Abstract The constant increase in temperatures under global warming has led to a prolonged aestivation period for Apostichopus japonicus, resulting in considerable losses in production and economic benefits. However, the specific mechanism of aestivation has not been fully elucidated. In this study, we first tried to illustrate the biological mechanisms of aestivation from the perspective of the gut microbiota and metabolites. Significant differences were found in the gut microbiota of aestivating adult A. japonicus (AAJSD group) compared with nonaestivating adult A. japonicus (AAJRT group) and young A. japonicus (YAJRT and YAJSD groups) based on 16S rRNA gene high-throughput sequencing analysis. The abundances of Desulfobacterota, Myxococcota, Bdellovibrionota, and Firmicutes (4 phyla) in the AAJSD group significantly increased. Moreover, the levels of Pseudoalteromonas, Fusibacter, Labilibacter, Litorilituus, Flammeovirga, Polaribacter, Ferrimonas, PB19, and Blfdi19 genera were significantly higher in the AAJSD group than in the other three groups. Further analysis of the LDA effect size showed that species with significant variation in abundance in the AAJSD group, including the phylum Firmicutes and the genera Litorilituus, Fusibacter, and Abilibacter, might be important biomarkers for aestivating adult A. japonicus. In addition, the results of metabolomics analysis showed that there were three distinct metabolic pathways, namely biosynthesis of secondary metabolites, tryptophan metabolism, and sesquiterpenoid and triterpenoid biosynthesis in the AAJSD group compared with the other three groups. Notably, 5-hydroxytryptophan was significantly upregulated in the AAJSD group in the tryptophan metabolism pathway. Moreover, the genera Labilibacter, Litorilituus, Ferrimonas, Flammeovirga, Blfdi19, Fusibacter, Pseudoalteromonas, and PB19 with high abundance in the gut of aestivating adult A. japonicus were positively correlated with the metabolite 5-HTP. These findings suggest that there may be potential biological associations among the gut microbiota, metabolites, and aestivation in A. japonicus. This work may provide a new perspective for further understanding the aestivation mechanism of A. japonicus. Keywords: Apostichopus japonicus, aestivation, gut microbiota, metabolites, 5-hydroxytryptophan 1. Introduction Apostichopus japonicus (A. japonicus) has become one of the core species of sea cucumber in China and around the world [[34]1]. With the increasing market demand for A. japonicus, the scale and breeding density of A. japonicus have constantly increased in China. Under the current global climate change, the sustainable development of the A. japonicus industry has been seriously affected. High-temperature stimulation leads to abnormal conditions, such as a decline in the growth rate, metabolic disorder, and prolongation of the aestivation period of A. japonicus [[35]1,[36]2]. During the aestivation period, A. japonicus stops feeding, its gut shrinks and degenerates, and its body weight significantly decreases, which prolongs the breeding cycle and greatly increases the breeding cost. Therefore, elucidating the aestivation mechanism of A. japonicus is of great significance for the breeding of superior varieties of A. japonicus and the sustainable development of A. japonicus aquaculture. Research regarding the aestivation mechanism of A. japonicus is increasing. Researchers have studied the internal regulatory mechanism of aestivation not only from the perspectives of not only behavior, physiological and metabolic characteristics, and critical temperature but also from differentially expressed genes and proteins, miRNAs, genome methylation, and protein modification [[37]3,[38]4,[39]5,[40]6,[41]7,[42]8,[43]9]. Among these studies, those including whole-genome sequencing and transcriptomic analysis of A. japonicus have provided important reference information for further revealing the aestivation mechanism of A. japonicus and promoting research progress [[44]10]. Nonetheless, the aestivation of A. japonicus is considered to be a very complex biological process, and it is necessary to explore the internal regulatory mechanism of aestivation from different perspectives. However, relevant studies on mammals may provide us with a reference, which found that gut microbes and metabolites were closely related to sleep or dormancy. The gut microbiota and metabolites can directly or indirectly affect sleep through the “microbiota-gut-brain” axis [[45]11]. There is significant evidence that the host gut microbiota not only affects digestive, metabolic, and immune functions but also regulates sleep and mental state through the “microbiota-gut-brain” axis [[46]11]. Available evidence suggests that the gut microbiota interacts with circadian rhythm genes, and the characteristics of gut microbiota and metabolism are associated with host sleep and circadian rhythms [[47]12]. Previous studies have found that the gut microbiota can affect sleep by regulating various factors, such as serotonin (a precursor of melatonin) synthesis or immune pathways [[48]13]. Disruption of the gut microbiota leads to the elimination of serotonin from the gut and affects serotonin levels in the brain, which in turn affects the sleep/wake cycle [[49]14]. However, during the aestivation period, the gut of A. japonicus degenerates and atrophies, and the metabolic rate decreases. This may result in the role of the gut and gut microbiota in aestivation being overlooked. Thus, the following question arises: is there a biological relationship between the gut microbiota, metabolites, and aestivation in A. japonicus? To answer this question, we analyzed the gut microbiota of aestivating adults, nonaestivating adults, and young A. japonicus growing under different temperature conditions. At the same time, a comparative analysis of gut microbiota host cometabolites was carried out. This work may provide a new perspective for further understanding the aestivation mechanism of A. japonicus. 2. Materials and Methods 2.1. Experimental Animals, Grouping, and Feeding Conditions The experimental A. japonicus were purchased from Weihai Xigang Aquatic Products Co., Ltd. The study included 24 young A. japonicus (4 months) with an average weight of 4.45 ± 1.14 g and 24 adult A. japonicus (24 months) with an average weight of 91.54 ± 3.63 g. Before the experiment, the animals were acclimated for one month in a 600 L tank at 15 °C, during which they were fed mixed feed once a day. The feed mainly included 30% sea cucumber compound feed, 40% fresh sea mud, and 30% Sargassum thunbergii (Haida, China). In the nonaestivating group, named AAJRT, the growth temperature of adult A. japonicus was controlled at 15 °C. In the aestivation group, named AAJSD, the growth temperature of adult A. japonicus was slowly increased from 15 °C to 26 °C at a rate of 0.5 °C per day, inducing it to enter the state of aestivation. In addition, considering that young A. japonicus do not aestivate, we named the young A. japonicus growing at 15 °C the YAJRT group. In the YAJSD group, the growth temperature of young A. japonicus was slowly increased from 15°C to 26 °C at a rate of 0.5 °C. During the feeding period, the salinity was maintained at 29–32, the dissolved oxygen content was approximately 6–8 mg/L, and the pH was 8.0–8.5. The whole experimental period was 60 days. 2.2. Analysis of Weight Change To clarify the effects of aestivation on the growth of adult A. japonicus and the effects of different growth temperatures on the growth of young A. japonicus, we recorded and analyzed the changes in the feed intake, movement status, and body weight of individuals in each group during the feeding process. 2.3. Microbial Diversity Analysis The gut microbiotas of adult A. japonicus in the AAJSD group and AAJRT group were analyzed by 16S rRNA gene high-throughput sequencing. Similarly, the gut microbiotas of the young A. japonicus in the YAJRT group and YAJSD group were compared and analyzed under different growth temperatures. The gut microbiotas of the AAJSD, AAJRT, YAJRT, and YAJSD groups were examined and comparatively analyzed by high-throughput 16S rRNA gene sequencing. Twelve A. japonicus were randomly selected from each group after seven days of feeding under different feeding conditions. The gut contents were collected, flash-frozen in liquid nitrogen, and then stored at −80 °C; half of these samples were used for microbial diversity analysis and the other half for metabolomic analysis. The detailed steps were as follows. 2.3.1. Sequencing The cetyltrimethylammonium bromide (CTAB) method was used to extract the total genomic DNA from six samples. One-percent agarose gels were used to evaluate DNA concentrations and purity. Using sterile water, the DNA was diluted to 1 ng/µL. The V3-V4 regions of the 16S rRNA genes were amplified using specific primers (338F-806R). PCRs and purification of PCR products were performed with reference to the literature [[50]15]. Following the manufacturer’s recommendations, sequencing libraries were generated with the NEB Next^® Ultra™ IIDNA Library Prep Kit (New England Biolabs, USA). The library quality was evaluated on a Qubit@ 2.0 Fluorometer (Thermo Scientific, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). Finally, the library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated [[51]16,[52]17]. 2.3.2. Data Analysis Paired-end reads were assigned to samples based on their unique barcodes and were truncated by cutting off the barcodes and primer sequences. Paired-end reads were merged using FLASH (version 1.2.11, [53]http://ccb.jhu.edu/software/FLASH/ (accessed on 20 December 2021)) [[54]18], and the splicing sequences were called raw tags. The filtering of the raw tags and comparative analysis of the clean tags were carried out with specific references to the literature [[55]19].