Thermal imaging of respiratory patterns during vocalisation

Our paper “Vocalization associated respiration patterns: thermography-based monitoring and detection of preparation for calling” was just published in the Journal of Experimental Biology! This was one of the most enjoyable research projects I have been part of lately, but also one of the more complex journeys for a research paper. Huge credit must go to Vlad Demartsev (Max Plank Institute of Animal Behaviour), lead author on the project! Congratulations, Vlad!

Here is the abstract of the study

Vocal emission requires coordination with the respiratory system. Monitoring the increase in laryngeal pressure, needed for vocal production, allows detection of transitions from quiet respiration to vocalization-supporting respiration. Characterization of these transitions could be used to identify preparation for vocal emission and to examine the probability of it manifesting into an actual vocal production event. Specifically, overlaying the subject’s respiration with conspecific calls can highlight events of call initiation and suppression, as a mean of signalling coordination and avoiding jamming. Here we present a thermal-imaging based methodology for synchronized respiration and vocalization monitoring of free ranging meerkats. The sensitivity of this methodology is sufficient for detecting transient changes in the subject’s respiration associated with the exertion of vocal production. The differences in respiration are apparent not only during the vocal output but also prior to it, marking the potential time frame of the respiratory preparation for calling. A correlation between conspecific calls with elongation of the focal subject’s respiration cycles could be related to fluctuations in attention levels or in the motivation to reply. This framework can be used for examining animals’ capability for enhanced respiration control during modulated and complex vocal sequences, detect “failed” vocalisation attempts and investigate the role of respiration cues in the regulation of vocal interactions.

Here is the supplementary video from the paper, demonstrating a thermal image video (taken with a FLIR T1030) of a basking and vocalisating meerkat. We also demonstrate the syncrhonisation procedure and show how the machine learning algorithm trained to identify the nostrils was used to obtain the coordinates from which we could go back to extract the median nostril temperature associated with inhalation and exhalation.

Technologically this was one of the most complicated studies I’ve worked on. It involved 3 weeks of some of most exciting field work in the Kalahari Desert (waiting for Meerkats to come out of their burrows in the morning), high resolution thermal imaging, high resolution audio recording, elaborate device synchronisation, ImageJ, R, machine learning, cigarette lighters, and more PERL code than I ever want to have to write again.

Long story short: we estimated the rhythmic pattern of inhalation and exhalation from the periodic changes in nostril temperature due to evaporative cooling (inhalation) and respiratory warming (exhalation). These breaths were tracked along with the morning “sunning calls” (i.e. vocalisations), and we were able to detect the subtle changes in their breathing patterns that emerge as a result of their calls.

Citation

Demartsev, V, Manser, MB, and Tattersall, GJ. 2022. Vocalization associated respiration patterns: thermography-based monitoring and detection of preparation for calling. Journal of Experimental Biology, In Press, https://doi.org/10.1242/jeb.243474.

Acknowledgements

This work was done while VD was funded by Minerva Stiftung and Alexander von Humboldt-Stiftung post-doctoral fellowships. Additional funding included Internationalization Initiative Start Up funding, University of Konstanz and Aharon and Ephraim Katzir Study Grant, The Israel Academy of Sciences and Humanities. The Natural Sciences and Engineering Research Council of Canada supported GJT’s research and thermal imaging camera (RGPIN-05814). MBM was funded by the University of Zurich. This article has relied on records of individual identities and/or life histories maintained by the Kalahari Meerkat Project, which has been supported financially by the European Research Council (Grant No 742808 to Tim Clutton-Brock, University of Cambridge since 1 July 2018) and the University of Zurich, as well as logistically by the Mammal Research Institute of the University of Pretoria.

Peripheral vasomotion and systemic inflammation

Congratulations to Dr. Simon Tapper for his publication in Physiological and Biochemical Zoology, entitled: “Changes in body surface temperature play an under-appreciated role in the avian immune response”. Simon worked on this as part of his PhD with Dr. Gary Burness at Trent University. He very kindly included Josh Tabh and myself in this paper, although the bulk of the work was done by Simon. A main take-away from the study is that when zebrafinches mount a profound change in peripheral vasomotion when they are immune challenge with LPS (a substance that mimics bacterial infection).

Summary and Abstract of the Study

Fever and hypothermia are well characterized components of systemic inflammation. However, our knowledge of the mechanisms underlying such changes in body temperature is largely limited to rodent models and other mammalian species. In mammals, high dosages of an inflammatory agent (e.g., lipopolysaccharide, LPS) typically leads to hypothermia (decrease in body temperature below normothermic levels), which is largely driven by a reduction in thermogenesis, and not changes in peripheral vasomotion (i.e., changes in blood vessel tone). In birds, however, hypothermia occurs frequently, even at lower dosages, but the thermoeffector mechanisms associated with the response remain unknown. We immune-challenged zebra finches (Taeniopygia guttata) with LPS and monitored changes in subcutaneous temperature and energy balance (i.e., body mass, food intake), and assessed surface temperatures of, and heat loss across, the eye region, bill, and legs. We hypothesized that if birds employ similar thermoregulatory mechanisms to similarly-sized mammals, LPS-injected individuals would reduce subcutaneous body temperature and maintain constant surface temperatures when compared with saline-injected individuals. Instead, LPS-injected individuals showed a slight elevation in body temperature, and this response coincided with a reduction in peripheral heat loss, particularly across the legs, as opposed to changes in energy balance. However, we note that our interpretations should be taken with caution due to small sample sizes within each treatment. We suggest that peripheral vasomotion, allowing for heat retention, is an underappreciated component of the sickness-induced thermoregulatory response of small birds.

Citation

Tapper, S, Tabh, J, Tattersall, GJ, and Burness, GP. 2021. Changes in body surface temperature play an under-appreciated role in the avian immune response. Physiological and Biochemical Zoology, doi: 10.1086/718410

Naked mole-rats rapidly decrease UCP1 in hypoxia

I’m happy to report on a paper from Matt Pamenter’s lab (U Ottawa) that has just been published in Nature Communications. Matt and colleagues teamed up to examine how naked mole rats show a remarkable capacity to rapidly down-regulate UCP1 levels in their brown fat. It might come as a bit of a surprise to some to hear that naked mole-rats even have functional UCP1, since they are often described as “poikilothermic” mammals, not capable of producing heat. This is actually not entirely accurate, as can be seen in thermal images of naked mole-rats (Figure 1 below from Cheng et al 2021), they have a substantial band of heat within their shoulder region, where the brown fat lies.

Figure 1. Thermogenesis ceases in acute hypoxia and body temperature drops to ambient levels.

Naked mole-rats are among the most hypoxia-tolerant mammals. During hypoxia, their body temperature (Tb) decreases via unknown mechanisms to conserve energy. In small mammals, non-shivering thermogenesis in brown adipose tissue (BAT) is critical to Tb regulation; therefore, we hypothesized that hypoxia decreases naked mole-rat BAT thermogenesis. To test this, we measure changes in Tb during normoxia and hypoxia (7% O2; 1–3 h). We report that interscapular thermogenesis is high in normoxia but ceases during hypoxia, and Tb decreases. Furthermore, in BAT from animals treated in hypoxia, UCP1 and mitochondrial complexes I-V protein expression rapidly decrease, while mitochondria undergo fission, and apoptosis and mitophagy are inhibited. Finally, UCP1 expression decreases in hypoxia in three other social African mole-rat species, but not a solitary species. These findings suggest that the ability to rapidly down-regulate thermogenesis to conserve oxygen in hypoxia may have evolved preferentially in social species.

This work was a team effort, lead by Dr. Matt Pamenter’s lab at U Ottawa and Dr. Mary-Ellen Harper (U Ottawa), and included colleagues from the University of Pretoria, and University of Shaqra, Saudi Arabia, and myself (Brock University).

Here is a link to the paper.

https://rdcu.be/cBR7d

Citation

Cheng, H, Rebaa, R, Malholtra, N, Lacost, B, El Hankouri, Z, Kirby, A, Bennett, NC, van Jaarsveld, B, Hart, DW, Tattersall, GJ, Harper, M-E, and Pamenter, ME. 2021. Naked mole-rat brown fat thermogenesis is diminished during hypoxia through a rapid decrease in UCP1. Nature Communications, 12: 6801. https://doi.org/10.1038/s41467-021-27170-2

Thermal Ethology: Staying Warm is not the Norm

I’m happy to report that our paper entitled “Staying warm is not the norm: Behavioural differences in thermoregulation in two snake species” is published in the Canadian Journal of Zoology at the following link:

https://cdnsciencepub.com/doi/full/10.1139/cjz-2021-0135.

Congratulations to the team in my lab for pulling this paper together.

In this study, we focus on laboratory measurements of behaviours (in two species of snakes) related to temperature regulation to highlight methodological approaches to studying thermoregulation in ectotherms.

Over the past few years, we have read a lot of papers that report on thermoregulation in ectotherms, but we have felt that critical information on whether the animals are purposely thermoregulating is missing. How do you know they are thermoregulating? Is it sufficient to simply examine their position within the thermal gradient? Perhaps the direction they orient is important to establishing their motivations? How do you know an ectotherm is thermoregulating rather than simply moving around at random? Maybe accounting for activity and exploration effects in these studies can help make a difference? These topics have been covered in a number of other papers from our laboratory (Wang et al 2019; Black and Tattersall, 2017; Black et al, 2019), but we test them here using two species of snakes with contrasting life histories, where we would expect different thermoregulatory preferences given the different microhabitats preferred in nature.

These are some of the questions we focus on in this study of the Eastern Garter Snake (Thamnophis sirtalis sirtalis) and the semi-fossorial Northern Red-bellied Snake (Storeria occipitomaculata occipitomaculata). While we do report that the semi-fossorial snakes appear to prefer cooler temperatures, please read the paper for some of the more subtle differences between these species.

Anyhow, we hope to convince fellow researchers to report on these sort of behaviours since they may likely be helpful in bolstering the case that the animal is motivated to select temperatures.

Video time lapse of a garter snake in a circular / doughnut shaped thermal gradient.

Thermal gradient used in the study.

Citation

Giacometti, D., Yagi, KT, Abney, CR, Jung, MP, and Tattersall, GJ. 2021. Staying warm is not always the norm: Behavioural differences in thermoregulation of two snake species. Canadian Journal of Zoology, Accepted, Aug 25 2021. http://doi.org/10.1139/cjz-2021-0135

Many thanks to the co-authors in this study. This research was originally part of Curtis Abney’s MSc thesis, supplemented with Matthew Jung’s Honours thesis (with input and guidance from Dr. Katherine Yagi), and brought together by the fine analytical and writing skills of Danilo Giacometti.

References

Black, IRG and Tattersall, GJ. 2017.  Thermoregulatory behavior and orientation preference in bearded dragons.  Journal of Thermal Biology. 69: 171-177.  https://doi.org/10.1016/j.jtherbio.2017.07.009; http://hdl.handle.net/10464/12875

Black, IRG, Berman, JM, Cadena, V, and Tattersall, GJ. 2019. Behavioral thermoregulation in lizards: Strategies for achieving preferred temperature. In: Behavior of Lizards: Evolutionary and Mechanistic Perspectives, Eds. Vincent Bels and Anthony Russell, CRC Press, 410 pp.

Wang, SYS, Tattersall, GJ, and Koprivnikar, J. 2019.  Trematode parasite infection affects temperature selection in aquatic host snails. Physiological and Biochemical Zoology. 92(1):71-79.  https://doi.org/10.1086/701236

Case of the shrinking salamanders

Congratulations to Patrick Moldowan for our publication in Global Change Biology on “Climate associated decline of body condition in a fossorial salamander”.

Abstract of the study below:

Temperate ectotherms have responded to recent environmental change, likely due to the direct and indirect effects of temperature on key life cycle events. Yet, a substantial number of ectotherms are fossorial, spending the vast majority of their lives in subterranean microhabitats that are assumed to be buffered against environmental change.

Here, we examine whether seasonal climatic conditions influence body condition (a measure of general health and vigor), reproductive output, and breeding phenology in a northern population of fossorial salamander (Spotted Salamander, Ambystoma maculatum). We found that breeding body condition declined over a 12-year monitoring period (2008–2019) with warmer summer and autumn temperatures at least partly responsible for the observed decline in body condition.

Our findings are consistent with the hypothesis that elevated metabolism drives the negative associa- tion between temperature and condition. Population-level reproduction, assessed via egg mass counts, showed high interannual variation and was weakly influenced by autumn temperatures. Salamander breeding phenology was strongly correlated with lake ice melt but showed no long-term temporal trend (1986–2019).

Climatic warming in the region, which has been and is forecasted to be strongest in the summer and autumn, is predicted to lead to a 5%–27% decline in salamander body condition under realistic near-future climate scenarios. Although the subterranean environment offers a thermal buffer, the observed decline in condition and relatively strong effect of summer temperature on body condition suggest that fossorial salamanders are sensitive to the effects of a warming climate.

Given the diversity of fossorial taxa, heightened attention to the vulnerability of subterranean microhabitat refugia and their inhabitants is warranted amid global climatic change.

This study resulted from the PhD research of Patrick Moldowan, working with Dr. Njal Rollinson (U of Toronto) and myself. The research emanated from a long-term monitoring project called BLISS (https://tattersalllab.com/bliss/) that was initiated at various times in the past, with an objective to monitor mole salamanders (Spotted Salamanders, Ambystoma maculatum) in a pristine environment, for potential changes over time in population, phenology, reproductive output, and morphology.

I first met the Bat Lake salamanders in 1993, being introduced to the field site by a generous Dr. James Bogart who trusted me enough to leave me alone for 4 months to conduct research on an NSERC USRA project. I really want to thank Jim for sending me to this place where the field site captured the imagination, was a retreat from the urban life, and a crash course in wildlife ecology.

Here is a link to the paper http://doi.org/10.1111/gcb.15766 or request access from researchgate

Citation

Moldowan, PD, Tattersall, GJ, and Rollinson, N. 2021. “Climate associated decline of body condition in a fossorial salamander”. Global Change Biology. http://doi.org/10.1111/gcb.15766 

Acknowledgements

Many thanks to all the folks at the Algonquin Wildlife Research Station for support over the years and to all the Salamanderers who took part in BLISS: DL LeGros, SP Boyle, O Butty, JWD Connoy, D Crawford, EA Francis, G H-Y Gao, N Hrynko, JA Leivesley, DI Mullin, S Paiva, D Ravenhearst, C Rouleau, M Terebiznik, H Vleck, L Warma, SJ Kell and T Wynia. There are so many others who have helped out over the years, and we hope we have acknowledged all their assistance in the paper acknowledgements!

Shape-shifting animals

Congratulations to Sara Ryding, Deakin University for the first chapter of her PhD thesis being published in Trends in Ecology & Evolution on “Shape-shifting: changing animal morphologies as a response to climatic warming”. Link to the paper here or here.

In this review, Sara writes about how animal appendages (ears, feet, limbs, bills, etc) are important morphological indicators of temperature and therefore potential signatures of changing climate.

Appendages have an important, but often undervalued, role in animal thermoregulation as sites of heat exchange.

This thermoregulatory role leads to geographic clines in animal morphology where animals at lower latitudes, in warmer climates, have larger appendages (a pattern known as ‘Allen’s rule’).

In this review, we discuss evidence for animals (mostly evidence in birds and mammals, although the field does extend to other animal taxa) that are shifting their morphologies to have proportionately larger appendages in response to climate change and its associated temperature increases.

A thermal image of a Galapagos sea lion, showing distinctly warm front flippers. Appendages tend to be variable in size and have capacity to vary peripheral blood flow, and thus may serve as sensitive indicators of changing climate.

It has been a real pleasure to work with Sara Ryding on this project. Full credit and thanks go to Sara for all her hard work on this paper. I helped out only a small bit, but she reviewed the field within which my lab has been conducting collaborative research since 2010. Hopefully more research will follow as she navigates the rest of the project us (Drs. Matthew Symonds and Marcel Klaassen and myself). Many late nights and early morning zoom meetings await us all. Many thanks to Deakin University and the Australian Research Council for supporting this project.

Citation

Ryding, S, Klaassen, M, Tattersall, GJ, Gardner, JL, and Symonds, MRE. 2021. Shape-shifting: changing animal morphologies as a response to climatic warming. Trends in Ecology & Evolution. DOI: https://doi.org/10.1016/j.tree.2021.07.006

News articles

https://www.vice.com/en_us/article/wx59p4/climate-change-is-forcing-animals-to-quickly-shape-shift-study-suggests

https://www.cnn.com/2021/09/07/world/animals-climate-change-shape-shift-scn/index.html

https://www.brisbanetimes.com.au/environment/climate-change/climate-change-means-bigger-bills-and-ears-and-tails-as-well-20210907-p58pg6.html

Reptilian Conversation: Let’s talk about pets.

Congratulations to my PhD student, Melanie Denommé for her article just published in the Conversation. Melanie recently attended (virtually) a SciComm conference and resulting from that meeting, she put together an opinion piece on the prevalence of reptiles as pets.

Here is a link to the article: https://theconversation.com/lizards-snakes-and-turtles-dispelling-the-myths-about-reptiles-as-pets-166257

It really is an honour to have graduate students pushing the boundaries and taking risks to get their ideas out there!

Citation

Denommé, M and Tattersall, GJ. 2021. Lizards, snakes and turtles: dispelling the myths about reptiles as pets. The Conversation. Published Online August 23, 2021.

Congratulations Leed McNabb, MSc

Leed McNabb successfully navigated the pandemic, and conducted his MSc on study subjects most difficult to get close to and obtain due to various restrictions.

His thesis was entitled “Quantifying the relationship of bilateral blood flow in glabrous skin at rest and during sympathetic perturbation”, co-supervised by Dr. Stephen Cheung (Kinesiology) and myself.

Here were are huddled in front of our computer screens enjoying Leed’s presentation and responses to our questions.

Many thanks to the external examiner, Dr. Derek Kimmerly (Dalhousie), examining committee member, Dr. Geoff Hartley (Nipissing University) and the chair, Dr. Nota Klentrou (Brock University) for their hard work and participation.

It was a distinct privilege to work with Leed, who I have known since his days as a Biological Sciences major, and an honour to be involved in the Kinesiology Department’s graduate program (Leed did all his research with Stephen Cheung’s lab). For me it was a time to learn how different schools think and train students, as well as an opportunity to learn how to work on a different study animal, Homo sapiens, an unusual species indeed for my lab.

Congratulations Leed!

Scaleless dragons evaporate more water than those with scales

The following is a guest blog by Nick Sakich


Nick Sakich here.  The first paper from my MSc has just been published in the Journal of Experimental Biology. The paper is entitled, “Bearded dragons (Pogona vitticeps) with reduced scalation lose water faster but do not have substantially different thermal preferences.”

In it, we examine both “wild-type” bearded dragons and two phenotypes unique to captivity (i.e. not found naturally): animals with scales of reduced prominence (known as “leatherbacks”) and completely scaleless animals (known as “silkbacks”). The following slideshow depicts the 3 variants:

There has long been speculation as to whether or not scales play a role in reducing evaporative water loss across the skin in reptiles. The seminal studies that most point to are by Licht and Bennett (1972) and Bennett and Licht (1975). Those authors looked at aberrant partially scaleless individual snakes found living in the wild and found that they did not have higher rates of water loss than “normal” snakes. However, these studies had some methodological issues, most notably sample sizes of only one (Licht and Bennett, 1972) and two (Bennett and Licht, 1975) partially scaleless snakes, respectively.

Furthermore, can reptiles (or lizards and snakes, at least) detect their rate of evaporative water loss and respond accordingly? If they can, animals with higher rates of evaporative water loss will perhaps choose cooler temperatures compared to animals with lower rates of evaporative water loss. The rate of evaporative water loss is partially thermally dependent, so for the animals this would be a way to compensate and bring their rate of evaporative water loss down.

In this study, we set out to test two hypotheses. First, we hypothesized that scales are indeed a barrier to evaporative water loss, and so leatherbacks and silkbacks would have higher rates of evaporative water loss than wild-types. Second, we hypothesized that, because of this increased rate of evaporative water loss, leatherbacks and silkbacks would have lower thermal preferences than wild-types.

We found support for our first hypothesis: both leatherbacks and silkbacks evaporated water faster than wild-types. It is likely that most of this occurs across the skin, rather than through changes in breathing or metabolism, given the simultaneous measurements we made of metabolism. This confirms what many who keep silkbacks as pets have long suspected. However, we didn’t find a statistically significant difference in thermal preference between the three phenotypes. This suggests that either leatherbacks and silkbacks can’t tell that they’re losing water faster than wild-types, or that they can tell, but they make a strategic decision to prioritize warmth over water.

I’d like to thank Arnold Liendo and Paula Rodriguez, Mandy Peck, and Kirk Riddle for supplying us with lizards for this study. I’d also like to thank Tom Eles and Wynne Reichheld, without whom keeping up with the nuts-and-bolts of animal acquisition and care would have been impossible.

Citation

Sakich, NB and Tattersall, GJ. 2021. Bearded dragons (Pogona vitticeps) with reduced scalation lose water faster but do not have substantially different thermal preferences. Journal of Experimental Biology. 224 (12): jeb234427.

A link to the pdf of the manuscript can be found here (limited to 50 clicks). Otherwise, requests for pdfs can be made on Researchgate.

References

Licht, P. and Bennett, A. F. (1972). A scaleless snake: tests of the role of reptilian scales in water loss and heat transfer. Copeia 1972, 702-707. doi:10.2307/ 1442730

Bennett, A. F. and Licht, P. (1975). Evaporative water loss in scaleless snakes. Comp. Biochem. Physiol. A Physiol. 52, 213-215. doi:10.1016/S0300- 9629(75)80155-1

Thermal imaging of stress: you won’t believe your eyes

The following is a guest blog by Dr. Joshua Robertson Tabh


In my short research career, I’ve come to accept (even relish) that there are some projects that endlessly surprise; projects with shifting objectives that find you running drive-by thermal camera hand-offs along the QEW at questionable hours. The project that I’m about to describe is one of “those”. And curiously, despite the innumerable twists and turns, it just so happened to be a project with some of the most useful outcomes I’ve helped to produce. In this guest post, I’ll describe those outcomes.

But first, let’s begin in 2016. I had just begun my PhD research in avian stress physiology, and mere months before, Paul Jerem and others had released a highly intriguing protocol which suggested that the physiological stress response could be detected, and possibly quantified, in birds by simply measuring changes in body surface temperature (https://www.jove.com/t/53184/thermal-imaging-to-study-stress-non-invasively-in-unrestrained-birds). The rationale behind their protocol was that following exposure to a stressor, the sympathetic nervous system triggers vasocontriction of blood vessels at the skin (among other things), which manifests as measurable changes in skin temperature. This idea isn’t new. Rather, it likely dates back to the early 20th century or previous (e.g. Wolff and Mittelman, 1937). However, Jerem et al’s protocol was the first to show that a stress-induced change in skin temperature could be detected at the eye region in a wild bird, using infrared thermography (see Edgar et al, 2013, for a study in chickens). A clever application of thermography.

Jerem et al’s work was exciting. But a few important questions seemed to linger:

(1) how well does this stress-induced change in eye region temperature reflect circulating changes in sympathetic nervous system markers (i.e. catecholamines, like adrenaline and noradrenaline)?

(2) Are changes in surface temperature at other bodily regions better indicators of the physiological stress response than the eye region (e.g. the bill: https://journals.biologists.com/jeb/article/223/8/jeb220046/223869/Body-surface-temperature-responses-to-food)?

And (3) how robust and reliably detectable are stress-induced changes in body surface temperature? More specifically, how resilient is this response to masking by changes in bird position (see: https://besjournals.onlinelibrary.wiley.com/doi/abs/10.1111/2041-210X.13563)?

So, being nagged by these questions, a team of ecophysiologists (Glenn Tattersall, Gary Burness, and Oliver Wearing), an endocrinologist (Gaby Mastromonaco), and myself sought answers.

To do so, we required an experimental approach that would allow us to measure both body surface temperature (here, at the eye region and bill) and circulating catecholamines in “stressed” and “unstressed” birds. However, measuring circulating catecholamines requires sampling blood. And since puncturing a vein with a syringe is surely sufficient to activate a physiological stress response on its own (thus rendering “unstressed” birds “stressed”) blood sampling by this standard method simply wasn’t possible. Ideally, we would fit a sample of birds with central venous catheters to permit blood sampling without capture and venipuncture.   This approach could work, however, even if blood samples were to be collected effectively, catecholamines can be a pain to quantify, even for contracted labs with high-end machinery. It’s for this latter reason that we accepted the reality of leaving our first research question unanswered. 

Fig. (1) Domestic pigeon being monitored during rest, before experimentation.

Nevertheless, we could persist with a simple experimental design to answer research questions (2) and (3); quite simply, thermographically image birds during rest (Fig 1) and during a stress exposure (for us, handling). To answer question (2), we would then quantify and compare the magnitude of stress-induced changes at the eye region and bill. And lastly, to answer question (3), we would aim to test the effect of head angle on our ability to detect stress-induced changes in eye region and bill temperature. In theory, a nice and clean approach.

Before I get to the answers of our remaining research questions,  a small note on how we estimated head angle (for the interested reader).

Estimating Head Angle from 2D Image

Estimating the orientation of a 3D object from a 2D angle has been a concern for humans since photography was invented. Among mathematicians, this challenge has since acquired a formal name: the “perspective-n-point” (or “PnP”) problem. All solutions to the PnP problem first require knowledge of where, in a 2D plane, at least 3 points in an imaged object lay. We’ll call these points “landmarks”. Of course, more than 3 landmarks are best to improve estimation accuracy, but most agree that 3 will do for a reasonable guess. Next, rough dimensions of the imaged object in 3D space are needed. Such dimensions must be sufficient for one to estimate where the chosen landmarks may lie, relative to each other, in a theoretical 3D co-ordinate system known as the “world co-ordinate system”.

Once this information is collected, several geometrical approaches may be used to calculate how the imaged object must have moved or rotated such that the landmarks in 3D space overlap with those observed in 2D space (after adjusted for lens distortion). Interestingly, there is one industry with considerable investment in creating efficient geometrical approaches: virtual reality (or “VR”) gaming. Why? Because using VR gaming requires that the system can estimate the gamer’s 3D position at all times (with, interestingly, tiny infra-red lights implanted in the headset as landmarks). Thanks to this investment by the VR industry, studies developing and comparing the accuracy of geometrical solutions to the PnP problem are flourishing. It’s a perfect time for biologists like us to start taking a peak at them.

For our study, we chose to use to an approach called the “EPnP” that was first proposed by Lepetit and others in 2009 (https://link.springer.com/content/pdf/10.1007/s11263-008-0152-6.pdf). We chose this approach because it permits one to use >4 landmarks for positional estimation (thus reducing error) with little cost to computational time relative to traditional solutions. Other approaches have been lauded for improving accuracy (e.g. P3P with RANSAC) and we encourage others to pursue those approaches. For our study, however, we were interested in balancing accuracy and efficiency.

To execute the EPnP approach, we estimated the 2D position of up to 9 landmarks on a pigeon’s head by loading our thermographic images into ImageJ (Fig 2). Building a 3D model turned out to be much less time consuming – simply draw on morphometric measurements of domestic pigeons reported in literature. From these data, and EPnP algorthims, we were thus able to estimate both a 3D translation and 3D rotation of an imaged bird’s head, relative to a virtual model of a perpendicularly facing individual.

Fig. (2) Thermographic image of a domestic pigeon with black dots marking 6 of 9 possible landmarks. The black line at the tip of the bill indicates the estimated direction in which the pigeon is facing.

Our results?

I’ll break them down by question.

Question (1): How well does this stress-induced change in eye region temperature reflect circulating changes in sympathetic nervous system markers (i.e. catecholamines, like adrenaline and noradrenaline)?

Answer: Yet unanswered.

Question (2): Are changes in surface temperature at other bodily regions better indicators of the physiological stress response than the eye region (i.e. the bill: https://journals.biologists.com/jeb/article/223/8/jeb220046/223869/Body-surface-temperature-responses-to-food)?

Answer: Our results suggest that, at least in our study species, surface temperature the bill is probably a better indicator of stress physiological state. I’ll explain why by referencing what we observed from data that did not control for head position. After stress exposure, bill temperature fell significantly by ~4°C after stress exposure (handling), while eye region temperature did not significantly change (Fig 3). Rather, temporal patterns in eye region temperature appeared remarkably similar between “stressed” and “unstressed” birds. Moreover, only stress-induced changes in bill temperature showed significant inter-individual variation, suggesting that if one wishes to build a metric of “stress-responsiveness” from changes in surface temperature, doing so at the bill is likely more effective than at the eye region.

Fig. (3) Changes in eye region and bill temperature across time in both stress-exposed and control birds. Time 0 (marked with a vertical dashed line) indicated that time that flight cages were opened to permit capture and handling of birds in the stress-exposed treatment group. Dots represent averages across birds per 5 seconds of observation, and lines of best fit represent trends estimated by generalised additive mixed-effects models. Ribbons represent 95% confidence intervals around trend estimates.

Question (3): How robust and reliably detectable are stress-induced changes in body surface temperature?

Answer: It depends on where you look. After correcting for changes in head position in our birds, a significant effect of stress-exposure on eye region temperature emerged (Fig 4). This was not the case for stress-induced changes in bill temperature, which were detectable regardless of whether head position was accounted for or not. This point, we think, is particularly important for two reasons:

(1) don’t correct for changes in object position and you risk missing out on detecting biological processes, and

(2) surface temperatures of some body regions might be better indicators of your biological process of interest than others.

Fig. (4) Change in eye region temperature of stress-exposed and control pigeons after correcting for changes in head orientation. Again, time 0 (marked with a vertical dashed line) indicated that time that flight cages were opened to permit capture and handling of birds in the stress-exposed treatment group. Dots represent averages across birds per 5 seconds of observation, and lines of best fit represent trends estimated by generalised additive mixed-effects models. Ribbons represent 95% confidence intervals around trend estimates.

Take Home Message

To conclude, drawing biological inference from thermographic images is tricky. Many sources of error can get in the way of your ability to meaningfully do so, and a common one is changes in object position. As such, biologist should always remember to correct for object position when working with their surface temperature data – perhaps by using our method or another. 

Read the full study here: https://physoc.onlinelibrary.wiley.com/doi/10.14814/phy2.14865

Citation

Tabh, Joshua KR, Burness, G, Wearing, OH, Tattersall, GJ, Mastromonaco, GF.  2021. Infra-red thermography as a technique to measure physiological stress in birds: body region and image angle matterPhysiological Reports, Accepted. https://doi.org/10.14814/phy2.14865

Acknowledgements

Dr. Joshua Robertson Tabh is a graduate of Trent University, co-supervised by Dr. Gary Burness and Dr. Gaby Mastromonaco. This research was made possible with the cooperation of the Toronto Zoo and by the watchful eye of Oliver Wearing. Since 2016, Joshua and Glenn have shared many conversations about avian physiology, imaging, and coding and Glenn invited Joshua to guest author this post after all these efforts finally reached the publication stage.

Funding for this research was provided by the Toronto Zoo Foundation, an NSERC Collaborative Research and Training Experience Program (Grant #: CREATE 481954-2016), a Howard P. Whidden grant to OHW, and an NSERC Discovery Grant to GJT (Grant # RGPIN-2014-05814).