Genetic integration of behavioural and endocrine components of the stress response

  1. Thomas M Houslay
  2. Ryan L Earley
  3. Stephen J White
  4. Wiebke Lammers
  5. Andrew J Grimmer
  6. Laura M Travers
  7. Elizabeth L Johnson
  8. Andrew J Young
  9. Alastair Wilson

  • Curated by eLife

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    Evaluation Summary:

    This is a timely paper on the genetic integration of behavioral and physiological components of the stress response in guppies. Using evolutionary quantitative genetic approaches, the authors show that genetic variation in the cortisol stress response is associated with genetic variation in stress-related behaviors. This result suggests that physiological and behavioral responses to stress should show correlated evolution in response to natural selection, which is of interest to evolutionary biologists and for animal welfare. The reviewers pointed out several conceptual and methodological issues with the definition of the phenotypes under study and and with the definition strong genetic integration.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The vertebrate stress response comprises a suite of behavioural and physiological traits that must be functionally integrated to ensure organisms cope adaptively with acute stressors. Natural selection should favour functional integration, leading to a prediction of genetic integration of these traits. Despite the implications of such genetic integration for our understanding of human and animal health, as well as evolutionary responses to natural and anthropogenic stressors, formal quantitative genetic tests of this prediction are lacking. Here, we demonstrate that acute stress response components in Trinidadian guppies are both heritable and integrated on the major axis of genetic covariation. This integration could either facilitate or constrain evolutionary responses to selection, depending upon the alignment of selection with this axis. Such integration also suggests artificial selection on the genetically correlated behavioural responses to stress could offer a viable non-invasive route to the improvement of health and welfare in captive animal populations.

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  1. Version published to 10.7554/elife.67126 on eLife
  2. Version published to 10.1101/770586v5 on bioRxiv
  3. eLife

    Reviewer #3 (Public Review):

    This manuscript characterizes the additive genetic variance-covariance of behavioural traits and cortisol level in a captive Trinidadian guppy population, in particular to test for the genetic integration of behavioural and physiological stress responses.

    The experimental design, trait definitions and statistical analyses appear appropriate. The main weakness of the study is a lack of clarity on the definition of genetic integration and the statistical ways to characterize, confirm or reject genetic integration (in particular, what defines and how to test for a "single major axis of genetic variation"?).

    The additive genetic variation-covariation is correctly estimated. The presence of additive genetic correlations and the eigen decomposition of G seem to support genetic integration, but the lack of clear predictions makes the the conclusion not completely clear. Another minor conclusion, that "correlation selection in the past has likely shaped the multivariate stress response" is not directly supported by the results as the argument ignores the possible role of other evolutionary forces (in particular mutational input which is likely to be pleitropic for behaviour and hormone levels).

    The nature of genetic (co)variation in behaviour and physiology is poorly known because most quantitative genetic studies of behavioural and physiological traits are still univariate, while it is clear that selection and evolution are better understood as multivariate processes. In addition to presenting some fresh results on the topic, this manuscript provides a mutivariate framework that could be applied in other populations. In particular, eigen decomposition of genetic variance-covariance matrices is not new but its application to the study of stress response integration is original and promising. As the authors mention, such methods could help improve health and welfare in captive animal populations via indirect artificial selection against stress, which is quite an original and stimulating idea.

  4. eLife

    Reviewer #2 (Public Review):

    This paper addresses a fundamental question regarding the evolution of the stress response, specifically that the action of natural selection on the stress response should promote the functional integration of its behavioral and physiological components. Therefore, the authors predict that genetic variation in the stress response should include covariation between its component behavioral and physiological traits. The results are intrinsically interesting and seem to provide a critical proof of principle that, if confirmed, will prompt future follow up research. However, there are some fundamental conceptual and experimental design issues that need to be addressed, in order to assess the conclusions that can be drawn from the results presented here.

    Conceptual issues:

    1. The authors selected multiple behavioral measures of the stress response but only considered the glucocorticoid response as a physiological trait. In my view this has several problems:

      A) Although, for historical reasons and because they are easier to measure, glucocorticoids have been perceived as a stress hormone, the fact is that they respond not only to threats to the organism (i.e. stressors) but also to opportunities (e.g. mating). In other words, glucocorticoids are produced and released whenever there is the need to metabolically prepare the organism for action. Therefore, glucocorticoids are probably not the best physiological candidate to look for phenotypic integration with stress behaviors, since they must have also been selected to be produced and released in other ecological contexts. In this regard it would have been interesting to measure the phenotypic integration of cortisol also with behaviors used in non-threatning but metabolically challenging ecological opportunities (e.g. mating), and to investigate the occurrence of an eventual trade-off (or of a "phenotypic linkage") between these two sets of traits (stress traits vs. mating traits).

      B) Sympathetic activation is a key component of the physiological stress response in vertebrates. It is thus odd not to consider the sympathetic response in a study that has the main aim of studying the evolution of a phenotypically integrated stress response. I understand that the sympathetic response in guppies is more difficult to study than measuring cortisol, but this technical challenge can certainly be overcome (e.g. techniques for measuring cardiac response to threat stimuli have been recently developed for other challenging model organisms, such as fruit flies; e.g. https://www.biorxiv.org/content/10.1101/2020.12.02.408161v1); or if not, then an alternative model organism should have been used to address this question.

    2. Typically, in vertebrates the behavioral response to a stressor has a passive (e.g. freezing) and an active (i.e. fight-flight) component. It would be very interesting to assess if these two components are phenotypically integrated with each other and each of them with the physiological response. Unfortunately, the authors did not use behavioral measures of each of these two components. Instead they have extracted 3 spatial behaviors from an open field test (time in the central part of the tank in an open field test (OFT); relative area covered; track length) and emergence latency in an emergence from a shelter test. It is not clear how each of the measured behaviors captures these two key components of the behavioral stress response. For example, a fish that freezes in the central part of the tank when it is introduced in the OFT will have a high time in the middle score and eventually a high relative area covered, but relatively low track length. However, if it darts towards the tank wall and freezes there, the result would probably be low time in the middle and low relative area covered. Thus, a fish that has spent approximately the same time in freezing may show very different behavioral profiles according to the variables used here. This could be avoided if explicit measures of fleeing and freezing behavior have been used. Given that the authors have video-tracked the fish, I suggest they can still extract such measures (e.g. angular speed is usually a good indicator of escape/fleeing behavior; and a swimming speed threshold can be validated and subsequently used to detect freezing behavior from tracking data) from the videos. The fact that variables of these two types of behavioral responses to stress have not been used in this study may explain to a large extent why the authors came to the conclusion that, "the structure of G is more consistent with a continuous axis of variation in acute stress responsiveness than with the widely invoked 'reactive - proactive' model of variation in stress coping style".

    3. The authors used a half-sib breeding design, which is the golden standard in evolutionary quantitative genetics. However, and this is not a specific critique of the present study but a general problem of this field, the extent to which estimates of G obtained with breeding designs reflect the G that would be obtained by actually sampling a natural population is questionable, because these designs create artificially structured populations with higher levels of outbreeding and concomitantly also with higher genetic variation than what is usually found in nature. This problem can be illustrated by analogy using the example of heritability estimates, which are typically lower when obtained from selection studies by comparing the generation after selection to the one before selection (aka realized heritability), than when computed from artificial breeding designs.

    Methodological issues:

    1. The authors considered the OFT, ET and ST testing paradigms to be behavioral assays that allow the characterization of the behavioral components of the stress response in guppies, because all these paradigms involve capturing and transferring the focal fish to a novel environment (tank) and in social isolation. Undoubtedly these procedures must have induced stress, however the stressor was not standardized because it consisted in the capture and transfer, and these may have varied from fish to fish (btw are there measures of handling time for each fish? And how to measure "handling intensity"?). In my view a standardized stressor, such as a looming stimulus (e.g. Temizer et al. 2015 Current Biology 25: 1823-34; Bhattacharyya et al. 2017 Current Biology 27, 2751-2762; Hein et al. 2018 PNAS 115: 12224-8), should have been used such that the behavioral measures could have been linked to the stressor in a more controlled way.

    2. Moreover, the authors have measured the "stress behaviors" and cortisol in response to two different stressors: the handling described above and the confinement and social isolation for the GC response. This is not the best experimental design, because the behavioral and physiological expression is expected to be linked and to be flexible, as shown by the data on cortisol habituation to repeated stressor exposure. Thus, when the goal of the study is to characterize the co-variation between traits it is critical to standardize the stimulus that triggers their expression in the two domains (behavioral and physiological) and behavior and physiological measures should have been obtained in response to the same stressor stimulus for each individual. In principle, the failure to do so will artificially decrease the observed co-variation between traits, due to environmental differences (i.e. test contexts and their specific stressors).

  5. eLife

    Reviewer #1 (Public Review):

    This paper uses a large breeding colony of guppies to measure genetic correlations between hormonal stress responses and behavior in an open-field test. Although we know a lot about the mechanisms of hormone-mediated behavior, we know less about variation in hormonal systems, particularly genetic variation. Understanding how hormones relate genetically to the behaviors they mediate is particularly important because it helps us understand how the entire hormone-behavior system evolves. A priori, we would expect genetic correlations between hormones and the behavior they underlie, such that selection on the hormone would lead to a response in the behavior and vice versa. However, evidence for this pattern is rare.

    Here, the authors show that stress-induced levels of cortisol are repeatable and heritable. Interestingly, they also show that individuals show a lower stress response to later stress and slightly less variation, indicating a G X E interaction. There was a significant genetic correlation between the hormonal response and one of the behaviors measured in the open field test, and the hormone loaded positively in the first genetic principal component along with all the behaviors. This is evidence of an correlated suite of traits that would evolve together in response to selection.

    This is an important study, because evidence of genetic variation in hormonal systems, not to mention genetic covariation with hormone-mediated traits, is rare. The results presented here provide insight into how a hormone-behavior complex might adapt to a changing environment. They are also relevant to ideas about the maintenance of variation in coping styles in natural populations.

  6. eLife

    Evaluation Summary:

    This is a timely paper on the genetic integration of behavioral and physiological components of the stress response in guppies. Using evolutionary quantitative genetic approaches, the authors show that genetic variation in the cortisol stress response is associated with genetic variation in stress-related behaviors. This result suggests that physiological and behavioral responses to stress should show correlated evolution in response to natural selection, which is of interest to evolutionary biologists and for animal welfare. The reviewers pointed out several conceptual and methodological issues with the definition of the phenotypes under study and and with the definition strong genetic integration.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  7. Version published to 10.1101/770586v4 on bioRxiv
  8. Version published to 10.1101/770586v3 on bioRxiv
  9. Version published to 10.1101/770586v2 on bioRxiv
  10. Version published to 10.1101/770586v1 on bioRxiv