al., Stress stimulates transepithelial macromolecular uptake in rat jejunum.American Journal of Physiology, 1998. 275: p. G1037–G1044.
  9. Dinan, T.G. and J.F. Cryan, Regulation of the stress response by the gut microbiota: Implications for psychoneuroendocrinology.Psychoneuroendocrinology 2012. 37: p. 1369—1378.
  10. Sanders, J.G., et al., Baleen whales host a unique gut microbiome with similarities to both carnivores and herbivores.Nature Communications, 2015. 6(8285): p. 1-8.
  11. El Gamal, A. Of whales and cows: the baleen whale microbiome revealed. Oceanbites 2016[cited 2018 07/31/2018]; Available from: https://oceanbites.org/of-whales-and-cows-the-baleen-whale-microbiome-revealed/.

 

Challenges of fecal hormone analyses (Round 2): finally in Seattle!

By Leila Lemos, Ph.D. Student, Department of Fisheries and Wildlife, OSU

In a previous blog of mine, you could read about the challenges I have been facing while I am learning to analyze the hormone content in fecal samples of gray whales (Eschrichtius robustus). New challenges appeared along the way over the last month, while I was doing my training at the Seattle Aquarium (Fig. 1).

Figure 1: View of the Seattle Aquarium.

 

My training lasted a week and I am truly grateful to the energy and time our collaborators Shawn Larson (research coordinator), Amy Green and Angela Smith (laboratory technicians) contributed. They accompanied me throughout my training to ensure I would be able to conduct hormonal analysis in the future, and to handle possible problems along the way.

The first step was weighing all of the fecal samples (Fig. 2A). Subsequently, the samples were transferred to appropriate glass tubes (Figs. 2B & 2C) for the next laboratorial step.

Figure 2: Analytical processes: (A) Sample weighing; (B) Transference of the sample to a glass tube; (C) Result from the performed steps.

 

The second conducted step was the hormone extraction. The extraction began with the addition of an organic solvent, called methanol (CH3OH), to the sample tubes (Fig. 3A & 3B). Hormones leach out from the samples and dissolve in the methanol, due to their affinity for this polar solvent.

Tubes were then placed on a plate shaker (Fig. 3C) for 30 minutes, which is used to mix the substances, in order extract the hormones from the fecal samples. The next step was to place the tubes in a centrifuge (Fig. 3D) for 20 minutes. The centrifuge uses the sedimentation principle, causing denser substances or particles to settle to the bottom of the tube, while the less dense substances rise to the top.

Figure 3: Analytical processes: (A) Methanol addition; (B) Sample + methanol; (C) Plate shaker; (D) Centrifuge.

 

After this process, the two different densities were separated: the high-density particles of the feces were in the bottom of the tube, while the methanol containing the extracted hormones was at the top. The top phase (methanol + hormones) was then pipetted into a different tube (Fig. 4A). The solvent was then evaporated, by using an air dryer apparatus (Fig. 4B), with only the hormones remaining in the tube.

The third performed step was dilution. A specific amount of water, measured in correlation with sample weight and to the amount of the methanol mixed with each sample, was added to each tube (Fig. 4C). Since the hormones were concentrated in the methanol, the readings would exceed the measurement limits of the equipment (plate reader). Thus, in order to prepare the extracts for the immunoassays, different dilutions were made.

Figure 4: Analytical processes: (A) Methanol transference; (B) Methanol drying; (C) Water addition.

 

The fourth and final step was to finally conduct the assays. Each assay kit is specific to the hormone to be analyzed with specified instructions for each kit. Since we were analyzing four different hormones (cortisol, testosterone, progesterone, and triiodothyronine – T3) we followed four different processes accordingly.

First, a table was filled with the identification numbers of the samples to be analyzed in that specific kit (Fig. 5A). The kit (Fig. 5B) includes the plate reader and several solutions that are used in the process to prepare standard curves, to initiate or stop chemical reactions, among other functions.

A standard curve, also known as calibration curve, is a common procedure in laboratory analysis for determining the concentration of an element in an unknown sample. The concentration of the element is determined by comparison with a set of standard samples of known concentration.

The plate contains several wells (Fig. 5C & 5D), which are filled with the samples and/or these other solutions. When the plate is ready, (Fig.5D) it is carried to the microplate reader that measures the intensity of the color of each of the wells. The intensity of the color is inversely proportional to the concentration of the hormone in both the standards and the samples.

Figure 5: (A) Filling the assay table with the samples to be analyzed; (B) Assay kit to be used; (C) Preparation of the plate; (D) Plate ready to be read.

 

Since this is the first fecal hormone analysis being performed in gray whales, a validation process of the method is required. Two different tests (parallelism and accuracy) were performed with a pool of three different samples. Parallelism tests that the assay is measuring the antigen (hormone) of interest and also identifies the most appropriate dilution factor to be used for the samples. Accuracy tests that the assay measurement of hormone concentration corresponds to the true concentration of the sample (Brown et al. 2005).

This validation process only needs to be done once. Once good parallelism and accuracy results are obtained, and we have identified the correct dilution factor and approximate concentration of the samples, the samples are ready to be analyzed. Below you can see examples of a good parallelism test (parallel displacement; Fig. 6) and bad parallelism tests (Fig. 7) that indicate no displacement, low concentration or non-parallel displacement; and a good accuracy test (Fig. 8).

Figure 6: Example of a good parallelism test. The dark blue line indicates the standard curve; the pink line indicates a good parallelism test, showing a parallel displacement; and the ratios in black indicate the dilution factors.
Source: Brown et al. (2005)

 

Figure 7: Examples of bad parallelism tests. The dark blue line indicates the standard curve; the light blue line is an example of no displacement; the pink line is an example of low concentration of the sample; and the green line is an example of non-parallel displacement.
Source: Brown et al. (2005)

 

Figure 8: Example of a good accuracy test while analyzing hormone levels of pregnanediol glucuronide (Pdg) in elephant urine. The graph shows good linearity (R2 of 0.9986) and would allow for accurate concentration calculations.
Source: Brown et al. (2005)

 

After the validation tests returned reliable results, the samples were also analyzed. However, many complications were encountered during the assay preparations and important lessons were learned that I know will allow this work to proceed more smoothly and quickly in the future. For instance, I now know to try to buy assay kits of the same brand, and to be extremely careful while reading the manual of the process to be performed with the assay kit. With practice over the coming years, my goal is to master these assay preparations.

Now, the next step will be to analyze all of the results obtained in these analyses and start linking the multiple variables we have from each individual, such as age, sex and body condition. The results of this analysis will lead to a better understanding of how reproductive and stress hormones vary in gray whales, and also link these hormone variations to nutritional status and noise events, one of my PhD research goals.

 

Cited Literature:

Brown J, Walker S and Steinman K. 2005. Endocrine manual for reproductive assessment of domestic and non-domestic species. Smithsonian’s National Zoological Park, Conservation and Research Center, Virginia 1-69.

Challenges of fecal analyses (Round 1)

By Leila Lemos, Ph.D. Student, Department of Fisheries and Wildlife, OSU

Fieldwork is done for the year and lab analyses just started with some challenges. This is not unexpected since no previous hormonal analysis has been conducted with any gray whale tissue, and whale fecal sample analysis is a relatively new technique. So, I have been thinking, learning, consulting, and creating a methodology as I go along. I am grateful to the expert advice and help from many great collaborators:

  • Kathleen Hunt (Northern Arizona University, AZ, United States)
  • Shawn Larson (Seattle Aquarium, WA, United States)
  • Amy Green (Seattle Aquarium, WA, United States)
  • Rachel Ann Hauser-Davis (Fiocruz, RJ, Brazil)
  • Maziet Cheseby (Oregon State University, OR, United States)
  • Scott Klasek (Oregon State University, OR, United States)

I have learned that an important step before undertaking fecal a hormonal analysis is the desalting process of the samples since salts can interfere in hormonal determinations, leading to false results. In order to remove salt content, each sample was first filtered (Fig. 1A), to remove a majority of the salt water content (Fig. 1B) that is inevitably collected along with the fecal sample. Each sample was then re-suspended in ultra-pure water, to dilute the remaining salt content in a higher water volume (Fig. 1C).

Figure 1: Analytical processes: (A) Filtration of the samples; (B) Result from filtration; (C) Addition of pure water to the samples.
Figure 1: Analytical processes: (A) Filtration of the samples; (B) Result from filtration; (C) Addition of pure water to the samples.

After these steps were completed for each sample, the samples were centrifuged (Fig. 2A) to  precipitate the fecal matter and leave the lighter salt ions in the supernatant (the liquid lying above a solid residue; Fig. 2B). After finishing these two phases, the water was removed with aid of a plastic pippete (Fig. 2C), and I was left with only desalted fecal at the bottom of the tubes (Fig. 2D).

Figure 2: Analytical processes: (A) Samples centrifugation; (B) Result from the centrifugation; (C, D) Results from separating water and sample.
Figure 2: Analytical processes: (A) Samples centrifugation; (B) Result from the centrifugation; (C, D) Results from separating water and sample.

The fecal samples were then frozen at -80°C (Fig. 3A & 3B) and then freeze-dried on a lyophilizer for 2 days to remove all remaining water content (Fig. 3C). Finally, I have what I need: desalted, dry fecal samples, ready for hormone analysis (Fig. 3D).

Figure 3: Analytical processes: (A) Freezing process of the samples; (B) Frozen samples ready to go to the lyophilizer; (C) Samples in the lyophilizer; (D) Final result of the lyophilizing process.
Figure 3: Analytical processes: (A) Freezing process of the samples; (B) Frozen samples ready to go to the lyophilizer; (C) Samples in the lyophilizer; (D) Final result of the lyophilizing process.

Writing this now, this process seems simple, but it was laborious, and took time to find the equipment needed at the right times. The end product is crucial to get a good final result, so my time investment (and my own increased stress level!) was worth it. This type of analysis is very new for marine mammals and our research lab is still in the learning the best methods. Along the way we were unsure of some decisions, some mistakes were made, and we were afraid of losing precious fecal material. But, this is the fun and challenge of working with a new species and new type of sample and, importantly, we have developed a working protocol that should make the process more efficient and reduce our stress levels next time around.

At the end of this sample preparation process, our 53 samples look great and are ready to be analyzed during my training at the Seattle Aquarium. We are also planning to analyze the water that was removed from the samples (Fig. 2D) to see if any hormone leached out from the poop into the water.

Results from this process will aid in future whale fecal hormone studies. Perhaps only the centrifugation step is needed and we can discard the water without losing hormone content. Or, perhaps we need to analyze both portions of the sample and sum the hormones found in each. We shall know the answer when we get our hormone metabolite results. Just another protocol to be worked out as I move ahead with the hormone analysis of these fecal samples. And through all these challenges I keep the end goal of this work in my mind: to learn about the reproductive and stress hormonal variation in gray whales and to link these variations to nutritional status and noise events. Onward!