Microbiome of Two Shrimp Species, Lebbeus groenlandicus and Pandalopsis japonica, Studied with Bacterial Systematics, Genomics, and Culture-independent Methods

Abstract

The majority of microorganisms coexist and interact within the digestive tracts of both humans and animals. Understanding the microbial community and its characteristics is crucial because microorganisms play various roles in the host body, particularly in relation to health. In this study, we investigated the microbial diversity and conducted genome analysis using culture-dependent methods in the intestines of two shrimp species, Lebbeus groenlandicus and Pandalopsis japonica. A total of 76 samples were collected from randomly selected individuals, and DNA was extracted from four different organs (stomach, hepatopancreas, anterior intestine, and posterior intestine). Barcode PCR and 16S rRNA gene amplification were performed using the Illumina Miseq platform, and the data were analyzed using QIIME2. The results revealed differences in microbial diversity between L. groenlandicus and P. japonica. P. japonica exhibited a dominant presence of class Gammaproteobacteria, particularly the genus Moritella, while L. groenlandicus showed similar proportions of class Gammaproteobacteria and Alphaproteobacteria. Additionally, the class Mollicutes was notable in several samples. The comparison of diversity between the two species revealed that L. groenlandicus had higher richness and evenness compared to P. japonica. Additionally, the richness varied among different organs, with L. groenlandicus showing lower richness in the hepatopancreas but higher richness in the intestine. In contrast, P. japonica displayed low richness across all organs. These findings suggest that the observed microbial diversity structure may be influenced by the characteristics of their deep and cold sea habitat, potentially impacted by the Moritella group. In a culture-dependent study conducted on L groenlandicus, gram-negative, aerobic bacterial strains M13 (KCTC 92916) and M17 (KCTC 92917) were isolated from the intestine and stomach. Phylogenetic analysis based on the 16S rRNA gene sequences confirmed that both M13 and M17 strains belong to a novel species within the genus Psychrobacter. Strain M13 displayed an irregular rod-shaped morphology, non-motility, weak positive oxidase reaction, and positive catalase reaction. This strain exhibited optimal growth at 20°C, pH 6.5, and a NaCl concentration of 1% (w/v). Strain M17 exhibited a diplococci shape, non-motility, and positive reactions in both oxidase and catalase tests. This strain showed optimal growth at 20°C, pH 7.5, and a NaCl concentration of 1% (w/v). The major polar lipids identified in M13 were phosphatidylethanolamine, phosphatidylinositol mannoside, unidentified glycolipid, and unknown phospholipid. In M17, the primary polar lipids observed were phosphatidylglycerol, sphingoglycolipid, and an unidentified phospholipid. The complete genome size of M13 was 3,207,175 bps, while the genome size of M17 was 3,260,452 bps. The average nucleotide identity (ANI) and average amino acid identity (AAI) values between M13 and M17 were 97.56% and 97.3%, respectively. Based on phylogenetic and genomic analyses, these strains are proposed as novel genera within the family Moraxellaceae. This study highlights the need for further research on the intestinal microorganisms of L. groenlandicus and emphasizes the potential for discovering novel strains.

Most of the microorganisms coexist and interact within the digestive systems of human and animals. The microorganisms play various roles in the host body, particularly in relation to health, so, it is crucial to understand the microbial community and its characteristics. Shrimp is one of the important marine products consumed worldwide, but not many studies have been reported on it. Therefore, we investigated the bacterial diversity of the intestinal microbial community of Lebbeus groenlandicus and Pandalopsis japonica, which are commonly called Dokdo shrimp. We randomly selected nine L. groenlandicus and ten P. japonica and collected four organs of the stomach, hepatopancreas, anterior intestine and posterior intestine for DNA extraction. A total of 76 samples were performed barcode PCR to proceed with 16S rRNA gene amplicon sequencing. The sequences were obtained from each sample via the Illumina Miseq platform. The raw data were analyzed using QIIME2 (Quantitative Insights Into Microbial Ecology 2). Results showed that the microbial diversity difference between L. groenlandicus and P. japonica. In case of P. japonica, the major class was Gammaproteobacteria, which dominated more than 80%. By contrast, L. groenlandicus had similar proportions of Gammaproteobacteria and Alphaproteobacteria. The class Mollicutes also accounted for a significant portion of several samples. At the genus level, the P. japonica showed a large portion of the genus Moritella of class Gammaproteobacteria, and family Moritellaceae, but L. groenlandicus has more various genera such as Psychromonas, Sphingomonas, Photobacterium et al. Comparing the diversity of L. groenlandicus and P. japonica by species, richness and evenness of L. groenlandicus are much higher than P. japonica. In the results by organ, the richness of L. groenlandicus was low in the hepatopancreas, but higher in the intestine, and the richness of P. japonica was low in all organs. It is assumed that the Moritella group influenced it because of the characteristics of living in deep and cold seas.

A gram-negative, aerobic bacteria, designated M13 (KCTC 92916) and M17 (KCTC 92917), were isolated from a intestine and stomach of Lebbeus groenlandicus, caught in the East Sea near Ulleung island. The strain M13 was coccobacilli-shape, non-motile, oxidase-weak reaction and catalase-positive. Growth of strain M13 was observed at 4–25 ℃ (optimum, 20 ℃) and pH 5.0–9.0 (optimum, pH 6.5) and in the presence of 0–6% (w/v) NaCl (optimum, 1%). The major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG), and unidentified glycolipids. The 16s rRNA gene sequences showed low similarity with the Psychrobacter luti NF11 (T) (98.55%), a member of Gammaproteobacteria. The strain M13 has a genome of total 3,207,175 bps (sum of contig 1: 3,177,391 bp and contig 2: 29,784 bp). The morphology of strain M17 was coccobacilli-shape, non-motile, and both of oxidase and catalase reactions were positive. As a result of finding out the optimal growth conditions for strain M17, it was revealed that 20 ℃, pH 7.5 and 1% (w/v) of NaCl concentration is the most appropriate conditions. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG), and unidentified glycolipids.constituted the primary cellular polar lipids. The strain17 has the low sequence similarity with its closely related genus, Psychrobacter arcticus 273-4 (T) (98.89%). The genome of the M17 strain was included a total of 3,260,452 bps, comprising contig 1 with a length of: 3,218,680 bp and contig 2 with a length of 41,772 bp. Average nucleotide identity (ANI) and average amino acid identity (AAI) values between strain M13 and M17 were calculated from whole genome sequencing. The values were 97.56%, 97.3% respectively. On the basis of phenotypic, phylogenetic, genomic analysis presented in this study, we suggest that the strain M13 and M17 were the novel genera which belongs to the family Moraxellaceae. In addition, it is assumed that several novel strains could be obtained within the digestive system of L. groenlandicus. This study showed the necessity for more research on the intestinal microorganisms that coexist with L. groenlandicus, regarded as an unexplored and valuable resource. This study will contribute to understand the characteristic of microbiome of the L. groenlandicus.