The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

Unveiling the early life core microbiome of the sea cucumber Apostichopus japonicus and the unexpected abundance of the growth-promoting Sulfitobacter.

BACKGROUND: Microbiome in early life has long-term effects on the host's immunological and physiological development and its disturbance is known to trigger various diseases in host Deuterostome animals. The sea cucumber Apostichopus japonicus is one of the most valuable marine Deuterostome invertebrates in Asia and a model animal in regeneration studies. To understand factors that impact on host development and holobiont maintenance, host-microbiome association has been actively studied in the last decade. However, we currently lack knowledge of early life core microbiome during its ontogenesis and how it benefits the host's growth. RESULTS: We analyzed the microbial community in 28 sea cucumber samples from a laboratory breeding system, designed to replicate aquaculture environments, across six developmental stages (fertilized eggs to the juvenile stage) over a three years-period to examine the microbiomes' dynamics and stability. Microbiome shifts occurred during sea cucumber larval ontogenesis in every case. Application of the most sophisticated core microbiome extraction methodology, a hybrid approach with abundance-occupancy core microbiome analyses (top 75% of total reads and > 70% occupation) and core index calculation, first revealed early life core microbiome consisted of Alteromonadaceae and Rhodobacteraceae, as well as a stage core microbiome consisting of pioneer core microbe Pseudoalteromonadaceae in A. japonicus, suggesting a stepwise establishment of microbiome related to ontogenesis and feeding behavior in A. japonicus. More interestingly, four ASVs affiliated to Alteromonadaceae and Rhodobacteraceae were extracted as early life core microbiome. One of the ASV (ASV0007) was affiliated to the Sulfitobactor strain BL28 (Rhodobacteraceae), isolated from blastula larvae in the 2019 raring batch. Unexpectedly, a bioassay revealed the BL28 strain retains a host growth-promoting ability. Further meta-pangenomics approach revealed the BL28 genome reads were abundant in the metagenomic sequence pool, in particular, in that of post-gut development in early life stages of A. japonicus. CONCLUSION: Repeated rearing efforts of A. japonicus using laboratory aquaculture replicating aquaculture environments and hybrid core microbiome extraction approach first revealed particular ASVs affiliated to Alteromonadaceae and Rhodobacteraceae as the A. japonicus early life core microbiome. Further bioassay revealed the growth promoting ability to the host sea cucumber in one of the core microbes, the Sulfitobactor strain BL28 identified as ASV0007. Genome reads of the BL28 were abundant in post-gut development of A. japonicus, which makes us consider effective probiotic uses of those core microbiome for sea cucumber resource production and conservation. The study also emphasizes the importance of the core microbiome in influencing early life stages in marine invertebrates. Understanding these dynamics could offer pathways to improve growth, immunity, and disease resistance in marine invertebrates.

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.

A Gram-staining-negative, oxidase-positive, strictly aerobic rod-shaped bacterium, designated strain PT1T, was isolated from the laboratory-reared larvae of the sea cucumber Apostichopus japonicus. A phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT1T was closely related to Neptuniibacter marinus ATR 1.1T (= CECT 8938T = DSM 100783T) and Neptuniibacter caesariensis MED92T (= CECT 7075T = CCUG 52065T) showing 98.2% and 98.1% sequence similarity, respectively. However, the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values among these three strains were 72.0%-74.8% and 18.3%-19.5% among related Neptuniibacter species, which were below 95% and 70%, respectively, confirming the novel status of PT1T. The average amino acid identity (AAI) values of PT1T showing 74-77% among those strains indicated PT1T is a new species in the genus Neptuniibacter. Based on the genome-based taxonomic approach, Neptuniibacter victor sp. nov. is proposed for PT1T. The type strain is PT1T (JCM 35563T = LMG 32868T).

Genome taxonomy of the genus Thalassotalea and proposal of Thalassotalea hakodatensis sp. nov. isolated from sea cucumber larvae.

The genus Thalassotalea is ubiquitous in marine environments, and up to 20 species have been described so far. A Gram-staining-negative, aerobic bacterium, designated strain PTE2T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PTE2T was closely related to Thalassotalea sediminis N211T (= KCTC 42588T = MCCC 1H00116T) with 97.9% sequence similarity. ANI and in silico DDH values against Thalassotalea species were 68.5-77.0% and 19.7-24.6%, respectively, indicating the novelty of PTE2T. Based on genome-based taxonomic approaches, strain PTE2T (= JCM 34608T = KCTC 82592T) is proposed as a new species, Thalassotalea hakodatensis sp. nov.

Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov.

A Gram-staining-negative, aerobic bacterium, designated strain PT3T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT3T was closely related to Amphritea ceti RA1T (= KCTC 42154T = NBRC 110551T) and Amphritea spongicola MEBiC05461T (= KCCM 42943T = JCM 16668T) both with 98.3% sequence similarity, however, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (in silico DDH) values among these three strains were below 95% and 70%, respectively, confirming the novelty of PT3T. Furthermore, the average amino acid identity (AAI) values of PT3T against other Amphritea species were on the reported genus delineation boundary (64-67%). Multilocus sequence analysis using four protein-coding genes (recA, mreB, rpoA, and topA) further demonstrated that PT3T, Amphritea ceti and Amphritea spongicola formed a monophyletic clade clearly separate from other members of the genus Amphritea. Three strains (PT3T, A. ceti　KCTC 42154T and A. spongicola JCM 16668T) also showed higher similarities in their core genomes compared to those of the other Amphritea spp. Based on the genome-based taxonomic approach, Aliamphritea gen. nov. was proposed together with the reclassification of the genus Amphritea and Aliamphritea ceti comb. nov. (type strain RA1T = KCTC 42154T = NBRC 110551T), Aliamphritea spongicola comb. nov. (type strain MEBiC05461T = KCCM 42943T = JCM 16668T), and Aliamphritea hakodatensis sp. nov. (type strain PT3T = JCM 34607T = KCTC 82591T) were suggested.

Tracking the dynamics of individual gut microbiome of sea cucumber Apostichopus japonicus during gut regeneration.

Sea cucumbers possess the remarkable capacity to regenerate their body parts or organs. Regeneration of host organs and/or body parts involves reconstruction of the host associated microbiota, however, the dynamics and contribution of microbiota to the regeneration process are largely unknown due to a lack of experimental models. To track the dynamics of individual gut microbiomes during gut regeneration, both caged mariculture and laboratory isolator systems of sea cucumbers (Apostichopus japonicus) were developed and longitudinal meta16S analyses were performed. Under natural environmental conditions in the caged mariculture system, both bacterial and eukaryotic communities in sea cucumbers' guts appeared to be reconstructed within 4 months after evisceration. Using the laboratory isolator, which can trace daily dynamics, we found that fecal microbiota collected before evisceration were clearly different from those collected after evisceration. We also identified eight key bacteria, belonging to Alteromonadaceae, Rhodobacteraceae, Oceanospirillaceae and family-unassigned Gammaproteobacteria, suggesting that these bacteria might interact with the host during the gut regeneration process. Six of the eight key bacteria were isolated for further bioassay using the isolator developed in this study to test whether these isolates affect gut regeneration.