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