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).
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.
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, Nick! We just heard from the Faculty of Graduate Studies that Nick has received the 2020 Fall Distinguished Graduate Student Award for his MSc project. A well deserved award and a credit to Nick’s hard work.
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)
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).
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.
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.
Measuring the lifezone. 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
The lab will be hosting a PhD student from Spain for the next 3 months.
Núria Playà Montmany from the University of Extremadura has just arrived (I’m a few days late, she arrived in late January!). I met Núria last summer defending her poster at the SEB meeting in Sevilla. She will be becoming a thermal imaging expert while she is here!
With overlapping interests in avian physiology and the lab’s interests in thermal biology and studying animal responses to climate change, we hope to have a productive visit. Here is a link to Núria’s blog:
It has been a busy January, and so my updates are out of date! From Jan 10th to the 24th, I hosted Dr. Agnes Jullian Vinet in the lab, mostly to learn thermal imaging for future human thermogenesis research.
Dr. Vinet was a welcome visitor, enduring the dark, but not so cold, Canadian winter with us! Thanks to Stephen Cheung’s lab, Gary Hodges and Leed McNab for helping to host Agnes.
Hopefully we can return the favour and visit her lab next summer!
