Impact of outdoor nature-related activities on gut microbiota, fecal serotonin, and perceived stress in preschool children: the Play&Grow randomized controlled trial

Due to rapid urbanization, children today have fewer opportunities to interact with nature and this may result in a greater risk for developing stress and depression. Outdoor nature-related activities can enhance general well-being. However, the underlying mechanisms are not fully delineated. Here we recruited 54 preschool children to participate in a 10-week structured nature-related “Play&Grow” program. Following the intervention, children were assessed for connectedness to nature and perceived stress levels using validated questionnaires. Moreover, fecal serotonin level and gut microbiota profiles were measured by ELISA and 16S rDNA amplicon sequencing, respectively. Children were significantly more connected to nature after the intervention. Their gut microbiota altered, especially by modulating the abundance of Roseburia and the fecal-serotonin level. Moreover, we also observed a reduction in the overall perceived stress, particularly in the frequency of anger among these children. This study is the first to demonstrate the impact of nature-related activities on gut microbiota, fecal serotonin and psychosocial behaviour of preschool children. However, further mechanistic studies are needed to confirm the functional role of gut microbiota in the association between connectedness to nature and improved psychosocial behavior.


Results
In total, fifty-four participant families were randomly assigned to the intervention (IG) (n = 30) or control groups (CG) (n = 24). Forty-five participants completed the pre-and post-intervention questionnaires and 84% of them provided the fecal samples (n = 27 in the IG and n = 18 in the CG) (Fig. 1). The demographics of the two groups are listed in Supplementary Table 1. Comparisons on demographics between study groups and their respective  Fig. 1a). Specifically, the scores for one of the factors, "Responsibility towards Nature" (RN) significantly increased from pre-to postintervention (p = 0.003, 3.33-3.67) ( Supplementary Fig. 1b).

Fecal serotonin & improvement of psychosocial behaviour.
A stable level of fecal serotonin was observed in the IG while a decreasing trend was found in the CG (p = 0.07, 249.36-220.60 pg/mg of feces) (Fig. 2a). Overall score related to the perceived Stress Scale for Children (PSS-C) and specific scores of anger frequency and prosocial behaviour improved significantly post-intervention (p = 0.05, p = 0.01 and 0.04, respectively, Fig. 2b-d) compared with the control group. We further separated the participants into three groups according to the changes in their PSS-C scores between pre-and post-intervention. A total of 65% of participants in IG (n = 13) have decreased PSS-C scores compared with 22% in the CG group (n = 2, Fisher's Exact Test p = 0.01). Especially, three children with the highest anger score improved (from level 4 to level 2) following the intervention. In addition, three other children who had higher anger scores of level 3 also decreased to level 2. Following the intervention, the two children with the lowest prosocial behaviour scores improved (Fig. 2d). Fecal serotonin was found to have a negative correlation with the overall PSS-C score (Repeated measures correlation, r = −0.45, p value = 0.02, Fig. 2e). It was also found to have an increasing trend of association with sleep frequency (Jonckheere-Terpstra test: JT = 832, p value < 0.01), and have a decreasing trend with anger frequency (Jonckheere-Terpstra test: JT = 475.5, p value = 0.01) ( Fig. 2f-g). The relationship of fecal serotonin with the frequency of anger was independent to the age of the children (Supplementary Table 2).
Changes in the gut microbiome. An average of 49,577 quality-filtered reads (per sample) were generated (Supplementary Table 3) and a total of 14 phylum, 176 genus, and 255 species were detected in gut microbiomes of the participants. The most abundant phyla of gut microbiota were Firmicutes, Bacteroidetes, and Proteobacteria (Median abundance: 0.44, 0.27, and 0.11%) ( Supplementary Fig. 2). Although no notable difference Item score (Frequency) Serotonin (pg/mg feces) Item score (Frequency) Serotonin (pg/mg feces)   www.nature.com/scientificreports/ in the overall alpha diversity between two time points both in IG and CG groups, the participants from the IG with decreased PSS-C score exhibited a significantly higher gut microbiota richness. (Supplementary Fig. 3). The richness of Bacteroidetes and Proteobacteria phyla changed significantly in the IG after the intervention. The Chao1 richness of Bacteroidetes significantly decreased (p < 0.01), while that of Proteobacteria increased (p = 0.03). On the other hand, there were no significant changes in the diversity of Actinobacteria and Firmicutes (both Shannon and Simpson indices) in the IG compared with the increased diversity in the CG following the intervention (Fig. 3). No significant difference in microbial community structure was observed between the IG and the CG at baseline; however, a clear difference between pre-and post-measurements in both IG and CG groups was observed based on the Canonical Correspondence Analysis (CCA) results. Several factors including completion of the intervention, serotonin levels, and anger frequency, were identified to influence the gut microbiota of children in the IG but not in the CG (Fig. 4a, b). Changes in certain species were found post-intervention ( Supplementary  Fig. 4). Interestingly, an increased abundance of Roseburia was shown in the CG but not in the IG (Supplementary Fig. 4g). The abundance of an unknown species of Bacteroides, Parabacteroides distasonis, an unclassified www.nature.com/scientificreports/ Acidaminococcus, an unclassified Dialister, and an unclassified Bilophila decreased in the IG, while an unclassified species of Blautia increased in the IG.

Alteration in predicted microbiota functions.
A total of 273 microbiota functional categories (at KEGG level-3, www.kegg.jp/kegg/kegg1 .html) were predicted using PICRUSt. Under the functional category of "Metabolism", 125 metabolic functions were detected with the highest abundance in "Purine metabolism. " Among those metabolic functions, we observed more functional changes in the IG (p < 0.05), in which 22 pathways decreased and 17 pathways increased. Comparatively, only a single pathway decreased ( Supplementary  Fig. 5) in the control group. At KEGG level-2, a significant correlation was observed between the overall PSS-C score and various amino acid metabolisms covering Phosphonate and phosphinate, d-Glutamine and d-glutamate, and beta-Alanine. Furthermore, the overall PSS-C score was positively correlated with the metabolism of carbohydrates including C5-Branched dibasic acid, Propanoate, Glyoxylate and dicarboxylate, Pyruvate, and Glycolysis/Gluconeogenesis. Betalain and Indole alkaloid biosynthesis, which have been increased by intervention, was negatively correlated with the total PSS-C score as well as the other sub-scores of the PSS-C questionnaire (Fig. 4d). However, none of these altered pathways showed a significant correlation to the fecal serotonin level in general.

Discussion
To investigate the impact of Connectedness to Nature (CN), we conducted a randomized trial and subsequently studied its impact on changes in gut microbiota and serotonin as well as the psychosocial behaviour of children. In this study, we observed that early environmental education intervention significantly reduced the overall perceived stress, particularly anger frequency among preschool children, modulated the abundance of certain gut microbiota, and in contrast to the respective control condition, did not reduce gut serotonin levels. The "Play&Grow" programme was successful in modulating the microbiome within the IG after regular exposure to nature over the course of 2 months. Similarly, a recent study in an adult population demonstrated that dipping hands in soil daily for 2 weeks changed the skin and gut microbiomes of the study's participants, indicating a correlation between natural interactions and the diversity of the gut microbiota 32 . In particular, our study discovered that exposure to natural bacteria moderated the diversity of the Actinobacteria and Firmicutes phyla, decreased the alpha diversity of the Bacteroidetes phylum, and increased the alpha diversity of Proteobacteria. We also observed inter-individual variation in changing gut microbiota diversity due to the intervention, and it might be related to the variation of exposure intensity among individuals.
Velles-Colomer et al. has recently reported an important association between depression, lower quality of life, and Bacteroides in the large Flemish Gut Flora Project 33 . Our controlled intervention allows us to suggest that exposure to bacteria from natural environmental could be beneficial to behavioural outcomes; however, this intriguing possibility needs further investigation. According to the natural history of gut microbiome development in early-life, the variation in the abundance of Bacteroides species among individuals is large due to different exposure levels and other factors associated with early life (e.g. birth mode and breastfeeding) 34,35 . Thus, the impact of our intervention on these particular taxa might be hindered by various factors. Despite this, we observed less individual variation of Bacteroides taxa following the intervention, which implies a movement toward modulation of the gut microbiome could be related to increased exposure to the natural environment and connectedness to nature 34,36 . During the first years of life, exposure to the natural microbial experience is therefore an important moderator for normal developmental patterns of gut microbiota and behaviour.
The current study showed positive changes in the psychosocial scores of participants in the IG after the "Play&Grow" intervention. This is in agreement with the systematic review of studies that found a beneficial impact of exposure to the natural environment on negative emotions (i.e., anger and sadness) 37,38 . Moreover, living in close proximity to forests is known to have salutogenic effects on the amygdala 39 . Likewise, the "Play&Grow" programme significantly improved the overall perceived stress, particularly anger frequency and www.nature.com/scientificreports/ prosocial behaviour of participants, which suggests a link between these behaviours and the bacterial load encountered by the participants. This is a new area of research, lacking the human data (although preclinical studies are promising); comparison of our results with other studies on similar age groups is therefore difficult. Studies in germ-free (GF) animals showed that GF mice were significantly socially impaired, yet this deficit could be repaired following bacterial colonisation, suggesting that gut microbiota affects psychosocial behaviour 40 .
Identification of the bacteria that is involved in these new mechanisms may help to understand the nature of the psychosocial consequences of bacterial alterations early in life. Our intervention decreased the prevalence of Parabacteroides distasonis, which has been positively associated with exposure to social stressors in mice 41 . A lower abundance of Roseburia has been reported in schizophrenics, but the opposite trend was found in people with mood disorders 42,43 . In this study, we also found that our intervention somewhat maintained Roseburia, whose abundance increased over time in the CG. Our results suggest that exposure to bacteria in nature may control the growth of Roseburia, resulting in a stabilization of gut serotonin levels. This indicates that the changes to the gut microbiota following exposure to natural environments may influence behaviours of preschool children. The gut microbiota related serotonin (5HT) pathway has already been proposed in animal models; but it has not yet been well-studied in human subjects, especially in children.
The "Play&Grow" programme's components align with biophilia theory. Both structured and unstructured nature experiences (i.e., playing with leaves and soil) and educational messaging (i.e., "it is ok to get your hands dirty with soil") increased children's contact with the natural environment and associated health outcomes. It strengthened the idea that nature is a "playground" and provided an opportunity for highly-urbanized children to reconnect to nature and interact with the microbiome of the natural environment.
Our intervention may be seen as an extension of the "Hygiene Hypothesis" also known as the "Microbiota Hypothesis", which suggests that exposure to bacteria and other pathogens may actually be beneficial to the education and development of the immune system [44][45][46][47][48][49] . Studies have reported that reduced exposure to microbes in modern urban societies may increase vulnerability to neurodevelopmental disorders (including autism spectrum disorders (ASD) and schizophrenia) and stress-related psychiatric disorders (including anxiety and mood disorders) 46,50-53 . Our study also showed this association, indicating children with the highest anger frequency had the least inter-individual variation in gut microbial community and the highest levels of Roseburia species.
This study investigated a possible relationship between fecal serotonin, the gut microbiome, and psychosocial behaviours. While a trend towards decreased fecal serotonin level was observed only in the CG, serotonin levels stayed stable in the IG. Moreover, serotonin is negatively correlated with the diversity of Bacteroidetes, Firmicutes, and Proteobacteria, as well as with the abundance of Roseburia. A recent study identified Roseburia as potential serotonin producers; however, the degradation pathway of serotonin by the bacteria is still not yet fully elucidated 33 . Hence, our study indicates that the Play&Grow intervention in preschool children increased connectedness to nature, improved children's moods, especially with respect to their anger levels, as well as changed the gut microbiome (especially the abundance of Roseburia), and in contrast to the respective control group, did not decrease gut serotonin levels. But due to the limitation of database and software, we didn't find the significant difference related to serotonin metabolism, so the associations need to be further confirmed by more targeted studies.
Besides the changes observed in microbial taxa, we also identified that our intervention significantly altered the microbiota functions within participants of the IG when compared with those in the CG. Among the functional changes, the most noteworthy one was a rise in Indole alkaloid biosynthesis, which also has a negative correlation with the PSS-C score, has been previously reported as a potential drug for depression and anxiety 54 . Another remarkable functional change was the decreased abundance of metabolic functions related to the biosynthesis of glycosphingolipid. A previous study also reported an increase in glycosphingolipid biosynthesis in Chinese children with autism spectrum disorders 55 , indicating a potential disadvantage to an abundance of glycosphingolipid. In general, the functional role of glycosphingolipids in the central nervous system have been detected 56 , but mechanistic study is needed to explore their roles in the behavioural development of children.
This is a pioneer study that links a nature-related intervention with psychosocial outcomes and modulating gut-microbiota-associated serotonin levels, suggesting a potential pathway to highlight the benefit of connectedness to nature. While our data are encouraging, certain limitations should be mentioned. Although there is a diurnal fluctuation of serotonin levels in the blood, this is still unobserved in fecal samples. Our study did not take account of the fecal sample collection time in order to have more flexibility for participants. Moreover, the sample size and the duration of the intervention may have significantly influenced the results of our study. Due to the limitation of the 16S rRNA reference database, our study could not provide species-level resolution for some Only the significant associated factors on gut microbiota in spearman correlation were listed. Colour in each cell of the heatmap indicated spearman correlation of serotonin and species abundance. Only the p value of association less than 0.05 were chosen for display in this figure. (d) Associations between gut microbiota functions and stress items, and changes in microbiota functions by the intervention. The heatmap indicated the spearman correlation between metabolic functions of gut microbiota and children's stress items. +p value less than 0.1; *p value less than 0.05; **p value less than 0.01. *in the front of the column name indicated we used the reversed score rather than the original score.

Conclusion
In our early environmental educational intervention study, we demonstrated the impact of nature-related activities on gut microbiota, fecal serotonin, and in improving psychosocial behaviour of preschool children. The impact included the changes in the diversity of microbiota, modulation of Roseburia abundance, prevention of a downregulation of fecal serotonin levels, and the improvement of psychosocial behaviours of children.
Our findings suggest that the gut microbiota can be a target for further studies on behavioural modifications and mental health interventions in a wider perspective. Further mechanistic studies are needed to confirm the mechanistic contribution of gut microbiota into the association between connectedness to nature and improved psychosocial behaviour.  Intervention design. A two-arm, randomized controlled trial (RCT) with masked outcome assessment-"Play&Grow"-was developed as a family-oriented, early environmental education programme for families with preschool children at the University of Hong Kong 29,30 . Primarily, the "Play&Grow" programme aims to reconnect preschoolers to nature and induce changes in health behaviours and outcomes by having outdoor activities that promote exposure to nature. On previous studies and calculation principles and methods 57 , the sample size (α = 0.05, power of 0.8) needed to detect the effect of the "Play&Grow" intervention on other related outcomes was 100 families in each group of RCT (including an allowance for 20% attrition). However, the sample size of this proof-of-principle study was not determined by a formal power calculation given the exploratory nature of the study. The assumption was that at least 30 children in total would be recruited from two groups: intervention group (IG) or Control group (CG). They were measured twice: before and after the intervention and the samples analysed for gut microbiota.
Participants. Children, aged two to five years, were recruited to participate in this programme, together with their main caregivers, via online advertisement at the beginning of 2018. The exclusion criteria were children from non-local families, children who were on antibiotic therapy in the two months prior to the start of the programme, and children with chronic health conditions.

Procedures.
Consenting families (n = 54) were randomly assigned to the Intervention (IG)(n = 30) or Control Groups (CG)(n = 24) using a random computer-generated number from 0 to 1 before the start of the trial, which was in April-June 2018. Participants were assigned to the IG if their number was > 0.5, and to the CG if their number was < 0.5. During the randomization process, no stratification or blocking were performed. All participants completed the pre-and post-intervention questionnaires, and 84% provided fecal samples (n = 27 in the IG (3 lost to antibiotic usage during the study, or failure to complete the study) and n = 18 in the CG (6 lost to antibiotic usage during the study, a failure to complete the study, or were lost to follow up) (Fig. 1). The demographics of the two groups are listed in Supplementary Table 1. The study aims and hypothesis were not discussed with any participants; they were only informed that they would be participating in a family health promotion programme. All measurements were done pre-and post-study on both groups, and all related research assistants, the questionnaire typist, and study statisticians were not informed of the group allocations, measurements and outcomes.
Intervention group (IG). The intervention was held once per week for ten consecutive weeks, from June 2018, in public parks throughout the Hong Kong SAR. Topics and elements of the programme were developed prior to the start of the intervention and were discussed in detail 29 . Each session, led by a pair of research assistants, included a guided nature activity that promoted "hands-on" experiences with materials found in nature. These activities were specifically designed for IG participants to provide them an opportunity to come into contact with the microbiome of the natural environment. Some additional "homework", such as collecting nature subjects, making nature art, and growing plants, was given to the families in the IG to further increase their interaction with the outdoor environment and its bacteria. A package of healthy lifestyle recommendations published by the Hong Kong government did not have any special dietary change recommendations and was available to all caregivers in Hong Kong 58 .
Control group (CG). In order to eliminate potential confound factors, i.e. changes in gut microbial composition as a result of any dietary changes, both groups were reminded of the resource mentioned above 58 .
Harms. This study focused on exposing children to the nature environment and did not contain any clinical procedure/intervention. As a result, no considerable harm or unintended effect were noted during the trial.
Outcome measurement. All measurements were conducted prior to and after the 10-week intervention.
Detailed procedures for children's psychosocial measurement, assessment for connectedness to nature (CN), serotonin measurement, and gut microbiota analysis were indicated in the Supplemental material.

Statistical analysis.
With the recommended pipeline in QIIME, the relative abundance of OTUs was summarised at phylum, genus and species level. Microbial alpha diversity was calculated by Chao1, Shannon, and Simpson indices. The differences between two time-points within groups were identified by the Wilcoxon signed-rank test (R package stats), including the difference of CN factor scores, microbiota diversity based on OTU, and species level, microbiota alpha diversity for each of the phylum, serotonin, and stress levels. Jonckheere-Terpstra test was used to test the trend (R package clinfun). The association between microbiota diversity, species, and stress levels with serotonin levels was measured by Spearman's rank correlation and logistic regression (R package stats) by category the continue number by the median. The association between PSS-C score Scientific Reports | (2020) 10:21993 | https://doi.org/10.1038/s41598-020-78642-2 www.nature.com/scientificreports/ with serotonin levels was measured by repeated measures correlation coefficient (R package rmcorr). CCA was performed based on species profile of the IG and the CG, separately (R package vegan).

Data availability
The sequence data under this study are publicly available in the European Nucleotide Archive under accession number PRJEB34058. Dr. Tun had full access to all subjects' meta-data and associated microbiota data in the study and takes responsibility of correspondence for data requests related to this study.
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