Laying hens are typically moved to a novel environment after rearing, requiring adaptability to cope with change. We hypothesized that the standard rearing of laying hen chicks, in non-changing environments with limited choices (a single variant of each resource), impairs their ability to learn new routines, use new equipment and exploit new resources. On the contrary, rearing in a changing environment that also offers a choice of resource variants could better prepare chicks for the unexpected. To explore this hypothesis, environmental change and choice were manipulated in a 2 × 2 factorial experiment. Compared to standard rearing, greater change during early rearing, through repeatedly swapping litter and perch types, reduced initial freezing when exposed to a novel environment suggesting a lower fear response. Greater choice during rearing, through simultaneous access to multiple litter and perch types, resulted in shorter latencies to solve a detour task, more movement in novel environments and less spatial clustering, suggesting improved spatial skills and higher exploration. However, combining both change and choice did not generally result in greater improvement relative to providing one or the other alone. We conclude that environmental change and choice during rearing have different positive but non-synergistic effects on later adaptability potential. Gallus gallus domesticus) are transferred from a relatively simple rearing environment to a more complex, multi-tiered adult housing system, they can suffer injuries and high mortality2. A recommendation for farm animals is to match the type of housing system used in the rearing and adult periods as a way to reduce potential negative outcomes from such transitions4. However, even if the housing system is similar, the transition from one facility to another still involves some environmental change. The difficulties encountered by animals following the transition may persist throughout adult life, affecting both welfare and reproductive performance6. Improved ability to cope with change would enable a smoother transition from the rearing facilities to the subsequent environments. Beside the practical relevance of reducing stress when transferred to a new environment, there is a need for improved understanding of general principles for promoting the adaptability potential of captive animals during rearing8.At certain points in their life, domestic animals and captive wildlife are often transferred between different environments. Such moves involve exposure to novel resources, routines and situations, which can be stressful and imply a welfare risk. For example, when young laying hens  access to perches during the first four weeks of life improves laying hens’ later ability to move in three dimensional space15. For example, aviary-reared hens are faster locating a reward in a spatial task and have greater working memory compared to individuals reared in a simple cage environment16. These results may be reflecting increased neural activation in the hippocampus, a brain region highly associated with spatial skills and navigation, which is seen already in day-old chicks exposed to spatially complex environments18. Thus it can be suggested that early exposure to mild stress and spatial complexity may contribute to an improved ability to cope with future stressful situations and greater ability to make the most of opportunities when they arise.Experience gained during the first weeks of life, a sensitive period for brain development, can have long lasting and often irreversible effects on behaviour and cognitive abilities21. It is possible that the positive effects of early stress and spatial complexity described earlier are a consequence of how young animals’ experience the predictability and controllability of their environment. This experience influences stress states and, in turn, behavioural22 and physiological adaptability24. Meagher25 suggests that some level of unpredictability in the environment could be beneficial for reducing boredom in captive animals and that any stress arising from this unpredictability could be reduced by adding choice to the environment. Effects of unpredictability early in life have been investigated following increasing levels of change in the home environment, by moving26 and exchanging objects27, or changing routines28. Choice, on the other hand, is tightly connected to the perception of controllability29. Increased choice can be given by setting up environmental gradients, thereby increasing spatial complexity30. Given these connections, it is possible that greater levels of environmental change and choice when young could be linked to improvements in coping ability.Two more aspects, predictability and controllability of resources available in the environment can play important roles in coping with stress4 and they differentiate between different types already from the first days of life31. Standard rearing environments for laying hen chicks usually offer only a single type of litter and a single type of perch throughout the rearing period. This results in a simple and non-changing environment with limited possibilities for individual choice , which is not optimal according to Meagher25. Environmental change and choice could be altered by manipulating the litter and perch types available to laying hen chicks across time and space. The level of environmental change can be increased compared to the standard rearing environment by repeatedly changing the type of litter and perch offered over time, producing a more unpredictable environment. The level of environmental choice could be increased compared to the standard rearing environment by offering multiple types of litter and perches in different locations of the pen, thereby increasing spatial complexity, and giving chicks more control over the types of substrates used for performing different activities. Both change and choice could be increased by simultaneously offering multiple litter and perch types, as well as changing their location over time. In this study, we explored effects of early exposure of domestic chicks to different levels of environmental change and choice in a 2 × 2 factorial experiment , comfort33 and agency34. Since agency reflects motivation for environmental engagement, it can be associated with behaviours connected to environmental interaction, such as foraging and movement. Physiological or psychological stressors activate the hypothalamo-pituitary-adrenal (HPA) axis, which plays an important role in regulating behavioural expression in bird35. The HPA-axis is already fully functional on day 1 post-hatch in laying hen chicks36. Thus, the behaviour of young chicks can reflect central stress mediation from an early phase of development. Our second goal was to evaluate the impact of the treatments on behavioural adaptability, which is relevant to understanding how the rearing treatments could affect chick ability to cope with a transition to a novel environment. To achieve this second goal, we conducted two standardised novelty challenge tests. The Novel pen test evaluated group-level acute behavioural responses following transition to a novel environment with unfamiliar resource types. The Multivariate behavioural test was used to investigate fearfulness, spatial abilities and exploration simultaneously at the individual level13.Our first goal was to assess how different levels of change and choice would affect chick welfare during imposition of the rearing treatments. This was necessary to evaluate the extent to which chicks were affected by the different treatments, as indicated using behavioural measures of stress  a higher level of environmental choice, achieved by simultaneously providing a variety of litter and perch types, would help to fulfil motivation for behavioural activities such as foraging and dustbathing leading to greater comfort and agency, along with enhancement of spatial skills37 including practicing the different bodily actions needed to land and balance on the different perch types31; while (3) higher levels of both environmental change and choice would enhance both adaptability and agency, and lead to the most advantageous outcomes in terms of both welfare during rearing and adaptability in response to novelty.We hypothesised that, when compared to a treatment representing standard rearing conditions; (1) a higher level of environmental change, achieved by repeated changes of litter and perch types, would result in mild stress during rearing but provide coping experience, thereby reducing fearfulness and leading to greater adaptability when challenged with noveltyPredictions arising from these hypotheses were that, compared to chicks in the standard rearing treatment; (1) chicks exposed to higher levels of environmental change would exhibit higher levels of behaviours indicative of mild stress , but also more rapid exploration and location of novel resources in the novelty challenge tests; and (2) chicks exposed to higher levels of environmental choice would exhibit higher levels of behaviours indicative of comfort (e.g. preening) and active engagement with their environment indicating higher agency ; while (3) chicks exposed to higher levels of both environmental change and choice would show signs of mild stress, comfort, and active environmental engagement during rearing, as well as being quicker to move and exploit new resources in the novelty challenges.Results are presented in graphs illustrating the interaction between the two levels of environmental change (Non-changing/Changing) and the two levels of environmental choice (Single/Multi-choice). First, we present behaviour observed in the rearing pens where differences were found between treatments during observation periods both under Undisturbed conditions Fig.  and withTreatment differences in spatial clustering . Differences were found in spatial clustering . Following disturbance, associated with changing the litter and perches in changing pens, or the mere entry of a person into non-changing pens, chicks having greater environmental change, choice, or change and choice showed more foraging behaviour. The responses of the chicks in the two novelty challenge tests, the Novel pen (group) and the Multivariate behavioural test , were intended to indicate how chicks from the various rearing environments might adapt to a change of environment later in life. Chicks reared with a higher level of change had a shorter initial freezing duration when placed in the Multivariate behavioural test. Chicks reared with more choice moved more in both novelty challenge tests, and were quicker to solve the detour task in the Multivariate behavioural test. This improved adaptability was not shown by chicks reared with higher levels of both change and choice, making their responses to the novelty challenges no different to those of birds reared in the standard pens.38. We had predicted that birds may experience the changing environment as somewhat stressful and that this would be reflected in increased vigilance, pecking at conspecifics and spatial clustering directly following changes. That this was not the case, and opposite effects to those predicted were observed for spatial clustering, suggests that the imposed level of change was perceived as stimulating rather than aversive. Further, the lack of differences in behaviour at times other than the first four hours after each change (i.e. during undisturbed observations) shows that the chicks rapidly adapted to the changes.Behavioural differences between chicks that had only one litter and perch type, but that varied in the level of environmental change (Non-changing*Single-choice versus Changing*Single-choice) were found during the disturbed period and in the Multivariate behavioural test. Following the first hours after the exchange of the litter and perch type, chicks showed less spatial clustering, less behavioural synchronization and more foraging, compared to chicks only having the human visit. This implies a higher level of environmental engagement, with chicks exploring the new litter and perch type and being more dispersed as a result of the increased activity39. This result supports the idea that the experience of novelty that is not excessive or uncontrollable leads to reduced fearfulness39, again suggesting that any stress arising from the changing environment was mild. For chicks living in a less complex environment, our results support the idea that regular changes in the environment (e.g. by swapping the type of litter and perch available) have welfare benefits. The increased environmental engagement, even if only short term, can be associated with a lower risk of boredom, as predicted by Meagher25. Reduced fearfulness of birds reared in a changing environment could lead to a smoother transition between rearing and adult layer facilities and may also lead to a lower prevalence of severe feather pecking40, a major welfare problem in today’s laying hen environments. It could also lead to reduced muscle and skeletal damage related to collision during sudden escape attempts41. It is possible that, according to our hypothesis, the changes we imposed resulted in mild stress that improved stress resilience12, leading to the reduced freezing response. Alternatively, the increased environmental engagement stimulated by the procedure of swapping litter and perch types reflected positive learning experiences about novelty leading to the reduced reaction when placed in the start box of the Multivariate behavioural test.In the Multivariate behavioural test, the lower freezing duration of chicks from the Changing*Single-choice treatment compared to the standard-reared chicks (Non-changing*Single-choice treatment) implies that they were less fearful42. Also, we saw very few aggressive pecks, and aggression is known to be low in chicks of this age44. Furthermore, no treatment differences were detected in severe feather pecking. Instead, the reduced spatial clustering, and that chicks more often crossed into parts of the open area in the Multivariate behavioural test where they had no visual contact with their pen mates, could indicate higher exploration connected to greater risk-taking46.Behavioural differences between chicks from the non-changing pens varying in the level of environmental choice (Non-changing*Single-choice versus Non-changing*Multi-choice) were found in all parts of this study. Chicks with a choice of four litter and perch types that were not relocated (Non-changing*Multi-choice) showed less spatial clustering in their pens compared to standard-reared chicks (Non-changing*Single-choice) throughout rearing. This, together with the increased foraging during the observations following disturbances, could support greater active engagement with the environment and thereby development of greater agency, as hypothesised. But contrary to our hypothesis, neither play nor dustbathing (indicating active engagement and a positive response to choice) was increased. This could be due to the relative rarity of these behaviours. We also found no support for the prediction of a higher level of the comfort behaviour, estimated by occurrences of preening. The reduced spatial clustering observed in the Multi-choice pens is unlikely to be explained by an increased need to avoid competition, feather pecks or aggressive behaviour in this treatment. All chicks could fit into the same litter tray or onto the same perch and, thus, exploit the same resource type at the same time48. The detour task has also been suggested to reflect inhibitory control, as chicks have to inhibit their tendency to directly approach their goal in order to reach it49. That a spatially more complex rearing environment results in improved spatial skills is supported by previous research32, with possible links to neural activity in the hippocampus18. However, the shorter latency to solve the detour task could also be linked to a generally improved ability to solve problems as a consequence of living in a more enriched environment . It is also possible that the usage of different variants of resources could have led to a positive feed-back loop whereby positive experiences from choosing different resource types resulted in a more optimistic outlook, as reported in animals living in more enriched or spatially complex environments50. This could explain the chicks’ higher likelihood of exploring a novel environment as well as their shorter latency to find their way out of the box in the detour task. However, since the detour task was a spatial task, we cannot exclude the possibility that the potentially improved problem-solving ability was limited to improved spatial skills. In the future, it would be interesting to test the ability of chicks reared in a multi-choice environment to solve other types of problems.The higher degree of movement in both the Novel pen and the Multivariate behavioural test by chicks reared with a higher level of choice did not reflect movement in their rearing pens, which did not differ from that in the standard treatment. Rather, movement can be related to exploration and further reflect increased environmental engagement according to our predictions. Furthermore, that chicks reared with choice had shorter latencies to solve the detour component of the Multivariate behavioural test supports our hypothesis about improved spatial skills, given that the outcome of a detour task reflects spatial abilities28 and are less willing to explore for hidden food sources51 implying an even greater need to promote these abilities. Exploration in laying hens has been associated with lower mortality, lower feather pecking levels and less fear of humans53, which would all be advantageous in a poultry farm setting. More risk-taking behaviour in a production setting where predators are absent would facilitate rapid adaptation as they would be more likely to explore and locate the resources. Ability to navigate in a novel multi-dimensional space and to locate feed, nests and water is crucial when birds are transferred to an aviary-style laying house54. That an environment with a higher level of environmental choice results in birds more able to acquire information and enhance skills fits with the predictions of 30. Our results suggest that a more spatially complex rearing environment better prepared chicks for future challenges by promoting behavioural adaptability that would be advantageous in a future spatially complex environment.Our results indicate that rearing chicks in an environment that provides multiple litter and perch types leads to increased exploration, possibly also involving greater risk-taking, as well as improved spatial skills. Compared to their wild ancestor, the red jungle fowl, domestic fowl show impaired spatial learning56. Play is generally considered an indicator of good welfare57 and, therefore, could imply that the provision of both environmental change and choice had a positive impact on the welfare of the chicks during rearing.Behavioural differences between chicks reared with higher levels of change and choice (Changing*Multi-choice) compared to standard-reared chicks (Non-changing*Single-choice) were only found in the rearing pen observations. The chicks reared with a combination of increased change and choice (Changing*Multi-choice), like those reared with only increased change (Changing*Single-choice) or only increased choice (Non-changing*Multi-choice), showed increased foraging in association with the disturbance compared to standard-reared chicks. Other than this, the only other behavioural difference from the standard-reared chicks was a higher number of sparring bouts, considered to be a type of play fighting behaviour, in the undisturbed rearing pen observations. Sparring in domestic fowl is most pronounced in chicks during the first weeks of life, before aggressive behaviour emerges58. Considering that a higher level of choice may increase the experience of controllability29, the repeated relocation of the resources may have decreased the sense of control following each relocation. Supporting this, we found more preening following disturbance in the treatment with change and choice than we did in the treatment with choice alone. Preening can either be connected with comfort and relaxation59 or be a displacement behaviour60 and could, in this case, indicate stress recovery following disturbance. There could also be possible counteracting effects of providing both choice and change. Campbell et al.54 obtained inconsistent results in a spatial task when chicks were exposed to repeated introduction of novel objects, relocation of structures, and unpredictable sounds and lights in the rearing environment between 4 and 21 days of age54. Disturbance from the relocation of resource variants in the Changing*Multi-choice treatment, as with the disturbance from multiple stimuli in Campbell et al.45, may have interfered with the enhancement of spatial skills otherwise expected in a spatially complex environment. In future studies investigating effects of change and choice, an option might be to exchange rather than relocate the resource variants in the Multi-choice environment, making the type of change comparable with the change in the Single-choice environment. This alternative design, however, will require a greater number of variants of each resource and, additionally, different groups of chicks in the Single-choice environment will need to experience each of these variants to avoid confounding change and choice with differences in the variant types used in each treatment.Contrary to our hypothesis, we did not find a generally beneficial synergistic effect of providing both change and choice together during rearing. The results from this treatment (Changing*Multi-choice) were either similar to providing only change (Changing*Single-choice) or only choice (Non-changing*Multi-choice), or intermediate between them, or could even be worse. This latter result occurred in the case of latency to solve the detour task in the Multivariate behavioural test, which was longer for chicks reared with both change and choice than it was for chicks reared only with a higher level of choice. This might be due to a difference in the manner in which environmental change was imposed in combination with choice. In the Changing*Multi-choice treatment, change was accomplished through relocation of the resource variants in space, whereas in the Changing*Single-choice treatment, variants were exchanged for new types over time. Lack of previous experience of repeated exposure to novel resource variants could explain why the freezing duration in the Multivariate behavioural test was not lower among chicks reared with both change and choice compared to the standard reared chicks. That the environmental choices were repeatedly relocated in the Changing*Multi-choice treatment may have also disrupted the positive effects of exposure to a variety of litter and perch types. Laying hens have a characteristic individual pattern of moving between different resources in the pen over the course of each day61 or at the same time54. Our findings suggest that these are two different types of inputs that affect chicks in different ways and, importantly, interact with each other. Further, previous studies compare the effects of these environmental enrichments to barren environments, whereas our treatments all provided basic resources such as litter and perches. This enabled us to focus specifically on the effects of environmental change and choice, thereby affecting predictability and controllability, respectively.Previous studies investigating effects of environmental enrichments or more complex early environments, have included novelty (corresponding to our changing treatment) and spatial structures (corresponding to our choice treatment), either aloneThe experience of environmental change, from exchanging litter and perch types, or of environmental choice, from presenting a variety of litter and perch types, could be advantageous for chick welfare during rearing because of an increase in active engagement with the environment. Further, a higher level of change reduced initial fear responses when first placed in a novel environment, while a higher level of choice stimulated exploration and spatial skills. However, an increase in both environmental change and choice, by relocating the resource types that provided choice, did not generally improve outcomes over those from change or choice alone. In fact, in some cases it resulted in intermediate outcomes or results no different from the standard condition offering neither change nor choice. These results suggest that change and choice have different but generally positive outcomes for the development of behavioural adaptability when provided in moderation. Based on these results, we encourage further studies on the benefits of providing change and choice during rearing for animals that will subsequently be transferred to a different environment.62.The study was approved by the Animal Research Ethics Committee in Uppsala SE (Protocol number 5.8.18-11549/2017). All methods were carried out in accordance with relevant regulations and following the ARRIVE guidelinesA total of 332 day-old female chicks of the white layer hybrid Bovans Robust from a commercial hatchery were allocated to 16 rearing pens (20 chicks/pen) in one room at the Swedish Livestock Research Centre (Lövsta). Average chick weight per pen was 33 ± 0.17 g. Four pens, balanced across room locations, were assigned to each of four treatments according to a 2 × 2 factorial design with two levels of environmental change (Non-changing/Changing) and two levels of environmental choice (Single-choice/Multi-choice). The treatments were, thus, Non-changing*Single-choice, Non-changing*Multi-choice, Changing*Single-choice and Changing*Multi-choice , along with ample litter and perch space per chick, made it possible for all chicks in the pen to use the same litter tray or perch at the same time if they chose to do so and also to better allow the expression of behavioural differences.Litter was presented in trays 70 × 35 × 3 cm) at four locations in each pen . Then the birds were disturbed by a person entering the pen, and either changing the litter and perches (Changing pens) or making no change (Non-changing pens). After this, there were 2–4 rounds under the relatively disturbed conditions (within the following 4-h time period). See Supplementary Information Fig. 2  for an oThis test was conducted when chicks were between 28–33 days of age. Chicks from each pen were randomly allocated to two groups of 10 chicks (from the same pen), and placed in two specially designed holding boxes. Four of these holding boxes (one per treatment combination) were then placed just inside the entrance of each of four novel pens Fig. a. At the13. It involved testing chicks individually in a detour task and open arena . Counts and latencies were modelled using a linear mixed model fit by REML and the Satterthwaite’s method (lmertest). Proportion data were analysed using generalized linear mixed models with a binomial distribution using the glmer-function of the lme4-package. All models included Change, Choice and the interaction between Change and Choice as fixed effects and Rearing Pen ID as a random effect. The effects of observer and observation order were not significant for any variables, and were therefore excluded from all models. Models for data from the rearing observations included the fixed effects of age (week), Disturbance and a three- (and two-) way interaction between Change, Choice, and Disturbance. Full models for data from the Multivariate behavioural test included chick and group test order. These were kept if having a significant effect (chick for freezing duration), or otherwise removed to simplify models . Assumptions were visually checked by residual plots; normal probability plot of residuals (QQ plot) and residuals versus fitted values. If models failed to converge, the random effect of pen was removed and weekly pen means were analysed with week as a fixed effect to control for repeated measures  or group means with group as a fixed effect . Overall pen means were analysed if necessary to achieve model convergence . If model assumptions were not met, a Kruskal Wallis test was performed on overall pen means (worm running). Effects were explored using a Type III Analysis of Variance Table with Kenward-Roger's method for linear mixed models, while the Anova-function in the car-package was used for linear models or generalized models. Interactions of Change and Choice were investigated using estimated marginal means from the emmeans-function. A significance level of 0.05 was used and trends were ignored. In the presence of significant pairwise-comparisons of interactions, significant main effects were ignored. All results from our fitted models are presented as estimated marginal means and standard error of the estimated mean, whereas data investigated using the Kruskal Wallis test are presented by means (Aggregate function) and standard error of the mean. Log-transformed values  are presented as back-transformed to the original scale.Supplementary Information.


R2 = .66, ∆R2 = .03) exploring the moderating effect of AAQ-II demonstrated a significant interaction term between AAQ-II and subjective burden. The second model , exploring the moderating effect of EACQ, demonstrated a significant interaction term between EACQ and subjective burden. These results provide evidence that carers with higher levels of experiential avoidance may be particularly prone to the negative effect of subjective burden on anxiety symptoms. Clinical implications for assessment of experiential avoidance and its treatment in carers of people with dementia are discussed.Anxiety remains understudied in family carers of people with dementia. Understanding factors that moderate the relationship between stressors and anxiety symptoms in this population is critical to inform interventions. This study examined whether generic experiential avoidance (AAQ-II) and experiential avoidance specific to caregiving-related thoughts and feelings (EACQ) moderate the relationship between subjective burden (ZBI-12) and anxiety symptoms (GAD-7) in carers of people with dementia. The first model ( This study provides evidence supporting that experiential avoidance may enhance the negative effect of carer subjective burden on anxiety symptoms.The strength of this effect seems to increase as levels of carer subjective burden rise.There is potential that generic experiential avoidance (measured by the AAQ-II) may have a stronger moderating effect than experiential avoidance specific to caregiving-related thoughts and feelings (measured by the EACQ).Early interventions to undermine experiential avoidance may be beneficial for carers of people with dementia.Monitoring the outcome of such interventions using the AAQ-II as the dementia progresses is recommended.It is estimated that one in three people will care for a person with dementia in their lifetime . Due to Nevertheless, the current dementia literature mainly focuses on carer depression as an outcome and carer anxiety is somewhat neglected. In addition, existing carer interventions, such as Cognitive Behavioral Therapy (CBT), are considered to be not as effective for anxiety as for depression in family carers of people with dementia . TherefoCarer subjective burden refers to personal appraisals of burden including the physical, psychological, social and emotional impact their caring role has on their life . A recenExperiential avoidance is the attempt to alter the form, frequency or intensity of private experiences such as thoughts or feelings, even when doing so is costly, ineffective or unnecessary . RecentlThe most widely used measure of experiential avoidance in research is the Acceptance and Action Questionnaire-II (AAQ-II). The AAQ-II is considered a generic measure of experiential avoidance and has been validated across various populations . The AAQTo overcome the limitation of the AAQ-II, researchers have developed several variations of instruments assessing experiential avoidance that are more specific to certain populations . These dConsidering the well-established moderating effect of experiential avoidance in non-carer populations between stressors and mental health outcomes, we hypothesize that both generic experiential avoidance and experiential avoidance in caregiving would moderate the relationship between carer subjective burden and anxiety symptoms in family carers of people with dementia. That is, higher levels of experiential avoidance would associate with greater anxiety symptomatology at higher levels of carer subjective burden. Carers who thus report high levels of carer subjective burden and present with high levels of experiential avoidance, are likely to report higher levels of anxiety symptomatology than carers with high levels of carer burden but low levels of experiential avoidance. Considering the possible differences in the moderating effects of general and domain-specific measures of experiential avoidance, we hypothesize that the domain-specific measure of experiential avoidance (EACQ) would account for more variance in anxiety symptoms than the generic measurement of experiential avoidance (AAQ-II).This is a secondary analysis of data from an intervention study assessing the feasibility and acceptability of an online self-help Acceptance and Commitment Therapy (ACT) programme . ScreeniPotential participants were contacted by the research team via telephone or email to check their eligibility. Participants meeting the eligibility criteria received an invitation letter and information sheet. Those who provided written consent were then invited to the screening session. Due to the COVID-19 pandemic, an appointment for the screening session was made remotely via video call or telephone. During this screening session, participants were asked to complete self-reported questionnaires using an online survey platform or hardcopies, which were sent home and returned via post, in the remote presence of a researcher.Demographic information including carer age, gender, relationship to the care recipient, and the length of care in months were collected to characterize the sample.not at all) to 3 (nearly every day). The total score indicates anxiety severity of minimal (0–4), mild (5–9), moderate (10–14), or severe (15–21). The GAD-7 has good psychometric properties with good internal consistency (Cronbach Alpha = .89)  . The Cronever) to 4 . The total score ranges from 0 to 48, with higher scores representing higher levels of carer subjective burden. The ZBI-12 has good psychometric properties with good internal consistency (Cronbach Alpha = .88)  . The Cronever true) to 7 . The items of the AAQ-II include statements such as “My painful experiences and memories make it difficult for me to live a life that I would value.” The total score ranges from 7 to 49, with higher scores indicating greater levels of experiential avoidance. The AAQ-II has good psychometric properties with good internal consistency (Cronbach Alpha = .88)  . The Cronot at all) to 5 (a lot). The total score ranges from 15 to 75, with higher scores indicating higher levels of experiential avoidance in caregiving. The EACQ has good psychometric properties with acceptable internal consistency (Cronbach Alpha = .70)  . The CroAll analyses were performed using IBM SPSS statistical software (Version 28). Data were examined for accuracy with no extreme outliers detected and most variables approximating normality. The percentage of missing data across the eight variables varied between 1.3% and 2.5%. Of the 79 participants, one respondent did not complete the Experiential Avoidance Caregiving Questionnaire (EACQ) while another respondent did not complete both the Zarit Burden Interview (ZBI-12) and the Experiential Avoidance Caregiving Questionnaire (EACQ). This resulted in a dataset of 77 family carers in the moderation model of EACQ and a dataset of 78 family carers in the moderation model of AAQ-II. To ensure consistency among both models, the participant that didn’t complete the EACQ was removed from the final moderation model of AAQ-II. Removing this participant did not result in any difference of significance.r correlations were conducted between control variables  and independent and moderator variables , and the dependent variable (anxiety symptoms). Correlations of r < .30 were considered small, r ≥ .30–.49 medium or moderate, and r ≥ .50 were considered strong  using the PROCESS computation macro Model 1;  to examiSD above the mean, the mean, and one SD below the mean . Participants’ age ranged from 32 to 85 with 43% of participants being older than 65 years. Forty-two per cent of participants showed minimal symptoms of anxiety, while 39% demonstrated mild symptoms and 8% and 12% of participants demonstrated moderate and severe symptoms, respectively. Other demographic information and means and standard deviations of measurements are shown in r correlations were conducted (see A series of Pearson’s cted see . Carer ab) and standard errors (SE) of the variables  and the model summary are presented in The unstandardized coefficients , as well as the conditional effect of experiential avoidance on anxiety symptoms . The interaction term  incrementally accounted for 3.4% of the variance in anxiety symptoms (∆R2 = .03). The total model explained 66% of the variance of anxiety symptomatology  = 27.15, p < .001). The effect size for this regression model (Cohen’s f2) was 1.91, suggesting a large effect size. These results confirmed that generic experiential avoidance measured by the AAQ-II is a significant moderator of the relationship between carer subjective burden and anxiety symptoms.The conditional effect of carer subjective burden on anxiety symptoms was significant  and at the mean level of the AAQ-II . However, the slope at one SD below the mean was not significant   see . When pab) and standard errors (SE) of the variables .The results of the moderation analysis are shown in β = .51, 95% CI , p < .001) and experiential avoidance in caregiving  on anxiety symptoms were significant. The total model explained 53% of the variance of anxiety symptoms  = 15.87, p < .001). This regression model showed a large effect size of 1.12 (Cohen’s f2). The addition of the interaction  did indicate a significant change beyond the main effect, incrementally accounting for 2.8% of the variance in anxiety symptomatology (∆R2 = .03). These results confirmed that experiential avoidance in caregiving assessed by the EACQ is a significant moderator of the relationship between carer subjective burden and anxiety symptoms.The conditional effects of both carer subjective burden , at the mean level of the EACQ , and at one SD below the mean   see . When paOur results confirmed that both, generic experiential avoidance (measured by the AAQ-II) and experiential avoidance in caregiving (measured by the EACQ) were significantly moderating the relationship between carer subjective burden and anxiety symptoms. The strength of this reverse buffering effect increased when participants presented higher levels of carer subjective burden, suggesting that experiential avoidance could worsen the impact carer subjective burden has on anxiety symptoms, particularly among family carers experiencing higher levels of subjective burden.Percentages of variance accounting beyond the main effects of experiential avoidance and carer subjective burden alone were significant in both models, with the interaction term of the AAQ-II and the EACQ accounting for 3.4% and 2.8% of the variance in anxiety symptoms, respectively. The interaction terms of the AAQ-II had a standardized coefficient of .208, whereas the standardized coefficient of the interaction term of the EACQ was .169. Although both interaction terms were statistically significant, there is potential that generic experiential avoidance may have a better moderating effect, which contradicts our hypothesis. This is also supported by visual inspection of slopes, which suggests that all slopes are steeper regardless of levels of the EACQ  comparedUnlike the AAQ-II, which is the generic measure of experiential avoidance, the EACQ assesses experiential avoidance towards thoughts and feelings related to the person with dementia and the care provided to them ” . Family The findings of this study provide important clinical implications in terms of assessment of experiential avoidance and interventions for preventing increased anxiety symptoms in family carers of people with dementia. Recent studies examining the association between carer subjective burden and Alzheimer’s disease severity and disease progression concluded that, as dementia progresses, carers are more likely to experience significant subjective burden . This suWhile this study successfully examined the moderation effects of generic experiential avoidance and experiential avoidance in caregiving, there are some limitations that need consideration. First, this study employed the AAQ-II as a general measure of experiential avoidance. Although AAQ-II has been widely used in research, recent studies raised some concerns about its convergent and discriminant validity , highligConsidering the explained variances (66% for the AAQ-II and 53% for the EACQ), there may be other factors affecting the proposed moderation models. For example, a recent study examining the moderating role of experiential avoidance and cognitive fusion in a non-clinical sample concluded that experiential avoidance indirectly contributed to depression and anxiety through cognitive fusion rather than experiential avoidance directly affecting depression and anxiety . CognitiThe current study collected data during the COVID-19 pandemic; therefore, results may be affected by the additional stress the pandemic brought to carers . In addiFinally, the sample size required for a regression model to achieve a power level of .80, a significance level of .05 and a medium effect size (.15) is 92 when five independent variables are used. This study had a sample size of 77, which is smaller than required although the effect size for both regression models was large in this study. The cross-sectional nature of this study also does not allow for any causal assumptions to be made, and thus, the findings need to be interpreted with caution. In conclusion, it is recommended that future studies replicate this study in family carers of people with dementia with higher levels of anxiety, using different measures of generic experiential avoidance  alongside the AAQ-II and in a longitudinal design.Despite limitations, this study provided evidence supporting that experiential avoidance may enhance the negative effect of carer subjective burden on anxiety symptoms in family carers of people with dementia. The strength of this reverse buffering effect seems to increase when carers present higher levels of subjective burden. The development of early interventions aimed at undermining experiential avoidance and monitoring the outcomes using the AAQ-II as the disease progresses may be beneficial for preventing increased anxiety symptoms among this population.


Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a “host-takeover module”, a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism—no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development. Bacillus subtilis (B. subtilis) [Staphylococcus phages K, G1 and Twort, Listeria phage P100, and Lactobacillus phage [B. subtilis DNA [Bacteriophage (phage) SPO1 is a lytic phage of ubtilis) . Besidesus phage . SPO1 haus phage . In partus phage . For exailis DNA .B. subtilis [The host-takeover module (HTM) in the terminal redundancy contains 24 genes A, all wisubtilis . PEIP  synthesis and iron metabolism. Thus, our study connects bacterial iron acquisition to the response from phage, and reveals a potential strategy employed by SPO1 to detoxicate iron from the environment or bacterial defense by upregulating bacterioferritin synthesis to acquire an excess amount of iron.gp48 in operon 4. However, gene products from adjacent operons shed light on its function. Among the neighboring genes whose functions have been annotated, gene product 44 is a non-specific RNAP binding protein, that is able to shut down RNA synthesis for both E. coli and B. subtilis protein [gp49, products of gene 50 and 51 were considered to bind to host RNAP for regulating host phage gene expression while shutting off host RNA synthesis [Okubovirus genus members that infects B. subtilis  groups showed the typical crystal violet for Gram-positive bacteria under the stain. As gp49 is a nonessential gene for the phage, the growth attenuation assay and the Gram stains suggest Gp49 may have a role for the phage in invading B. subtilis.A previous report suggested overexpressing plasmid-borne Gp49 in ondition , it was ry phase A. Inspiracterium , we then00 = 1.4 B. Intere15N and 13C labeled Gp49 (We thus attempted to elucidate the function of Gp49 by solving the structure of Gp49 using standard triple resonance nuclear magnetic resonance (NMR) spectroscopy with recombinantly expressed led Gp49 A,B. The led Gp49 C. Echoinled Gp49 D. Furtheled Gp49 E. NotablD = 1.86 ± 0.04 μM. In conclusion, Gp49 is a novel RNA binding protein, whose structure does not resemble any other published RNA binding protein.To verify if Gp49 is indeed a nucleic acid binding protein, we performed electrophoretic mobility shift assays (EMSA) with agarose gel. Using the oligos listed in gp49 regulated by SPO1 early promoter [B. subtilis using RNA-seq to check if the interaction between Gp49 and RNA affect the global transcription of the bacterium. The number of differentially expressed genes (DEGs) with a threshold of log2 fold change >1.0 and padj values ≤ 0.005 are 318, including 146 upregulated and 172 down regulated among 4900 total genes. According to the gene ontology (GO) categorization, most of the upregulated genes are involved in tetrapyrrole biosynthesis and metabolism in the biological process category, with heme and tetrapyrrole binding the molecular function category  was stained safranin color instead of violet during Gram staining in the iron-supplemented condition (300 μM) [B. subtilis. As bacteriophage inactivation was shown to correlate with the concentration of ferrous iron or Fe (II) at low doses [As Gp49 is an RNA binding protein and expressed during the early stage of phage development as promoter , the hoscategory A. These uisition . Similaruisition C. Thus, and hemH E. Meanwhsubtilis D. This c(300 μM) . The 11-B. subtilis and its relatives have been considered gut commensals, with features being advantageous to survival within the human gastrointestinal tract (GIT), compared with soil strains [Bacillus-based probiotics have been shown to improve digestive health by strengthening the intestinal barrier and limiting inflammatory responses [B. subtilis in the GIT, phage-mediated horizontal gene transfer (HGT) has profound consequences for human health and disease [ strains . Bacilluesponses . Remodulesponses . In addi disease . Thus, l disease ,18,25. AIron is an essential element for almost all living organisms, including bacteria. Bacteria have evolved sophisticated mechanisms to regulate iron uptake, storage, and utilization in order to maintain cellular homeostasis and ensure survival in various iron environments ,26. The hem operon, SPO1 would gain advantage under certain external conditions, such as with an excess amount of environmental iron. Thus, our study revealed that Gp49 is a novel RNA binding protein that regulates host iron metabolism at transcriptional level. We propose that Gp49 would provide survival advantage for the phage while encountering excess iron from either cellular or external sources.While iron can also trigger prophage induction , phages B. subtilis 168 strains. The cultures were then grown overnight at 37 °C in LB liquid medium supplemented with 25 μg/mL chloramphenicol. Then, 1% of the cultures were transferred to 2 × YT medium containing 2% (w/v) glucose (to prevent leakage of pHT01 vector), 25 μg/mL chloramphenicol, and either water or 1mM IPTG. Cultures were conducted in 96-well plates  with a volume of 200 μL per well. The 96-well plates were assessed for OD600 using a Cytation 5M enzyme marker . The cultures were incubated at 37 °C with continuous shaking at 573 rpm for a duration of 12 h. Three biological and technical replicates were performed.Gp49 was amplified from SPO1 genome by PCR using the primers listed in At the end of the 12 h growth curve, 10 μL of each bacterial solution from the four groups was absorbed onto a slide and rapidly fixed at high temperature. Following primary staining, mordanting, decolorization, and counterstaining, bacterial morphology was observed under an oil immersion microscope at 200× magnification .600 of 0.6–0.8 and induced with 1 mM of isopropyl-b-D-thiogalactopyranoside (IPTG) in LB media. Soluble Gp49 was purified by immobilized metal-ion chromatography with a Ni-NTA column. After purification, the C-terminal 6xHis-tagged Gp49 was immediately dialyzed overnight at 4 °C into the buffer . For NMR study, the 15N and 13C labelling of Gp49 was achieved using M9 minimal medium containing 0.07% 15NH4Cl and 0.2% 13C-glucose (CIL).SPO1 gene 49 was amplified by PCR and ligated into pET-28a expression vector. The plasmid was transformed into BL21 (DE3) cell strain, and the cells were grown at 37 °C to ODα and Hβ assignments were obtained using HBHA(CBCACO)NH and the full side-chain assignments were extended using HCCH-total correlation (TOCSY) spectroscopy and (H)CCHNH-TOCSY. Three-dimensional 1H-15N/13C NOESY-HSQC experiments provided the distance restraints used in the final structure calculation. Three-dimensional 1H-15N/13C NOESY-HSQC (mixing time 100 ms at 800 MHz) experiments provided the distance restraints used in the final structure calculation. The ARIA protocol was used for completion of the NOE assignment and structure calculation. The structural statistics are shown in The structural determination for Gp49 was performed in the buffer described above with a final concentration of 0.7 mM. The spectra were collected at 298 K on a Bruker 800 MHz (Avance NEO), Billerica, MA, USA, spectrometer equipped with cryo-probe. The backbone assignment was completed using our in-house, semi-automated assignment algorithms and standard triple-resonance assignment methodology using HNCACB/CBCANH, and HNCO/HN(CA)CO. HThe RNA was synthesized in vitro using RiboMAX Large Scale RNA Production System . The template DNA containing 21-nt complementary strands were obtained by mixing equivalent molar amounts of each oligonucleotide, then adding the enzyme mix, RiboMAX™ Express T7 2X Buffer, linear DNA template , nuclease-free water, mixing gently, and incubating at 37 °C for 3 h. The RNA was then purified using gel extraction purification.These assays used RNA, ssDNA, and dsDNA fragments  was fixed on the COOH sensor chip (CM5) by capture-coupling, the interactions of fixed Gp49 with ssRNA detected by Biacore T100  at 25 °C. The binding time was ~85 s and the disassociation time was approximately 120 s. The flow rate was 30 μL/s. A 1:1 diffusion corrected model was fitted to the wavelength shifts corresponding to the varied protein and ligand concentration. The data was retrieved and analyzed with Biacore T100 Evaluation Software.B. subtilis (stain 168) containing empty pHT01 and pHT01-gp49 were induced with 0.5 mM IPTG for 4 h at 37 °C at OD600 = 0.6. Then, 1 mL cell culture was taken from each sample for RNA extraction. The bacteria RNA was extracted from uninduced and induced samples by RNA Extraction Kit . RNA quality and quantity were checked using Bioanalyzer  and RNA 6000 Nano kit . For RNA-seq experiments, rRNA was removed using the Ribo-Zero rRNA removal kit. Tophat2 and Cufflinks 2.2.1 were used to analyze the RNA-seq data and identify differential genes [al genes . The bacB. subtilis 16S ribosomal RNA as the internal control. Total RNA was extracted from each sample using RNA fast200 Kit , according to the manufacture’s instruction. Then, 5 μg of total RNA was reverse transcribed with qPCR RT Master Mix Kit  for complementary DNA synthesis. RT- qPCR was performed in a 20 μL volume consisting of 10 μL 2X QuantiNova SYBR Green PCR Kit , 8 μL RNA-free water, 1 μL cDNA template, and 0.5 μL of each primer pair according to the manufacturer’s procedure using CFX Connect Real-Time PCR System . Three biological replicates were performed each with three technical replications. Relative differential expression levels of msyB were calculated based on the cycle threshold (CT) values normalized with the control gene using the 2−△△CT method.RT-qPCR used


RNA is integral to gene expression as messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) each play roles to transmit information from DNA into synthesis of functional proteins. During their lifespan, these nucleic acids may be chemically modified by alkylation, oxidation and the removal of bases, which alters their activity. Though much research has been devoted to the detection and repair of damaged DNA, RNA is viewed as a short-lived molecule that is quickly degraded upon damage. However, recent studies indicate that RNAs that become modified, particularly during stress, function as important signalling molecules. In this review, we focus on the effects of abasic RNAs and the modifications that lead to the loss of a base, as RNAs that are initially methylated or oxidized often become abasic. We describe how these chemical changes occur and cite recent work showing that in addition to being indicators of damage, abasic RNAs function as signals that mediate downstream cellular responses to stress. Various chemical agents, ranging from environmental pollutants to metabolites from endogenous sources, may alter the composition of nucleic acids, thereby affecting their functions. Due to the reactivity of their oxygen and nitrogen atoms , nucleicin vitro studies characterized the resistance to depurination of RNA compared to DNA. Spontaneous generation of abasic RNA is slower than DNA due to the electron-withdrawing nature of the 2’-OH group of ribose which lowers the rate of hydrolysis [RNA is challenging to study because of its relatively transient nature, and its multiple copies suggest that any damage to individual molecules would not likely be permanently detrimental to the cell. Still, there are important biological consequences of abasic lesions in RNA. RNA is more abundant than DNA  and servdrolysis ,8. Howevdrolysis . The incIn this review, we describe how abasic sites form in mRNA, tRNA and rRNA and we examine their effects in cells. In addition to being damaged RNAs that must be detected and degraded, abasic RNAs are also important signalling molecules that communicate cell status and control gene expression.Abasic sites are formed in RNA by the hydrolysis of the glycosidic bond connecting the nitrogenous base to the ribose sugar. The process begins with the protonation of a nitrogen atom leaving a positive charge on the base. Usually, this occurs on purines rather than pyrimidines as the pyrimidine’s glycosyl bonds are 20-fold more stable than purinic bonds . PurinesIn a typical depurination setting enhanced by acidic conditions, the N-glycosidic bond is under threat by the protonation of the N−7 of purines. This protonation will create a rearrangement of electrons starting with the neighbouring double bond that will alleviate the localized positive charge on N−7 . The chaThis mechanism is very similar to spontaneous hydrolysis described above in that a positive charge is induced on the nitrogenous ring . The N−72•-), hydrogen peroxide (H2O2) and hydroxyl radical (OH−). The mechanism of RNA depurination begins with the sequestration of the C1’ hydrogen atom from the ribose sugar by a superoxide radical which yields a peroxyl radical (Reactive oxygen species (ROS) are commonly produced by normal aerobic metabolic processes and can be increased by environmental stressors such as ionizing radiation and pathogen attack . They ar radical . The supE. coli, A3027 yeast) within a GAGA tetraloop on the SRL. The conserved amino acids within the active site of glycosylases include a glutamic acid and an arginine which are the principal actors in depurination while amino acids valine and serine help to stabilize the adenine base [RNA abasic sites can also be generated by RNA N-glycosylases (EC 3.2.2.22). These enzymes are produced in many plants, fungi and bacteria where they are described as toxins involved in pathogen defence . These Rine base . Similarine base ,21.Abasic sites in RNA from biological samples are often detected using an aldehyde reactive probe. The ribose lacking a base forms an unequal equilibrium between two conformations: the ‘closed’ ring structure of the ribose and the ‘open’ structure revealing an accessible aldehyde group . An alde2O2 through NADPH oxidase and the second accumulates superoxide radicals. The third wave involves the peroxidation of membranes from superoxide radicals leading to cell death. Chmielowska-Bak et al. [Abasic sites on mRNAs arise largely from hydrolysis of oxidized bases, which are formed from reaction with reactive oxygen species during normal cellular metabolism. For example, superoxide anion is produced by reaction of molecular oxygen with the reduced form of nicotinamide adenine dinucleotide (NADH), a component of the electron transport chain in mitochondria . The supk et al.  observedk et al. .mRNA can also be oxidized by cytochrome c-mediated peroxidation resulting in the loss of a guanosine residue to create an abasic site . With cyAbasic mRNAs have also been observed in the R-loops of humans and yeast . R-loopsFollowing initiation of translation, protein synthesis relies on three repeating steps: 1) eEF1A, with GTP, binds an aminoacyl-tRNA to the ribosomal A site; 2) peptidyl transferase forms a peptide bond between the peptide at the ribosomal P site and the aminoacyl-tRNA at the A site, shifting the peptide to the A site; 3) eEF2, with GTP, translocates the peptidyl-tRNA with its mRNA codon to the P site leaving the A site empty for a new aminoacyl-tRNA. The processivity of translation involves the movement of mRNA through the ribosome and decoding of its sequence, one codon at a time (reviewed in ). Abasicin vitro translation assay [To investigate the impact of abasic sites on mRNA translation, a synthetic mRNA containing an abasic site in the middle of a coding sequence for five amino acids was tested in an on assay . Translain vitro bacterial system, 95% of the peptides were isoleucine (Ile) and only 5% were methionine (Met). By comparison, the eukaryotic HEK293T cells were unable to translate this modified codon, suggesting a different level of tolerance for abasic templates between prokaryotes and eukaryotes. Termination of translation has also been investigated by the insertion of abasic nucleotides at the stop codon [The effect of the position of a non-standard nucleotide within a codon was also investigated. Specifically, abasic nucleotides were introduced in the mRNA and investigated using mass spectrometry (MS) to identify the decoded amino acid . When anop codon . Under nop codon ,46. Howeop codon . TherefoDecrease in translation caused by modifications to mRNA has broader effects on gene expression. Specifically, accumulation of oxidized RNA, primarily 8-oxo-rG, correlated with a decrease in mRNA translatability which preferentially reduced levels of short-lived transcription factors. Some of these factors were repressors of transcription and their reduced levels resulted in enhanced transcription from genes controlled by these repressors . TherefoRecently, Ochkasova and colleagues ,49 showePhe contains wybutosine, a hypermodified guanine residue at position 37 that affects translational fidelity by stabilizing the codon-anticodon base pairing in the decoding step of translation [Phe is subsequently cleaved at the abasic site by aminoglycoside antibiotics [Abasic sites are also found in some tRNAs as an intermediate step in their degradation. For example, yeast tRNAnslation . The modnslation . tRNAPheibiotics . Cleavagibiotics . EndonucMature tRNAs that are not properly post-transcriptionally modified or contain sequence mutations are subject to rapid tRNA degradation (reviewed in ). In yeain vitro assays testing purified glycosylase activity in cell-free translation systems showed complete translation inhibition, which supported the idea that these enzymes were toxins synthesized to kill infected hosts or invading pathogens by inhibiting their translation [Abasic rRNA is best exemplified by the activity of RNA N-glycosylases on the α-sarcin/ricin loop of the large 23S−28S rRNA. RNA N-glycosylases, often referred to as ribosome inactivating proteins, depurinate the rRNA at A4324  of the large ribosomal subunit ,75. The nslation ,78.Recent evidence suggests that cells detect damage to rRNA in advance of whole-scale translation inhibition. Specifically, expression of ricin in yeast resulted in only 10% depurination of rRNA which was sufficient to trigger cell cycle arrest . TherefoSince ribosomes play a central role in protein synthesis, conserved stress response signalling pathways have evolved to respond to obstructions to translation, which include detection of rRNA damage. Ribotoxic stress response (RSR) is a cellular signalling cascade in prokaryotes and eukaryotes that is activated following loss of a base or structural damage to the α-sarcin/ricin loop (SRL) of the 28S rRNA  Figure . The MAPRecent results show that the MAPKKK ZAKα signals rRNA depurination through autophosphorylation , but howHow abasic mRNAs are detected and degraded needs further investigation. Though No-Go decay pathway appears integral to the process and may involve collision of ribosomes upstream of the ribosome at the abasic site, the endonucleases that cleave abasic mRNAs remain partially identified. Specifically, the yeast endonuclease Cue2 was recently shown to cleave mRNA at the ribosome upstream of the stalled ribosome . HoweverThe discovery of abasic RNA within R-loops that form during transcription is the first to show that this RNA modification can affect the elongation of RNA polymerase II . Abasic mRNAs, tRNAs and rRNA may become abasic during their lifetime and though this damage can be a consequence of normal metabolism, their levels increase during periods of cell stress. Cells have ways to detect this damage and even though RNAs do not seem to be repaired like DNA, the time between loss of a base and degradation of the RNA is enough to elicit signalling pathways in cells. Specifically, we have seen how abasic mRNAs may cross-link to ribosomal proteins or cause ribosomes to stall when they encounter missing bases at their A site. In addition, abasic mRNAs within R-loops can pause the RNA polymerase and determine which messages get transcribed. Though abasic mRNAs reduce levels of their individual protein products, damage to rRNA in the form of α-sarcin/ricin loop depurination will have wider impact on cellular translation. Known as the ribotoxic stress response, rRNA damage triggers a kinase cascade resulting in inflammation and potentially cell death. Though we have some understanding of how abasic RNAs are detected, we are just beginning to ask how this damage is communicated within cells and what controls the balance between cell recovery and adaptation versus apoptosis. Several interesting questions remain to be answered.Continued research will provide answers to these and other questions about how abasic RNAs are detected, how they function to communicate cell status and control gene expression before they are eventually degraded. However, it is clear that abasic RNA levels change during cell stress and given the chemical stability of this modification, abasic RNAs may act as signalling molecules that allow cells to either adapt or succumb to environmental change.


Technological innovations that improve the speed, scale, reproducibility, and accuracy of monitoring surveys will allow for a better understanding of the global decline in tropical reef health. The DiveRay, a diver-operated hyperspectral imager, and a complementary machine learning pipeline to automate the analysis of hyperspectral imagery were developed for this purpose. To evaluate the use of a hyperspectral imager underwater, the automated classification of benthic taxa in reef communities was tested. Eight reefs in Guam were surveyed and two approaches for benthic classification were employed: high taxonomic resolution categories and broad benthic categories. The results from the DiveRay surveys were validated against data from concurrently conducted photoquadrat surveys to determine their accuracy and utility as a proxy for reef surveys. The high taxonomic resolution classifications did not reliably predict benthic communities when compared to those obtained by standard photoquadrat analysis. At the level of broad benthic categories, however, the hyperspectral results were comparable to those of the photoquadrat analysis. This was particularly true when estimating scleractinian coral cover, which was accurately predicted for six out of the eight sites. The annotation libraries generated for this study were insufficient to train the model to fully account for the high biodiversity on Guam’s reefs. As such, prediction accuracy is expected to improve with additional surveying and image annotation. This study is the first to directly compare the results from underwater hyperspectral scanning with those from traditional photoquadrat survey techniques across multiple sites with two levels of identification resolution and different degrees of certainty. Our findings show that dependent on a well-annotated library, underwater hyperspectral imaging can be used to quickly, repeatedly, and accurately monitor and map dynamic benthic communities on tropical reefs using broad benthic categories. Local and global stressors have caused a continued decline in reef health over recent years11. Amidst this global trend of declining reef health and rapid environmental change, the need for fast, efficient, and accurate assessments of reef communities is more pressing than ever before12. Many methods to optimize reef community monitoring have been developed and implemented over the years, each with their own tradeoffs, advantages, and disadvantages17. Reef monitoring has historically been conducted through repetitive identifications and annotations of benthic categories by investigators. One of the more common methods is the phototransect methodology, which is still widely used and often regarded as the baseline standard for the evaluation of new techniques19. Although photoquadrat surveys typically cover small expanses of reef, they require extensive post-collection processing and analysis. Moreover, it is difficult to investigate several characteristics that could drive reef community dynamics by using photoquadrats alone, such as species/taxa distributions, habitat patchiness and clustering, or environmental context.Shallow-water tropical reefs harbor the highest biodiversity of marine ecosystems on the planet20. However, the emergence of new technologies has enabled the development of a suite of alternative approaches to benthic monitoring21. The flexibility of remote sensing has led to a wealth of alternative methods using a variety of instruments including satellites14, aircrafts22, drones23, submersibles24, and handheld sensors25. Some approaches included the use of active remote sensing via radar26 or LiDAR27, while others employed passive remote sensing using RGB or multispectral imagery29, and hyperspectral imaging35. These studies generated massive spatial and temporal datasets36 that required novel analysis methods. Machine learning is one such method that has been used to effectively analyze these large datasets37. Machine learning has been employed in multiple ecological studies and has produced positive results for species identification, predictive modelling, biomonitoring, and habitat mapping44. It has also allowed researchers to process hyperspectral data in reef monitoring studies45.Globally, in-situ reef surveys are estimated to have only been carried out in approximately 0.01% to 0.1% of the world’s coral reef regions, and much of the data comprise observations at a resolution of one to ten meters25. This initial study served as a proof-of-concept for the use of this methodology. Though conducted on biodiverse reefs in Papua New Guinea, the habitat map was generated for a single transect that primarily consisted of scleractinian corals and a few other functional groups25. Subsequent studies have produced promising results suggesting that this methodology could serve as a viable method for fast and reliable reef monitoring and mapping47. However, many of these surveys were conducted on Caribbean reefs with much lower taxonomic diversity compared to the biodiverse reefs in the tropical Pacific.A diver-operated hyperspectral imager, the HyperDiver, was developed by the Max Planck Institute  with the aim of leveraging hyperspectral imaging and machine learning to automatically map benthic communities48, Guam is home to more than 5000 documented marine species52, making it one of the most species-rich ecosystems of all US jurisdictions. Much like other reef systems globally, the health of Guam’s reefs have seen a steady decline since the 1960s53. However, this decline has accelerated since 2013, resulting in an estimated 34–37% decline in total coral cover56 and substantial changes in reef composition57. Surveys were conducted on eight reefs in Guam with the aim of evaluating the utility of a diver-operated hyperspectral imager and machine learning as a proxy for benthic reef monitoring. This was done by comparing the hyperspectral results to traditional photoquadrat surveys, focusing primarily on accuracy across two levels of benthic category identification. The advantages and considerations of both survey methodologies for reef mapping and monitoring were evaluated and discussed.To increase the scope and utility of underwater hyperspectral imaging, PlanBlue GmbH  started to develop the DiveRay as of 2017. The DiveRay was used to conduct reef surveys in Guam , the largest and southernmost island of the Mariana Archipelago. Positioned just outside the Coral TriangleBenthic surveys were conducted in October 2019 at eight fringing reefs (8–12 m in depth) along the west coast of Guam  organisms to the highest taxonomic resolution possible  and (2) abiotic reef substrates . Photos of 0.25 m59. The same categories were also aggregated into 52 groups consisting primarily of genera, species, species complexes, and abiotic reef substrates. This was done to group related organisms that are difficult to distinguish from one another without in-depth morphological or genetic analysis , taxa that are difficult to identify in photoquadrat images , or taxa for which many species are undescribed . These groups, considered “Level 3” categories by the GCRMN, were used to compare the reef communities at a high taxonomic resolution59.The 128 categories were aggregated into 10 taxonomic or functional groups: Scleractinia, Corallinophycidae, Peyssonneliales, other red macroalgae, green macroalgae, brown macroalgae, Porifera, octocorals/hydrozoans, other benthic invertebrates, and turf algae/cyanobacteria/bare substrate. These groups were used to compare the two survey methods using broad benthic categories that could be used in rapid macroscale reef monitoring or ecological assessments, comparable to either “Level 1” or “Level 2” community data as outlined by the Global Coral Reef Monitoring Network (GCRMN)2 of reef at each survey site. The resulting scans are large (upwards of 4–6 gigabytes per scan), as each recorded pixel contains complete spectral information about the visible and near-infrared part of the spectrum.A beta version of an underwater hyperspectral imager, the DiveRay, was used to scan transects at each of the eight study sites . Spectra were then normalized to account for incident light using the recorded signal of a 10 × 10 cm matte grey reference plate that was placed in the survey area of each transect. Assuming incident light does not change significantly during the survey, this normalization of spectra allows for a comparison between transects at a site. To account for depth, surveys followed the topography of the reef, where the DiveRay was oriented approximately one meter above, and parallel to, the varying reef terrain at any given time. This was done primarily to reduce the inaccuracies and errors resulting from large variations in reef height because the quality and range of spectral data are influenced by the optical environment and the height from which hyperspectral images are capturedAcropora species). However, these categories were consistent between the photoquadrat and hyperspectral survey methods, as they were used to compare the predicted benthic communities across two distinct degrees of taxonomic resolution , which also served as the platform to annotate hyperspectral images and discern benthic categories. 91 benthic categories were recognized in the hyperspectral analysis, as some benthic categories were visually indistinguishable from one another in the hyperspectral scans  to the highest threshold of 99% . The benthic cover estimates derived from the hyperspectral scans were then compared to those of the photoquadrat surveys to evaluate using the DiveRay as an alternative to photoquadrat surveys.64. By ranking dissimilarities, ANOSIM compliments non-metric multidimensional scaling (nMDS) approaches to evaluating community data64. For the communities identified using broad benthic groups, the significance of differences in the estimated cover of Scleractinia between survey methods were tested using a one-way ANOVA and the Tukey’s honest significant difference (Tukey’s HSD) post hoc test. Differences in community assemblages between survey methods were visualized using nMDS plots based on Bray–Curtis dissimilarity matrices and overlaid with hulls around each site. Broad group community differences were plotted without transformation in order to best compare survey methods. However, communities described using high resolution categories were typically dominated by few taxa and contained a high number of categories with (very) low cover. To reduce the large discrepancies in cover between groups, high resolution groups were visualized using square root transformed cover data.All statistical analyses were done using R version 4.1.3  and RStudio version 2022.02.1 + 461 . Analysis of similarities (ANOSIM) tests were performed using the ‘vegan’ package to test whether there were statistical differences between the communities estimated using the two surveys at each site. This was done by using a ranked dissimilarity matrix with defined groups (survey method) to compare the mean rank within groups (transects) to the mean rank between groupsPorites species, Porites rus/monticulosa, and Leptoria phrygia. Five sites were dominated by pavement/turf algae/cyanobacteria, which made up between 59 ± 9% (Tumon NFSAS) and 80 ± 6% (Orote FSAS) of the substrate cover on these reefs. These two categories accounted for over 78% of the total benthic cover across all sites, while the presence of other abundant organisms (≥ 5% average cover) varied between sites. One site, Orote NFSAS, possessed a high abundance of green macroalgae, which was mostly comprised of Halimeda spp. (20 ± 7% cover). Sponges were present in high abundance at Finger Reef FSAS (9 ± 3%), Gab Gab NFSAS (8 ± 2%), and Orote FSAS (5 ± 2%), but the species composition differed between these sites. The sponge communities present on Finger Reef FSAS and Gab Gab NFSAS were primarily comprised of Melophlus sarasinorum, Rhabdastrella globostellata, and Xestospongia carbonaria, while other Porifera species  occurred in high abundance at Orote FSAS. Lastly, members of the reef-building and cementing Corallinophycidae were present in relatively high abundance at Asan NFSAS (8 ± 2%), Tumon FSAS (6 ± 2%), and Tumon NFSAS (6 ± 2%). No other substrate categories occurred in abundances greater than 2.5% at the surveyed reefs.Scleractinian corals represented the highest benthic cover at three sites: Finger Reef FSAS (54 ± 5%), Gab Gab NFSAS (55 ± 5%), and Orote NFSAS (42 ± 6%) but covered much less reef at the other five sites, ranging from 8 ± 4% (Tumon FSAS) to 25 ± 11% (Tumon NFSAS). While coral communities were species rich (over 50 identified taxa separated into 26 high resolution ID categories), they were largely dominated by a combination of massive Prior to running further analyses, the optimal certainty threshold needed to be determined. This was done by examining the ANOSIM results of the comparison between the photoquadrat-derived community metrics and those predicted from the hyperspectral scans for a series of certainty value thresholds  were retained. Lower certainty thresholds retained less abundant taxa, which was apparent when comparing the community composition between sites , many of the other benthic organisms present at these sites occurred in small heterogenous patches that were often situated between columns or plates of P. rus. This made identifying the small heterogenous patches of turf algae/cyanobacteria and other benthic organisms on the hyperspectral scans difficult at these sites. In addition, contrary to the other sites, these reefs also occur on a relatively steep slope, adding a significant vertical component to the reef structure. The reef pictured in Fig. The community estimates showed significant differences across all analyses for two survey sites: Finger Reef FSAS and Gab Gab NFSAS. This emphasizes the need to consider initial site selection carefully when using the DiveRay system. These two sites, located within the sheltered Apra Harbor Fig. e, are unP. rus and small heterogenous patches of turf algae/cyanobacteria at Orote NFSAS likely contributed to the difference in community estimates between the two methods. Orote FSAS was characterized by low coral and high turf algae/cyanobacteria cover (Supplementary Table Cliona and Dysidea spp.) were small and difficult to identify on hyperspectral scans, which likely contributed to their underestimation in the model-predicted communities.Broad group community estimates differed significantly between survey methods for two other sites: Orote FSAS and Orote NFSAS. The reef community at Orote NFSAS was unique due to the high abundance of green macroalgae  compared to the 47 identified in the Curaçao study47. The significantly higher marine biodiversity in Guam suggests that a much more robust annotation library might be needed, which could be accomplished through increased sampling and annotation efforts.DiveRay data from 2019 did not sufficiently characterize benthic composition using high-resolution categories. The communities of only two sites (Asan NFSAS and Tumon NFSAS) were found to not differ significantly between both survey methods. The significant differences observed at the other four sites cannot solely be attributed to the machine learning model. A model can only be as effective as the input data from the annotation library. Limitations to delineate multiple ROIs in the hyperspectral scans for all high resolution categories might have been insufficient to account for the high biodiversity of Guam’s reefs. A similar study from Curaçao examined a subset of 31 hyperspectral transects from a dataset containing 147 hyperspectral transects from 8 nearshore reefsPorites spp., Porites rus/monticulosa, and pavement/turf algae/cyanobacteria) contained more pixels  than the other 49 ID categories combined . This imbalance in ROIs was also observed in the hyperspectral dataset of the Curaçao study, where the authors concluded that this could be avoided by prioritizing biodiversity coverage rather than spatial coverage across all transects46. The identification of ROIs used in our study was directed toward prioritizing both spatial and biodiversity coverage, suggesting that this imbalance could be better explained by other factors such as rarity of occurrence , small size , or difficulty of identification on hyperspectral scans despite a pixel resolution of less than one centimeter . Consequently, the model-predicted identification of less abundant and smaller benthic categories was inconsistent and, somewhat paradoxically, tended to overpredict the occurrence of rarer categories. This could be due there not being enough pixels (ROIs) available to sufficiently train the model to distinguish these taxa from others, causing the model to confuse them with other more dominant categories. The identification accuracy of these categories is expected to improve with increased annotation effort, more surveying, or targeted scanning of less common taxa. Predictions based on hyperspectral data performed better when just comparing scleractinian coral communities using the high-resolution categories, where the communities at four sites  were found not to differ significantly. This is similar to results reported by Schürholz and Chennu47 and indicate that this methodology is effective in differentiating corals at a high taxonomic resolution. The coral communities at these four sites were dominated by massive Porites species, Porites rus/monticulosa, and Leptoria phrygia. These three categories are abundant on Guam’s reefs, easily identifiable, and were well-represented in the annotation library. On the other hand, the coral communities of the other two sites (Asan FSAS and Orote FSAS) were more species rich, with 18 and 20 of the 26 scleractinian categories being found at each site respectively, while no more than 16 coral categories were recorded at the other four sites. It is also worth noting that the area of reef surveyed using the DiveRay was more than quadruple the area surveyed using photoquadrats, which could account for some differences in cover estimates.While there were 91 benthic categories identified in the annotation libraries, the proportion of ROI identifications were unintentionally skewed toward the most dominant taxa. The annotation imbalance was so pronounced that the three most recorded ID categories . Moreover, the photoquadrat methodology has been shown to underestimate the percentage cover of substrate categories73, which could also explain some of the discrepancies between the traditional survey method and the DiveRay surveys. Lastly, the surveys conducted for this study used a beta version of the DiveRay, which has undergone continuous hardware and software development since 2019. Studies have reported improved results as sensor technology improves47, and updates to the DiveRay and the HyperSuite software would likely improve classification success.Photoquadrat and hyperspectral surveys require similar field expertise. For non-repetitive small-scale survey campaigns, photoquadrat surveys are favored as they can provide a comprehensive baseline and the equipment is readily available. The same is true for collecting high taxonomic resolution benthic community data. These photoquadrat surveys have long been used for marine benthic studies75. Employing the traditional photoquadrat method for such expansive surveys would demand considerable time, effort, logistic, and financial resources. Though underwater hyperspectral surveys require less time in the field than photoquadrat surveys, building annotation libraries demands for an initial investment of time and effort . However, once established, the availability of annotation libraries is beneficial for longer term monitoring efforts as additional imagery can be analyzed within minutes to hours through the automated data processing pipeline. This is considerably faster than the 1–3 weeks needed to analyze the photoquadrats of one site. In addition, once an annotation library is built, the analysis of hyperspectral scans can be executed immediately after data collection. So, while the initial expenditure to conduct hyperspectral surveys seems higher than those of photoquadrat surveys, cost savings are to be expected for repetitive monitoring efforts that cover large areas25.Hyperspectral surveys of benthic reef communities could improve the speed of data collection and processing. It is often necessary to survey vast reef areas to properly assess biodiversity and spatial metrics such as patchiness and clustering47. As such, a more robust library of ROIs that better account for rare taxa and the high biodiversity in Guam could significantly improve model prediction accuracy, and thus drastically improve overall time-to-data in subsequent surveys. Scans can also be re-analyzed as annotation libraries are updated over time, which could further increase the prediction accuracy and temporal comparison with previously conducted surveys. Further, libraries built using survey data from a broad range of habitats over a large geographical area could standardize and reduce observer bias in monitoring efforts with a regional or even global focus.Results from hyperspectral surveys in Curaçao suggest that annotation libraries can be exchanged between survey efforts of similar reef environments47. The results of this study suggest the same, as benthic cover estimates based on hyperspectral surveys were most reliable when using broad benthic categories such as those often used by reef managers or in large-scale rapid ecological assessments76. The average community similarity ([1 − Bray–Curtis dissimilarity] × 100) between the different broad category estimates was high for all sites  were not addressed in this study, hyperspectral imaging and machine learning algorithms can be used to track and quantify temporal changes in reef health after storms (or other damaging disturbance events), shifts in biodiversity , or coral bleaching86. Given the often time-sensitive nature of coral reef surveys, the rapid data turnover enabled by underwater hyperspectral imaging can make this a valuable tool in future monitoring efforts.The use of machine learning for automated annotation of benthic surveys has been shown to be effective when discerning broad or well-defined benthic categoriestes Fig.  suggests35 . However, this capability is likely to improve as hardware technology advances (particularly sensor resolution and accuracy) and more extensive annotation libraries become available47. Hyperspectral imaging was already effective when predicting community composition using broad categories, particularly in estimating scleractinian coral cover. A strength of underwater hyperspectral imaging is its ability to generate detailed habitat maps at higher resolutions than afforded by above water techniques. So, when mindful of the type of habitat investigated and pending the availability of well-documented annotation libraries, underwater hyperspectral imagery is a promising tool for rapid, frequent, and accurate monitoring of reef communities.As is the case with other survey techniquesSupplementary Figure S1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.