Can a dragon overheat?

We’ve been studying gaping behaviours in bearded dragons for a while and one of Ian Black’s (former MSc student) thesis chapters has just been published! A link to the article is here: http://link.springer.com/article/10.1007/s00360-020-01332-y

We devised a simple way to prevent gaping (i.e. temporary and rapidly reversible) and examined how strongly this influenced thermoregulatory behaviours. Interestingly, although it did significantly lower thermal selection / thermal preference behaviour the effect was quite small. We also saw some interesting changes in heat orientation behaviour. Animals that were not able to gape behaved more randomly with respect to postural orientation, whereas the control lizards tend to shy away from orienting to hot temperatures (i.e. the definition of thermoregulation is to exhibit a corrective response when moving outside the set-point range).

Alas, we don’t have any cool images to share from this study, but consider looking at some of our other papers here and here where we have examined evaporative water loss and thermal imaging in bearded dragons.

The article is part of a special issue honouring Dr. Peter Frappell, a friend and colleague in respiratory and thermoregulatory physiology. Thanks Frapps for all your input and support!

Congratulations to Ian Black for getting this published and thanks to Dr. Laura Aedy for her early work on this project.

Citation

Black, IRG, Aedy, LK, and Tattersall, GJ. 2021. Hot and covered: how dragons face the heat and thermoregulate. Journal of Comparative Physiology B, In Presshttps://doi.org/10.1007/s00360-020-01332-y

Secrets of field thermography revealed?

We’re happy to share news that our paper (shareable link) was recently accepted in Methods in Ecology and Evolution! As for the nitty gritty secrets of the study, that headline was to get your attention! I’ll point out some highlights here, with some visuals to summarize the main results.

The study resulted from a visiting PhD student (Núria Montmany-Playà, @NuriaBeachy) to the lab in January 2020, immediately prior to the pandemic and lock-down. Sadly, some of the research we planned to do was not possible due to travel restrictions and embassies immediately calling back their citizens. So, this represents about the only kind of research my lab is capable of doing during a lockdown.

So, what is the paper about? Technically, it’s a methods (thus the journal choice!) and resource paper (information on empirical measurements of emissivity which I often get from colleagues) with a warning to field thermographers to “check your distance“! (Actually, Faye et al 2016 already advised this, but given various interactions I have had over the past few years, some field thermographers might not be reading all the literature – so please read Faye et al’s paper cited at the bottom of this blog).

Distance Effects

It has become increasing common in some animal thermography studies to capture the maximum temperature from a specific region of the body (often the face or the eye) and use this as an estimate of body temperature (it is not a good estimate!) or as a proxy for vasoreactivity related to stress (which it is a better indicator of, although depends on species and body part). This effect of distance was earlier studied by Faye et al (2016), although we focus on the challenges in animal thermography in the field where animals control how close you can get! Watch the video below for the effect in action, paying attention to the maximum temperature in the eye region and how it changes as this Trumpeter swan walks closer (starting from ~15 m) and closer (~2 m) to the camera:

Thermal video of a Trumpeter swan walking toward the camera. Max refers to the maximum temperature being captured (see plus sign) in the face/eye region.

Here are the results plotted over time as an animated plot:

Animation of the estimated maximum eye region temperature as the Trumpeter swan walks closer to the camera. Distance estimates are calculated based on an empirical relationship based on the camera’s focus distance estimate and vary depending on the focus and region being assessed. The major drop in temperature ~9 metres was due to a brief period of time where the image was out of focus (showing how crucial focus effects can be!!).

As the bird gets closer, the maximum temperature of the same animal rises from a low of ~25C to reach closer to a more realistic value of ~33C. This is an enormous range and cannot reasonably be based on vasoreactivity, when the physics of thermal optics can explain this. Read the paper for the explanation behind how spot size and distance effects interact to produce fairly large errors in thermography. Controlling for distance and/or being aware of its influences is important to any field application of thermography. I doubt too many people are trying to image animals at 10 m away given the low resolution of many thermal cameras, but as prices come down and people take their devices into the field, I am sure researchers will run into these situations.

We measured the technical error using a blackbody calibration source placed at different distances from the camera, and tested under different conditions. Below, Tb refers to the blackbody temperature (i.e. true temperature) and Ta refers to prevailing ambient temperature. Delta T refers to how the thermal camera estimates the same black body temperature (where 0 refers to the closest distance measurement). We tested this with different camera/lens combinations to give insight into how the devices function even under well controlled conditions, far different from most field applications. You can see below that the error in temperature can be as poor as 6C below actual temperature at 10 m distance, and these results are for measurements of an electronic calibration source.

Error (Delta T = offset in estimated temperature from true temperature) as a function of distance from a calibration source as a function of camera/lens and background radiation difference from blackbody calibration temperature, Ta – Tb).

Angle Effects

Thermal image of an Atlantic puffin taken 0.97 seconds apart after the bird changes head position. This sudden change in the angle of the bill leads to a decline in estimated surface temperature, unlikely to result from vasoreactive physiological events.

We also published some simple results on how the angle of incidence influences thermography measurements (see image above of the puffin). This is also well known by thermal imaging engineers and physicists, but not likely well appreciated by biologists, so we used a Leslie cube (see image below) and adhered various bits of biological materials to a copper surface, heated the surface up and painstakingly measured the temperature, allowing us to calculate the apparent emissivity. Objects at steep angles of incidence to the camera will have a lower emissivity, which means that the apparent radiation we measure is actually a lot of background reflected radiation, and thus is a source of possible error in field thermography.

A Leslie cube is typically made of copper or a highly conductive metal, onto which a surface paint or test material is adhered. The copper allows the material to be heated up to a constant temperature, while the angle of incidence can be adjusted and the apparent radiation (or temperature) of the surface of interest be estimated. The image above shows the same surface measured at different angles; at steeper angles the surface (dotted lines) appears cooler because it is reflecting more of the cooler room radiation back to the observer.

So the main message of the paper is to keep good records of how far your camera is from your object of study. Correcting for this effect is complex and beyond the scope of our study, although we report that the potential error of being 10 meters away from an object can be as high as 4 to 6C, with similar errors for measuring objects at angles greater than 50 degrees incidence.

Acknowledgements

Many thanks to Núria for her hard work on this project. Without her visit, we would not have done this. And we also want to thank the two reviewers for their hard but fair questions but also for listening to our response.

Lockdown science had me borrowing plant material from my parents, Clifford and Brenda Tattersall, so their help was crucial to the final acceptance of the manuscript!

Citation

Montmany-Playà, N. and Tattersall, GJ. 2021. Spot size, distance, and emissivity errors in field applications of infrared thermography. Methods in Ecology and Evolution. https://doi.org/10.1111/2041-210X.13563

References

Faye, E., Dangles, O. & Pincebourde, S. (2016) Distance makes the difference in thermography for ecological studies. Journal of Thermal Biology, 56, 1-9.

Lend us your thermal images!

Please consider taking part of an open repository initiative of thermal images hosted at the following website: https://trench-ir.azurewebsites.net/. If you are acquiring thermal images of plants, animals, or their environment using FLIR cameras, we would like you to share your images as part of this initiative. We welcome images from research grade cameras or from hand-held mobile phone provided the images are radiometric jpgs.

Infrared imagery offers a unique opportunity to see biophysical properties in real time. We can watch organisms heat up, cool down, and generally transfer heat back and forth throughout their environment. In the TrEnCh-IR Project, we use infrared imagery to help people see the world from a thermal perspective because we believe it’s an intuitive first step to understanding microclimate and the impacts of warming.

The TrEnCh-IR project is part of a larger initiative interested in Translating Environmental Change into organismal responses. Our goal is to build case studies of how animals are impacted by climate change to improve our approach to climate change biology education, policy, and research.

Mission

FLIR cameras are extensively used, increasingly so with the availability of FLIR thermal cameras that attach to phones. However, the cameras produce images in a non-standard format (radiometric jpgs) and analyzing the images requires purchasing expensive FLIR software. Project collaborator Tattersall has produced an open source R package (ThermImage, https://github.com/gtatters/Thermimage) that converts the images into standard formats and extracts additional data to allow analysis in commonly used and open source software such as ImageJ. Our web service makes these tools more accessible. We aim to empower more people to view the world from a thermal perspective.

Our thermal image repository will allow researchers to analyze the surface temperatures of disparate organisms in diverse environments. Education and outreach resources promote understanding how organisms experience their environment. We aim to maintain the repository long term, but can not guarantee longevity at this point. An ongoing aim is to use initial AI algorithms, potentially combined with crowd sourced landmarking, to distinguish organisms, particular body parts, and backgrounds. Our interface will allow users to explore the images to understand how organisms interact with their environments.

Motivation

Currently most analyses of the impacts of climate change on organisms are based on air temperatures, but body temperatures of ectotherms can differ from air temperatures by tens of degrees. Additionally, the characteristics and behaviours of organisms can result in their experiencing different body temperatures even in the same environment with repercussions for species interactions. Moving beyond air temperatures to consider body and surface temperatures may thus be essential to accurately forecasting climate change impacts. Thermal images provide compelling visual examples of why we need to move beyond air temperatures in examining climate change impacts as well as data that can inform approaches for modelling how organisms interact with their environment.

Team

Dr. Lauren Buckley, University of Washington, Professor

Abigail Meyer, Lead Developer & University of Washington Research Scientist

Dr. Glenn Tattersall, Brock University Professor & Thermal Biologist

Site development based with University of Washington, in the Department of Biology.

We are funded by AI for Earth, a Microsoft initiative.

Forced Hibernation on the CBC

My former MSc student and colleague, Anne Yagi’s research is featured on this CBC – Radio Canada link!

https://ici.radio-canada.ca/tele/la-semaine-verte/site/segments/reportage/211434/serpent-massasauga-espece-voie-disparition

This speaks to ~8 years of winter efforts to test this technique out in the lab and in the field! Hopefully we will publish soon. I’m not used to the CBC scooping us, but they don’t have to write the manuscripts and do the stats, so maybe I can excuse them.

These efforts are all related to a head starting project initiated by Anne Yagi on the Massasauga, taking physiological and behavioural data performed in careful lab experiments, testing these for 1-2 winters, then expanding to larger sample sizes in subsequent years to lead to the 9 minute videos above.

Many thanks to 8Trees Inc, Anne Yagi, Dr. Katharine Yagi and all of the animal care and field assistants that make Anne’s work possible (Theresa Bukovics, Tom Eles, Shawn Bukovac, Matt Jung, and many others).

Fish get the “rotten egg gas” chills.

At long last, resulting from herculean efforts of a number of former students, our paper is published. Out today in Royal Society Open Science, our paper entitled: “Hydrogen sulfide exposure reduces thermal set point in zebrafish” represents the efforts of two honours students (JC Shaw and CD Dobell) and the writing and analytical skills of a great PDF and colleague (DA Skandalis).

Here is a link to the study and full citation:

Skandalis DA, Dobell CD, Shaw JC, Tattersall GJ. 2020 Hydrogen sulfide exposure reduces thermal set point in zebrafish. R. Soc. Open Sci. 7: 200416.

https://royalsocietypublishing.org/doi/10.1098/rsos.200416

We tested whether dissolved H2S in the water will alter thermal preference. Previously, work in mice has suggested that mice could be induced to adopt a “hibernation-like” state, although there was some doubt (in the literature) as to whether H2S signalled a change in thermoregulatory state or simply acted as a metabolic inhibitor. By testing this in zebrafish, we could test formally whether they prefer cooler temperatures with H2S exposure, and they did. Not only did they choose to cool down, but they continued to make thermoregulatory decisions, swimming back and forth between cool and warmer water, suggesting they are still making thermoregulatory decisions and not simply caught in the cold water. So…yeah, complicated. H2S might induce a behavioural anapyrexia (a lowered thermal set-point). We discuss the potential environmental and neurophysiological context in the paper for those interested. The rotten egg reference is to the smell of H2S gas.

To conduct this study, we used a system built by Brock University’s Technical Services and employed in our research lab that allows us to track fish in a two chamber thermal shuttle box:

Schematic of the Shuttle Box System (see Figure S1 in the paper).

This setup allows us to heat and cool a tank and track the fish’s choices over time. Here is a thermal image depicting an earlier version of the shuttle box (correcting the spill over of warm-water in the centre can be corrected using baffles and a circular chamber system, but I haven’t taken a new picture with the thermal camera during the pandemic lockdown):

There was some considerable interest in developing H2S as a therapeutic to put mammals and/or tissues/organs into a suspended state. It is intriguing that animals like zebrafish that can behaviourally regulate body temperature continue to do so under this exposure. Anaprexic stategies are commonly seen in ectotherms and perhaps by hijacking an innate signalling system, H2S evokes this response.

Congratulations Anne Yagi, MSc!

Anne successfully defended her MSc research on “Flood Survival Strategies in Neonatal Snakes”. Congratulations!

Anne’s MSc research represents an amazing amount of research into overwintering snake behaviour and physiology, and what is in her thesis is still only a fraction of the research she has pursued while a Masters student in my lab!

Some highlights from her (online) defence:

With a teary eye for the closing of this particular chapter in our collaboration (as student and advisor), I am very proud for her achievement. I still expect many more years of conversations and collaborations!

We owe a lot of gratitude to many people, including the Yagi family, for their support of Anne while she pursued her MSc as full-time employee and for their help with data extraction from behaviour videos. Katharine Yagi in particular needs acknowledgement for her herculean efforts with statistical analyses and patiently working with us!

Thank you for the examining committee:

Dr. Cheryl McCormick, Chair (and microphone manager)
Dr. Bruce Kingsbury (external examiner)
Dr. Liette Vasseur (committee member)
Dr. Fiona Hunter (committee member)

Video based quantification of activity

Although not an inspiring or catchy title, our study has just been published, demonstrating that fixed-frame video capture can provide a quantitative assessment of energetics (citation below).

Summary of the study

Infrared thermal imaging is a passive imaging technique that captures the emitted radiation from an object to estimate surface temperature, often for inference of heat transfer.

Infrared thermal imaging offers the potential to detect movement without the challenges of glare, shadows, or changes in lighting associated with visual digital imaging or active infrared imaging.

In this paper, we employ a frame subtraction algorithm for extracting the pixel-by-pixel relative change in signal from a fixed focus video file, tailored for use with thermal imaging videos.  

A greyscale video of a metronome. Inanimate objects are still visible to the thermal camera due to slight differences in thermal equilibration, emissivity, and reflectivity across the different surfaces.
Same video as above, but as an “absolute difference image”, where each frame is subtracted from the previous frame and positized (i.e. the absolute value taken for each pixel difference). During zero motion, the image space is black with low variability based on sensor noise (sd number in upper left). After motion starts (~0:11), you clearly see the moving metronome as each frame difference depicts how pixels in a fixed frame differ.

By then cumulatively summing the sd for each frame across an entire video, we are able to assign quantitative activity assessments to thermal imaging data for comparison with simultaneous recordings of metabolic rates. We tested the accuracy and limits of this approach by analyzing movement of a metronome (see above) and provide an example of the approach to a study of Darwin’s finches.

Fixed frame Different Image Thermography video of a bird filmed over a long period of time within a viewing chamber. The cumulative difference image provides a slope value that corresponds to an indication of movement. Scale is relative to the degree of motion.

Simultaneous measurements of oxygen consumption (dark black lines) match up well with smoothed estimates (blue lines) of the activity score (grey lines) derived from the difference image thermography videos. Resolving fast respirometry data is difficult and best left to the experts at Sable Systems, but the average trends are informative for energy expenditure.
Correlations between 5 minute average oxygen consumption measurements and the activity values line up well at the individual level.

In principle, this “Difference Imaging Thermography” (DIT) would allow for activity data to be standardized to energetic measurements and could be applied to any radiometric imaging system.

Caveats

Fixed frame is required. Changing the reference frame or using a camera without a tripod would not work unless you do a lot of motion correction. Also, we used infrared thermal imaging because we were collecting data for a different purpose (still writing those up!), but we think that any sort of imaging should work, provided it produces a simple, ratiometric or radiometric image. Usually monochrome cameras or near infrared cameras produce a signal that is a simple greyscale image. Reflected light might interfere with the approach, so this is why we argued this might be an addition use of thermal imaging videos.

Behind the scenes

This paper took a long time to put together, but was the beginning of my lab’s journey into R, ImageJ, and open source coding. A lot has changed since I started the data analysis. Combing through 500 Gb of video files, extracting, processing, converting them into something manageable took the better part of 2 weeks on a supercomputer, until I realised that there were more efficient routes!

We have since created routines in ImageJ that help facilitate the conversions and have placed those routines in a github repository, where we will write them up as a methods paper in the future. The principles outlined in the paper are not themselves novel. Sliding average and frame subtraction routines are common in video processing software. Assessing whether the motion captured is correlated with meaningful biological information is what we hoped to capture with the study.

Citation

Tattersall, GJ, Danner, RM, Chaves, JA, and Levesque, DL. 2020. Activity analysis of thermal imaging videos using a difference imaging approach. Journal of Thermal Biology.  91 102611 https://doi.org/10.1016/j.jtherbio.2020.102611

Link to the full text here (first 50 clicks can access the paper).

Congratulations, Nick Sakich, MSc!

In a first for the lab, we just held a completely online MSc defence. Valiantly, Nick defended his MSc with grace, precision, insight, humour, and interesting anecdotes.

His thesis is entitled: “The Physiological and Behavioural Consequences of Reduced Scalation in Captive-bred Phenotypes of the Bearded Dragon (Pogona vitticeps Ahl 1926)“.

Here he is giving his presentation (sorry for the screen cap, Nick)

speaking about one of our favourite study animals:

The 3 phenotypes of captive-bred Pogona vitticeps studied in Nick’s thesis. a) wildtype, b) leatherback, and c) silkback.

Many thanks to the examining committee:

External Examiner, Dr. Chris Oufiero, Towson University
Chair, Dr. Cheryl McCormick
Committee Member, Dr. Jeff Stuart
Committee Member, Dr. Robert Carlone
Supervisor, Dr. Glenn Tattersall

Virtual congratulations to Nick are insufficient expression of gratitude for his hard work and devotion to his research. When and if we can safely congregate in small groups, we will celebrate appropriately! I really owe Nick my thanks for joining my lab. He has helped educate me through his efforts. It cannot be fun wrapping up one’s MSc during a pandemic, and Nick did a brilliant job.

A few highlights from Nick’s presentation below.

Thanks as well to A&A Dragons for their support over the years.

Massassauga Rattlesnake Overwintering Lifezone

Congratulations to lab member, Anne Yagi on publishing a life’s work of research on the overwintering lifezone of the Eastern Massassauga rattlesnake! The final proofs have been sent back to the publisher and we are anxiously awaiting it to make it to press:

Summary of the study here, with links to the paper below.

Temperate snakes occupy overwintering sites for most of their annual life cycle. Microhabitat characteristics of the hibernaculum are largely undescribed yet are paramount in ensuring snake overwintering survival.

We hypothesized that snakes survive hibernation within a vertical subterranean space that we termed a “life zone”, that is aerobic, flood, and frost-free throughout winter and did this by studying an isolated, endangered population of Massasaugas (Sistrurus catenatus) inhabiting an anthropogenically-altered peatland and monitored the subterranean habitat during a period of environmental stochasticity.

Lifezone concept (Credit: A Yagi)

Initial radio telemetry confirmed that snakes moved between altered and natural habitats during the active season and showed hibernation site fidelity to either habitat. We used a grid of groundwater wells, and frost tubes installed in each hibernation area to measure lifezone characteristics over 11 consecutive winters.

The lifezone within the impacted area was periodically reduced to zero during a flood-freeze cycle, while the lifezone in the natural area was maintained.

Sample figure from the paper showing year by year changes in the winter lifezone size (cm = depth or size underground that remains frost and flood free). Mined sites refer to an anthropogenically disturbed site where surface peat extraction had historically occurred. Unmined site is a peat bog. Flood events refer to a period of time when large regions of the site experienced sustained surface flooding.

Soil-depth and flood status best predicted lifezone size. Thermal buffering and groundwater dissolved oxygen increased with lifezone size, and annual Massasauga encounters were significantly correlated with lifezone size.

This analysis suggests a population decline occurred when lifezone size was reduced by flooding. Our data give support to the importance and maintenance of a lifezone for successful snake hibernation.

Our methods apply to subterranean hibernation habitats that are at risk of environmental stochasticity, causing flooding, freezing, or hypoxia, and speak to the issues regarding management of sensitive watersheds inhabitated by species-at-risk.


Snake well installation in the field to test the overwintering lifezone.

Citation

Yagi, A, Planck, RJ, Yagi, KT, and Tattersall GJ. 2020. A long-term study on Massasaugas (Sistrurus catenatus) inhabiting a partially-mined peatland: presenting a standardized method to characterize snake overwintering habitat. Journal of Herpetology. 54: 235-244. https://journalofherpetology.org/doi/full/10.1670/18-143

For further information, please see 8Trees.ca.