1. Introduction
The complex community of rumen microbiota, including bacteria, protozoa, fungi, and archaea, contributes substantially to the powerful ability of ruminants in digesting plant materials into exploitable nutrients [
1]. Rumen bacteria have been regarded as the most diverse microbial community and have been studied extensively over the past decades [
2,
3,
4], especially when next-generation sequencing technologies coupled with “omics” approaches became available and popular. These studies mainly focused on the interactions of diet–microbe, host–microbe, and environment–microbe [
1,
5,
6]. However, ruminal bacteria are generally cooperative in the microbial fermentation of carbohydrates, with bacterial species contributing uniquely to maintaining normal and ecological balance [
1,
7]. Therefore, a deep understanding of the bacteria–bacteria network is urgently required to generate potential strategies for improving the productivity of ruminants.
Quorum sensing (QS) is a cell-to-cell communication mediated by signal molecules to regulate their phenotypes, such as biofilm formation, motility, and virulence [
8,
9]. The signal molecules include acyl-homoserine lactones (AHLs), autoinducer peptides (AIPs), and autoinducer-2 (AI-2), which are primarily secreted by gram-negative bacteria, gram-positive bacteria, and both gram-positive and gram-negative bacteria, respectively [
10]. Moreover, the signal molecule AI-2 was proposed to be the universal language for interspecies communication [
9]. Once the concentration of signal molecules secreted by bacteria reaches its threshold, other bacteria will sense the change and facilitate cooperative defense against an unfavorable environment via altering gene expression and phenotype [
10]. Therefore, the QS system is actually a self-defense strategy to enhance bacterial adaptability and survival to the various habitat. Numerous studies have reported on bacteria-to-bacteria communication from a single species (e.g.,
Escherichia coli,
Salmonella typhimurium, and
Vibrio harveyi) of non-ruminant origin [
11,
12,
13]; however, these communications in the rumen microbiome are limited. Since Erickson et al. [
14] detected AHLs in ruminal contents, many reports have revealed the existence of AHLs in rumen bacteria [
12,
15,
16]. Data from expression levels in rumen metatranscriptome datasets showed that LuxS/AI-2 QS was the most abundant in the rumen, and the genus of
Prevotella expressed the highest LuxS synthase [
17]. Similar data from rumen microbial metagenomics also revealed that AI-2-mediated QS system may participate in biofilm formation and fiber degradation [
8]. However, Ran et al. [
10] detected the presence of the gene
luxS, but not the AI-2 signaling molecules, both in vivo and in vitro, probably due to the limitations of detection techniques or pre-adapted diets. Environmental changes, such as initial pH, carbon sources, and glucose concentration, were reported to stimulate AI-2 production and
luxS transcription [
18]. These results indicated that bacterial LuxS/AI-2 QS may be opportunistic in rumens of ruminants.
A modern ruminant production system generally involves several growth or finishing stages, and each stage requires a unique nutritional feeding strategy to meet its requirement for maintenance and production [
19]. A diet shift is the most popular manner in which to adjust a feeding strategy, and a rapid shift leads to a perturbed microbial community, reduced performance, and poor host immune response [
20,
21]. Many reports have revealed the strong associations between improved host health and production and stable ruminal microbiomes [
20,
22]. Therefore, less time to achieve the stability of microbiomes will improve host productivity, and the regulation of ruminal bacteria communications, such as QS, may be a potential strategy for shortening the adaptation time before stability.
This study attempted to uncover the possibility of a diet shift on the triggering of LuxS/AI-2 QS, and provide further insight into a new strategy for relieving stress upon a diet shift. We hypothesized that a diet shift would trigger the LuxS/AI-2 QS of rumen bacteria, and that cooperative behavior would be activated to fight against adversity due to the dietary shift.
4. Discussion
When the dietary concentrate to forage ratio shifted abruptly from 75:25 to 49:51, serum physicochemical characteristics changed accordingly. As a sensitive hormonal indicator of stress, the level of cortisol increases remarkably by activating the hypothalamic–pituitary–adrenocortical (HPA) axis when animals and humans suffer from stressors [
35]. A higher concentration of cortisol indicated that the diet shift caused stress successfully. It is widely known that, once stress occurs, the body initiates an immune response to withstand adversity stress. As the main immunoglobulin class among five distinct immunoglobulins, Ig G plays a decisive role in maintaining normal life [
36]. This study found that the concentration of Ig G increased after the diet shift, indicating that a defensive response was activated to fight against nutritional stress. Glucose concentration is a critical indicator for energy metabolism, and its value increased with greater intake of digestible energy or more concentrate [
37,
38]. The serum glucose before the diet shift showed higher levels, which could be attributed to the higher density of energy in Pre when compared with the energy density in Post. Another possible explanation for the lower concentration of glucose in Post would be the fact that glucose could be synthesized from rumen propionate via hepatic gluconeogenesis [
39], because numerically higher molar concentration and higher molar proportion of propionate were observed in Pre.
As the primary QS in rumen bacteria revealed by both metagenomics and metatranscriptome dataset analysis, LuxS/AI-2 QS plays a vital role in the interspecies communication of rumen bacteria [
8,
17]. AI-2 is the core and featured signaling molecule in LuxS/AI-2 QS system, and its concentration could be perceived by bacteria to coordinate collective behavior, such as cell density and biofilm formation [
8,
9]. In the current study, the concentration of AI-2 increased after the diet shift; meanwhile, increased microbial density and more biofilm formation were observed, indicating that bacteria had exhibited collective behaviors when the signaling molecule concentration reached its threshold to defend against the abrupt diet shift. These phenotype variations were regulated by their corresponding gene expressions, namely
luxS for AI-2 synthesis and
ftsH for biofilm formation. The gene
luxS encodes autoinducer 2 synthase (LuxS), which is responsible for the synthesis of AI-2 [
8,
17]. The sequences of the
luxS gene were found to be abundant in
Prevotella,
Butyrivibrio,
Ruminococcus,
Pseudobutyrivibrio, and
Eubacterium [
8,
17]. In this study, a decline in the trend of
luxS gene expression was observed in
Prevotella after the diet shift even with a higher AI-2 concentration, probably due to the uncharacterized relationships between diverse
luxS-containing bacteria and AI-2 secretion [
26]. Another possible explanation for the inconsistent results is that they may be attributed to the lower serum glucose in Post, because a previous study found that the level of
luxS-mRNA decreased when deficient glucose was supplied to
Streptococcus bovis [
40]. Biofilm is a complex three-dimensional structure of bacterial aggregates, mainly composed of polysaccharides, proteins, nucleic acids, and lipids [
41,
42]. Its existence proved more resistant to stress when compared with its planktonic counterpart, and mixed-species biofilm exhibited more resistance to environmental stress than single-species biofilm [
42,
43]. The gene
ftsH was proposed to be involved in biofilm formation to protect against stress, and its mutant strain showed a reduced biofilm formation capacity [
44]. Higher gene expression of
ftsH, as well as more biofilm formation, was observed in Post when compared with Pre, indicating that the diet shift stimulated more biofilm formation via up-regulating the gene expression of
ftsH. However, extracellular polysaccharide and protein concentrations did not show a significant difference in rumen fluid between Pre and Post, which is probably due to the distinct development, structure, and function of biofilm between mixed species and single species [
43]. This study revealed a positive association between AI-2 concentration and biofilm formation, which is in line with previous reports [
26,
45]. Moreover, the current results also revealed that the
ftsH gene was more sensitive to the diet shift than to the
luxS gene, indicating that biofilm formation may be prioritized over AI-2 synthesis when undergoing a diet shift. These results suggest that the diet shift triggered rumen bacterial LuxS/AI-2 QS, and ruminal bacteria cooperated together to defend against the diet shift by improving microbial density, AI-2 synthesis, and biofilm formation.
The diet shift altered ruminal fermentation patterns and efficiency, but did not affect the absolute concentrations of VFA and individual VFA, except for valerate. Generally, VFA is considered as the primary energy utilization form of ruminants, and a higher NGR indicates a lower FE of dietary energy from carbohydrates to VFA [
31]. In this study, higher NGR and lower FE were observed after the diet shift, indicating that the diet shift impeded rumen fermentation. Liu et al. [
46] reported that an increase in dietary concentrate feeding yields a higher concentration of valerate; similar results were observed for the valerate concentration of Post regarding both concentration and proportion, probably due to the reduction in dietary concentrates in Post. It is easy to explain why Post showed a decreased proportion of propionate and a higher acetate to propionate ratio because of the well-established theory that structural carbohydrates produce more acetate and less propionate when compared with non-structural carbohydrates [
47]. A higher proportion of butyrate was found in Post, which is inconsistent with previous reports that a high-density diet produced more butyrate [
32]; this is probably explained by the fact that the VFA of Pre decreased faster due to its high-grain composition [
48]. Branched-chain volatile fatty acids, including isobuyrate, valerate, and isovalerate, are the main products of crude protein degradation and are commonly used to monitor protein fermentation [
49]. Therefore, it is reasonable to expect the increments in the trend of concentration and proportion of BCVFA in Pre when compared with Post due to differences in dietary crude protein (16.14% vs. 14.12%).
The diet shift altered the rumen bacterial richness, which was indicated by Chao 1 and observed species. A previous study revealed that a high-density diet decreased ruminal bacteria richness and evenness [
32], and the current study found similar results, since the dietary energy and protein in Pre were higher than in Post. Zhao et al. [
50] reported higher Actinobacteriota abundance when the concentration of oxygen decreased, probably explaining the trending decline of this phylum in Post because more roughage could carry more oxygen into the rumen [
51]. The genus
Prevotella is involved in fermenting starch and degrading protein [
52], which partly explains its higher abundance in Pre due to its higher dietary protein and energy density. However, as a genus belonging to the family Prevotellaceae,
Prevotellaceae UCG-001 showed opposing results to
Prevotella, indicating not all genera in the same family share similar responses to dietary change. Scott et al. [
53] reported that
Roseburia was identified as a producer of butyrate, and the current results revealed a higher abundance of this genus in Post, which corresponded well to the higher molar proportion of buyrate in Post.
Megasphaera is a predominant gram-negative commonly found in the rumens of cattle fed high-grain diets [
54], and its main function is to ferment lactate into acetate and propionate [
55]. Therefore, it is expected that the higher abundance of
Megasphaera was observed in Pre because of the high dietary concentrate to forage ratio. Similar to
Megasphaera,
Dialister possesses the capability of utilizing simple sugar [
56], probably providing evidence for the higher abundance of this genus in Pre due to the higher content of non-structure carbohydrates. The family Lachnospiraceae is commonly isolated from the rumens of cattle fed high-fiber diets and plays a vital role in facilitating forage degradation [
57], and Liu et al. [
58] found higher abundances of genera in this family when goats were fed an all-forage diet. These findings explain the current higher abundance of
Lachnospira in Post because sheep in this treatment received more fiber (
Table 1).