Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome

Abstract

Climate change is one of the most significant challenges facing the sustainability of global poultry production. Stress resulting from extreme temperature swings, including cold snaps, is a major concern for food production birds. Despite being well-documented in mammals, the effect of environmental stress on enteric neurophysiology and concomitant impact on host-microbiome interactions remains poorly understood in birds. As early life stressors may imprint long-term adaptive changes in the host, the present study sought to determine whether cold temperature stress, a prominent form of early life stress in chickens, elicits changes in enteric stress-related neurochemical concentrations that coincide with compositional and functional changes in the microbiome that persist into the later life of the bird. Chicks were, or were not, subjected to cold ambient temperature stress during the first week post-hatch and then remained at normal temperature for the remainder of the study. 16S rRNA gene and shallow shotgun metagenomic analyses demonstrated taxonomic and functional divergence between the cecal microbiomes of control and cold stressed chickens that persisted for weeks following cessation of the stressor. Enteric concentrations of serotonin, norepinephrine, and other monoamine neurochemicals were elevated (P < 0.05) in both cecal tissue and luminal content of cold stressed chickens. Significant (P < 0.05) associations were identified between cecal neurochemical concentrations and microbial taxa, suggesting host enteric neurochemical responses to environmental stress may shape the cecal microbiome. These findings demonstrate for the first time that early life exposure to environmental temperature stress can change the developmental trajectory of both the chicken cecal microbiome and host neuroendocrine enteric physiology. As many neurochemicals serve as interkingdom signaling molecules, the relationships identified here could be exploited to control the impact of climate change-driven stress on avian enteric host-microbe interactions.

Keywords: avian; gut; microbiome; neurochemical; stress.

Figure 1

Microbial diversity in control and cold stress group chickens across time-points. Chickens were cold stressed at 4, 5, and 6 d of age as described in Methods: (A) Principal coordinate analysis plot based on Aitchison distances exhibits significant separation (**P < 0.01) between the gut microbiomes of control and cold stress chickens at 7, 9 and 21 d. (B) Chao diversity significantly decreases (*P < 0.05) in cold stress chickens at 7 and 9 d before recovering at 21 d. (C) Shannon and (D) Simpson diversity do not significantly differ between groups at any time-point.

Figure 2

Differentially abundant taxa between control and cold stress group chickens at (A) 7 d, (B) 9 d and (C) 21 d. Chickens were cold stressed at 4, 5, and 6 d of age as described in Methods. Effect size is defined as the between group differences divided by the within group differences, an effect size cut-off of absolute 1 is suggested for reproducible results.

Figure 3

Associations between cecal bacterial relative abundances and neurochemical or short chain fatty acid concentrations in the ceca. (A) CCA of microbiota composition regressed on neurochemical concentrations in cecal tissue. The canonical variates explain 26.5% and 12.31% of the total explainable variance in the first and second axis respectively. (B) CCA of microbiota composition regressed on neurochemical concentrations within cecal content. The canonical variates explain 23.75% and 10.84% of the total explainable variance in the first and second axis respectively. (C) CCA of microbiota composition regressed on short chain fatty acid concentrations within cecal content. The canonical variates explain 25.24% and 12.13% of the total explainable variance in the first and second axis respectively. Filled circles represent samples while grey crosses represent bacterial taxa (genus level) with the top 5% of taxa which best fit the axes being indicated by name. Neurochemical or SCFA vectors point to the direction of taxa with which they exhibit the strongest association with while their magnitude indicates the strength of the variable in explaining the bacterial dispersion observed (only significant vectors, P < 0.05, are displayed). 5-HT: 5-hydroxytryptamine.

Figure 4

Metagenomic diversity in control and cold stressed chickens at 21 d. Chickens were cold stressed at 4, 5, and 6 d of age as described in Methods. (A) Principal coordinate analysis plot based on Aitchison distances exhibits significant separation between gene family abundances of control and cold stress chickens at 21 d. (B) Differentially abundant gene families between control and cold stress chickens at 21 d. Effect size is defined as the between group differences divided by the within group differences, an effect size cut-off of absolute 1 is suggested for reproducible results. (C) Functional enrichment analysis of pathways based on differentially abundant gene families.

Figure 5

Heat trees of differentially abundant genes. Heat trees displaying differentially abundant genes encoding for enzymes between cold stress and control groups across the BRITE functional hierarchy for enzymes.

Figure 6

Serotonergic gene expression is altered in the chicken ceca following cold stress. Chickens were cold stressed at 4, 5, and 6 d of age as described in Methods. mRNA fold change of (A) 5-Hydroxytryptamine Receptor 7 (HTR7), (B) 5-Hydroxytryptamine Receptor 2C (HTR2C), and (C) Sodium dependent serotonin transporter (SLC6A4) were determined in full thickness chicken ceca tissue (n = 8-10 chickens per group) using RT-qPCR as described in Methods. Outliers were assessed using ROUT (q = 1%), and data analyzed using 2-way ANOVA with outliers removed followed by Sidak post-hoc test. Data are presented as mean ± SEM. Statistical significance was set at P < 0.05; * = P < 0.05; *** = P < 0.001.

Figure 7

Short chain fatty acids concentrations are not significantly affected in chicken cecal content following cold stress. Chickens were cold stressed at 4, 5, and 6 d of age as described in Methods. Short chain fatty acids (SCFA) (A) Acetic, (B) Propionic, and (C) Butyric acids were determined in chicken ceca content (n=8-10 chickens per group) using GC-MS as described in Methods. SCFA concentrations are reported in mg of SCFA per g of cecal content. Data are presented as mean ± SEM. Data was analyzed using 2-way ANOVA followed by Sidak post-hoc test. Statistical significance was set at P < 0.05. * = P < 0.05.