Abstract
Intestine regeneration in the sea cucumber Apostichopus japonicusis a fascinating biological event and a typical example for studying host-intestinal microbiota interactions. The intestinal microbiota may play important roles in developing intestine promotion, but the underlying mechanism remains unclear. Notably, altered microbiota abundance may be a key marker of the observed ecosystem. To understand the role of the developing intestinal microbiota in intestine regeneration via quantitative data, we developed a germ-free sea cucumber model and analyzed the intestinal microbial differentiation of faster and slower regenerating A. japonicus individuals during intestine regeneration. The results revealed that depletion of the intestinal microbiota resulted in elevated abundance of the potential key players Flavobacteriaceae and Rhodobacterales during intestine regeneration and thus promoted the intestine regeneration rate ofA. japonicus . These results first revealed a direct link between intestinal microbial quantity and microbiome features and the intestinal regrowth rate of A. japonicus . Metagenomic analysis revealed that the increased abundance of Flavobacteriaceae elevated the enrichment of genes associated with carbohydrate utilization, whereas the abundant Rhodobacteraceae -enriched genes were associated with polyhydroxybutyrate production. We identified microbiota abundance as a key driver of microbial community alterations, especially beneficial microbiota members, in the developing intestine of A. japonicus . This study provides new insights into the mechanism of host-microbiota interactions related to intestine development, and the understanding of molecular diversity to questions within intestinal ecology.
KEYWORDS: Apostichopus japonicus , intestine regeneration, intestinal microbiota, host-microbiota interactions, regeneration rate
1 | INTRODUCTION
The intestinal microbiota is increasingly recognized for its major roles in host health, growth, and mucosal and systemic immunity and for the important effect of resident intestinal bacteria promoting cell proliferation in the developing intestine. For instance, variation in the gut microbial structure can correlate with digestive enzyme activity and aid predigestion of the host nutrition (Holt, van der Giezen, Daniels, Stentiford, & Bass, 2019). Mite infection is significantly associated with altered microbial communities in Urocyon littoralis catalinae (DeCandia, Brenner, King, & vonHoldt). The intestinal microbiota can promote hematopoietic recovery after bone marrow transplantation (Staffas et al., 2018), affect the bone marrow niche (Y. Luo et al., 2015), and promote the generation of hematopoietic stem cells (Josefsdottir, Baldridge, Kadmon, & King, 2017). The gut microbiota influences skeletal muscle growth and function in mice (Lahiri et al., 2019) and significantly influences the larval growth rate of Melitaea cinxia (Ruokolainen, Ikonen, Makkonen, & Hanski, 2016). In addition, intestinal microbiota can promote cell proliferation in the developing vertebrate intestine (Cheesman, Neal, Mittge, Seredick, & Guillemin, 2011). Moreover, the gut microbiota can increase Paneth cell proliferation in the small intestine (Schoenborn et al., 2018). It is thus well established that functional interactions between the gut microbiota and the host are important for host physiology, growth, and sustained health.
Apostichopus japonicus is a temperate sea cucumber species, and has been exploited as an economically valuable fishery resource in many Asian countries, especially China. A. japonicus ingests organic matter, protozoa, microbes, algae and aquatic animal detritus, and thus plays an important role in benthic biogeochemical cycles (Choo, 2008; Yamazaki et al., 2016). In particular, sea cucumber possess a unique defense mechanism called evisceration. The organs, including the intestine, hemal system and respiratory trees can be eviscerated when they are subjected to natural or induced stimulation (Dolmatov & Ginanova, 2009; X. Li et al., 2017; L. Sun et al., 2017). The lost organs can concurrently regenerate within a few weeks (Shukalyuk & Dolmatov, 2001; L. Sun et al., 2011). Thus, it is an excellent model to study host-microbiota interactions in developing intestine. Actually, a lack of studies on factors affecting the intestine regeneration rate of A. japonicus is an obstacle to further success in this field. Extreme gaps of intestine regenerating rate among sea cucumbers are also a common problem in the study of animal organ regeneration. As a result, there is a right-skewed intestine length distribution among regrowth animals even when cultured in the same tank under identical conditions (e.g., temperature and animal density). This issue can be seen in Figure 1, in which the faster individuals are 1.83-3.03 times longer than the slower individuals. The cause of this regeneration gap is unknown.
In the past decade, research on the intestinal microbiota of the sea cucumber A. japonicus has rapidly accumulated and has been accompanied by increased interest in host-microbiota interactions as a means to modulate the animal’s health and growth. Previous studies have focused on intestinal bacterial community structures (Gao et al., 2014; Gao, Li, Tan, Yan, & Sun, 2014), the physiological characterization of culturable bacteria in the intestine of the sea cucumber (L. Wang, Li, Hu, Lai, & Shao, 2015; X. Zhang et al., 2013), and the effects of intestinal microbiota on A. japonicus growth and health (Sha et al., 2016; Yamazaki et al., 2016; Z. Zhang et al., 2018). They found that the intestinal microbiota might play an important role in sea cucumber growth. However, few studies have addressed the question of the development of the intestinal microbiome composition and function ofA. japonicus during intestine regeneration stages (Luo Wang et al., 2018; H. Zhang et al., 2019). Moreover, the effects of the intestinal microbiome and microbial quantity on intestine regeneration of A. japonicus are still unclear. Therefore, we are interested in the functional interactions between the intestinal microbiota and intestinal regrowth rate of A. japonicus . Understanding the regulatory mechanisms by how the intestinal microbiota might influence the process is an important objective in improving outcomes during intestine regrowth, and may help in the development of a theoretical basis for further understanding intestinal ecology and the role of the microbiome in the process of intestine development.
We observed a regeneration gap during intestine regeneration in sea cucumbers A. japonicus . To our knowledge, there have been no studies investigating the possible effects of the intestinal microbiota on intestine regeneration of A. japonicus . Our hypothesis is that the intestinal microbiota plays an important role in intestinal regrowth of A. japonicus during intestine regeneration. To explore any possible contributions of the gut microbiome to its host’s intestinal regrowth, individual taxon abundance and microbiome comparisons (both taxonomic and functional) of both the faster and slower regenerating sea cucumbers A. japonicus were performed. Deriving germ-free (GF) animals is a powerful experimental approach to investigating the function of intestinal microbiota. To further gain mechanistic insight into the interaction between the intestinal microbiota and the intestinal regrowth rate, we developed a GF sea cucumber model, quantified the intestinal bacterial 16S rRNA abundance by qPCR, and taxonomically analyzed the microbial diversity. The results will demonstrate the link between the microbiota quantity and microbial community variation and intestine development, which is of great theoretical and practical significance to the understanding of the dynamics of host-microbiota interactions, and to the cultural ecology and conservation of fishery resources.
2 | MATERIALS AND METHODS