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Article

Structure and Diversity of Microbiomes Associated with the Gastrointestinal Tracts of Wild Spiny Lobsters and Profiling Their Potential Probiotic Properties Using eDNA Metabarcoding

by
Muhamad Amin
1,*,
Hussein Taha
2,
Laila Musdalifah
3,
Muhamad Ali
4,
Alimuddin Alimuddin
4,
Sahrul Alim
5 and
Takaomi Arai
2
1
Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C UNAIR Mulyorejo, Jl. Mulyorejo, Surabaya 60115, Indonesia
2
Environmental and Life Sciences Program, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, Bandar Seri Begawan BE1410, Brunei
3
Research Center for Fishery, National Research and Innovation Agency of the Republic of Indonesia, Jakarta 10340, Indonesia
4
Laboratory of Microbiology and Biotechnology, Faculty of Animal Science, University of Mataram, West-Nusa Tenggara, Mataram 83115, Indonesia
5
Aquaculture Study Program, Faculty of Agriculture, University of Mataram, West-Nusa Tenggara, Mataram 83115, Indonesia
*
Author to whom correspondence should be addressed.
Fishes 2024, 9(7), 264; https://doi.org/10.3390/fishes9070264
Submission received: 25 April 2024 / Revised: 18 June 2024 / Accepted: 26 June 2024 / Published: 4 July 2024
(This article belongs to the Topic Sustainable Development of Natural Bioactive Compounds/Products in Animal Resource and Agriculture Science: Volume II)
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Abstract

:
Microbial communities have been documented as playing many pivotal roles, and contributing to the growth or health performance of animal hosts. Thus, many studies are currently looking for potential beneficial bacteria “probiotics” from diverse environments, including wild species. The present study aimed to investigate the diversity and potential metabolic functions of bacterial communities in the gastrointestinal tract of wild spiny lobsters. The gastrointestinal (GI) tracts of two wild lobster species (Panulirus ornatus and Panulirus homarus) were dissected aseptically and analyzed through high-throughput sequencing, followed by PICRUSt analysis. The results exposed that the most dominant phyla inhabiting both lobster species at the post-puerulus and juvenile stages were Proteobacteria, Firmicutes, Bacteriodota, Patescibacteria, and Verrucomicrobiota, while at the genus level, the GI tracts were mostly dominated by Photobacterium, Candidatus Bacillopora, Vibrio, and Catenococcus at the post-peurulus stage, and Vibrio, Catenococcus, Acanthopleuribacter, Acinetobacter, Pseudoalteromonas, Grimontia, and Photobacterium at the juvenile stage. Further metagenomic prediction analysis discovers many potential probiont properties indicated by the detection of marker genes corresponding to many important metabolic activities, such as antimicrobial compounds (streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, and neomycin), antioxidants (e.g., flavonoids and carotenoids), and several important digestive enzymes (e.g., lipase, protease, and amylase). These results suggest that GI tracts of wild spiny lobsters are potential sources to discover novel probionts for aquaculture purposes. Further studies, such as the isolation of the natural product-producing bacteria, or cloning of the beneficial compound-identified genes, are highly recommended to develop novel probiotic strains for aquaculture purposes.
Keywords:
GI tract; probiont; spiny lobster; metabolic functions; microbiomes
Key Contribution: The results showed that Proteobacteria, Firmicutes, Bacteriodota, Patescibacteria, and Verrucomicrobiota are the most dominant phyla, inhabiting both lobster species at the post-puerulus and juvenile stages. Metagenomic prediction analysis discovers genes relating to production of antimicrobial compounds (streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, and neomycin), antioxidants (e.g., flavonoids and carotenoids), and several important digestive enzymes (e.g., lipase, protease, and amylase).

Graphical Abstract

1. Introduction

The benefits of probiotic bacteria are now of great importance in the aquaculture sector due to their important functions related to the health or growth of aquatic organisms [1]. Therefore, many researchers are making great efforts to produce new probiotic strains from different aquatic environmental conditions, including aquatic sediments [2], pond sediment [3], or pond water [4]. Several studies have shown that probiotic strains isolated from these aquatic environments have improved viability and performance in applied aquaculture species [5].
The most common way to obtain new probiotic strains is by using culture-based methods. However, this method is not considered to be very effective because many environmental microbiologists estimate that there are few bacteria (0.01%) that can be grown in the laboratory [6]. Similarly, Alam et al. [7] concluded that only tiny amounts of bacteria can be cultivated under standard laboratory conditions, which restricts our knowledge of these uncultured microbial metabolisms. As a consequence, potential probionts in certain environments are frequently underestimated, and their functions remain unknown [8]. To respond to this issue, a culture-independent metagenomics study has been developed in the last few decades. The latest advancements in next-generation sequencing have been enabling us to study uncultured bacteria, and have broadened the scope of metagenomics. In addition, developments in bioinformatics analysis, such as the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), have enabled us to determine predictive metagenome functions of the uncultured bacteria, such as antimicrobial or digestive enzyme synthesis [9]; furthermore, the results might be used identify potential probionts without culturing.
Wild lobsters feed on various diets in wild environments and may harbor various microbial species which enable them to tolerate environmental conditions in the gastrointestinal tracts. These microorganisms may be beneficial to lobster by either helping diet digestibility, and/or acting antagonistically to pathogens or parasites found in the wild [10]. Thus, screening for bacteria associated with the gastrointestinal tracts of wild lobster using a metagenomics or metabarcoding approach for finding potential probionts is the proper approach. Understanding the microbial structure and its potential metabolic activities may provide insight into the digestion processes in spiny lobsters [8]. Thus, the present study aimed at profiling the community structure and potential metabolic functions of the microbiomes in the gastrointestinal tract of wild ornate spiny lobsters (Panulirus ornatus) and wild scalloped spiny lobsters (Panulirus homarus), which may contribute to the development of lobster hatcheries in future.

2. Materials and Methods

2.1. Sample Collections

Ten ornate spiny lobsters (five at the post-puerulus and five at juvenile stages) and ten scalloped spiny lobsters (five at the post-puerulus, and five at juvenile stages) were widely caught from Prigi Bay, East Java, Indonesia. All spiny lobster samples were placed in plastic bags with portable aeration, and transported to the Laboratory of Microbiology, Faculty of Fisheries and Marine, Universitas Airlangga. Thereafter, gastrointestinal (GI) tracts were dissected out aseptically under a laminar flow as previously described by Amin et al. [11], pooled into four groups (post-puerulus stage of ornate spiny lobster, juvenile stage of ornate spiny lobster, post-puerulus stage of scalloped spiny lobster, and juvenile stage of scalloped spiny lobster), placed into a sterile 5 ml falcon tube containing 99% absolute ethanol, and stored at −20 °C.

2.2. DNA Extraction, Amplification, and Sequencing of 16S rRNA Genes

Bacterial DNA in the gastrointestinal tract (GIT) samples was extracted according to a factory instruction manual of the ZymoBIOMICSTM DNA kit (ZYMO RESEARCH, Orange, CA, USA). In brief, the hypervariable V3–V4 region of the bacterial 16S rRNA gene was amplified using locus-specific sequence primers (5′–CCT ACG GGN GGC WGC AG–3′ and 5′–GAC TAC HVG GGT ATC TAA TCC–3′) with overhang adapters [12]. The overhang adapter sequences were as specified by Illumina for compatibility, with Illumina index and sequencing adapters as previously described in Amin et al. [1].

2.3. Bioinformatics

Bioinformatic analysis was performed as previously described by Amin et al. [11] with some modifications. Quality assessment of raw reads was performed using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 25 April 2024), followed by the removal of primer and adapter sequences using Cutadapt 3.5 [13]. Then, paired-end reads were merged using DADA2 V1.18, and taxonomy assignments were performed using the SILVA nr database V138.1. Merged reads were clustered de novo, using UPARSE v11.0.667, into amplicon sequence variance (ASV). Thereafter, each ASV was aligned, and a phylogenetic tree was constructed using QIIME V1.9.1 against the Silva database 16S rRNA database.

2.4. Profiling of Predictive Metagenomic Functional Contents

The metagenomic function of bacteria associated with the GITs of spiny lobster was predicted using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), according to Langille et al. [9]. In brief, the metagenomic functions of bacterial communities were predicted and categorized with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, as previously described.

3. Results

3.1. Number of Reads

A total of 800,196 raw reads were obtained, which were as follows: 171,114 reads from the post-puerulus stage of ornate spiny lobster, 244,337 reads from the post-puerulus stage of scalloped spiny lobster, 148,021 raw reads from the juvenile stage of the ornate spiny lobster, and 236,724 raw reads from the juvenile stage of scalloped spiny lobster. After bioinformatics analysis, we obtained 777,883 reads (170,805 reads for the post-puerulus stage of ornate spiny lobster, 243,887 reads for the post-puerulus stage of scalloped spiny lobster, 134,072 reads for the juvenile stage of ornate spiny lobster, and 229,119 reads for the juvenile stage of scalloped spiny lobster) (Figure 1).

3.2. Taxonomic Composition of Bacteria

The gastrointestinal tract of both spiny lobsters and both life stages were dominated by phylum Proteobacteria (Figure 2). In addition, bacterial composition in the GI tract seemed to be grouped based on the lobster life stages, and not by the lobster species. At the post-puerulus stage, the GI tract of the ornate spiny lobster was predominated by Proteobacteria (68.85%), followed by Firmicutes (22.92%), Bacteriodota (7.48%), Patescibacteria (0.32%), Verrucomicrobiota (0.23%), Bdellovibriota (0.09%), Campylobacterota (0.08%), Actinobacteriota (0.02%), Acidobacteriota (0.02%), and Deinococcota (0.01%). Similarly, the GI tract of the scalloped spiny lobster at post-puerulus stages was dominated by Proteobacteria (83.53%), followed by Firmicutes (983%), Bacterioidota (4.71%), Patescibacteria (0.65%), Verrucomicrobiota (0.44%), Planctomycetota (0.19%), Fusobacteriota (0.15%), and Actinobacteriota (0.12%). Furthermore, seven other phyla, including Chloroflexi, Deinococcota, Acidobacteriota, Bdellovibrionota, Campylobacterota, Cyanobacteriota, and Desulfobacterota, accounted for less than 0.01% each.
At the juvenile stage, GI tracts of ornate and scalloped spiny lobsters were predominated by Proteobacteria and Bacteroidota (Figure 2). The most dominant phylum in the GI tract of the juvenile ornate spiny lobster was Proteobacteria (80.41%), followed by Bacteroidota (9.72%), Acidobacteriota (5.79%), Verrucomicrobiota (2.24%), Desulfobacterota (0.72%), Firmicutes (0.60%), Campylobacterota (0.20%), and Bdellovibrionota (0.14%). The other two phyla (Fusobacteriota and Planctomycetota) were counted at 0.07% each. Accordingly, GI tracts of the juvenile scalloped spiny lobster were also predominated by Proteobacteria (88.57%), followed by Bacteroidota (7.08%), Firmicutes (0.96%), Cyanobacteriota (0.93%), Patescibacteria (0.38%), Actinobacteriota (0.34%), Planctomycetota (0.33%), Bdellovibrionota (0.28%), Myxococcota (0.27%), Verrucomicrobiota (0.26%), Campylobacterota (0.16%), Acobobacteiota (0.11%), and Desulfobacterota (0.11%). Furthermore, the other two phyla (Chloflexi, and Fusobacteriota) were counted for 0.03 and 0.01%, respectively.
At the genus level, 49 bacterial genera were identified from the GI tract of ornate spiny lobsters at the post-puerulus stage. The five most dominant genera were the genus Photobacterium (38.52%), followed by Candidatus Bacilloplasma (20.91%), Neptunomonas (6.30%), Vibrio (3.91%), and Catenococcus (3.80%) (Figure 3). Additionally, there were 89 bacterial genera identified from the GI tract of the post-puerulus stage of scalloped spiny lobster. The five most dominant genera were Photobacterium (61.52%), followed by Vibrio (11.66%), Candidatus Bacilloplasma (9.76%), Catenococcus (1.97%), and Tenacibaculum (0.67%).
At the juvenile stage, 49 genera were identified from the GI tracts of the ornate spiny lobster (Figure 4a). The five most dominant genera were Vibrio (35.51%), followed by Catenococcus (18.11%), Acanthopleuribacter (5.79%), Acinetobacter (3.31%), and Pseudoalteromonas (2.61%). Additionally, there were 168 genera identified from the GI tracts of scalloped spiny lobster (Figure 4b). The five most dominant genera were Catenococcus (37.71%), followed by Vibrio (6.64%), Pseudoalteromonas (3.08%), Grimontia (2.11%), and Photobacterium (1.86%).

3.3. Predicted Metagenomic Functional Contents

To infer the probiotic functional potential of the microbiomes in the GIT tracts of lobster samples, the predicted ORFs were aligned against KEGG databases. The result showed that a total of 6605 predicted genes referred to in KEGG Orthology (KO) terms were identified, as follows: 4476 KO ids identified from PP_OSL, 5286 KO ids from PP_SSL, 4496 KO ids from J_OSL, and 6445 KO ids from J_SSL. Further analysis found that the 6605 KO ids were categorized into 11 metabolic activities (Figure 5). Thereafter, functional potencies of the microbial communities identified in the GI tract of the lobster samples were analyzed with a particular focus on antimicrobial production and digestive enzyme production. The obtained predicted functions were therefore selected based on their relevance to the two metabolic activities.

3.4. Predicted Biosynthesis of Secondary Metabolites

Deeper analyses indicated that GI tract microbiomes of the wild spiny lobster had the potential capacity to synthesize many important secondary metabolites, including antimicrobial compounds or antibiotics such as streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, neomycin, and antioxidants (flavonoids and carotenoids). For instance, streptomycin synthesis was indicated by the detection of marker genes belonging to 12 KO ids, (Table 1). The 12 KO ids were detected from a total of 1672 bacterial ASVs. Among these ASVs were some identified as members of Firmicutes (Bacillus tropicus, Blautia hominis, Kineothrix alysoides, and Caldicellulosiruptor bescii), Proteobacteria (Roseicella frigidaeris, Teredinibacter turnerae), and Actinobacteria (Gordonia rubripertincta, Mycolicibacterium chlorophenolicum, Mycolicibacterium montmartrense, Rhodococcus phenolicus, Rhodococcus gordoniae, Intrasporangium mesophilum, Allokutzneria oryzae), and several members of Chloroflexi (Litorilinea aerophila).
Potency of penicillin and cephalosporin syntheses was indicated by the detection of two marker genes, which were D-amino-acid oxidase [EC:1.4.3.3; K00273] and isopenicillin-N epimerase [EC:5.1.1.17; K04127]. In all, there were 275 pathways from J_SSL, with 27 from PP_OSL and 145 pathways from J_SSL. These marker genes were detected in twenty ASVs, of which eight ASVs belonged to Proteobacteria (Candidatus Pelagibacter ubique, Azospirillum brasilense, Nisaea nitritireducens, Nisaea denitrificans, Oceanibaculum pacificum, Plesiocystis pacifica (two ASVs), and Hydrogenophilus thermoluteolus). Furthermore, sixteen ASVs were identified as members of Planctomycetota (Gimesia maris, Rubinisphaera brasiliensis, and Symmachiella macrocystis (four ASVs), and Thalassoglobus neptunius). Additionally, four ASVs belonged to Actinobacteria (Gordonia rubripertincta, Blastococcus capsensis, Intrasporangium mesophilum, and Nocardioides fonticola). The other ASV identified was Prochlorococcus marinus subsp. pastoris (member of Cyanobacteria).
In addition, the potency of tetracycline biosynthesis was indicated by the presence of seven marker genes encoding acetyl-CoA carboxylase carboxyl transferase subunit alpha [K01962; EC:6.4.1.2 2.1.3.15], acetyl-CoA carboxylase biotin carboxyl carrier protein (K02160), acetyl-CoA carboxylase, biotin carboxylase subunit [K01961; EC:6.4.1.2 6.3.4.14], acetyl-CoA carboxylase carboxyl transferase subunit beta [K01963; EC:6.4.1.2 2.1.3.15], acetyl-CoA/propionyl-CoA carboxylase, biotin carboxylase, biotin carboxyl carrier protein [K11263; EC:6.4.1.2 6.4.1.3 6.3.4.14], acetyl-CoA/propionyl-CoA carboxylase carboxyl transferase subunit [K18472; EC:6.4.1.2 6.4.1.3 2.1.3.15], and tetracycline 7-halogenase/FADH2 O2-dependent halogenase [K14257; EC:1.14.19.49 1.14.19.-]. These genes were detected in 1748 bacterial ASVs; among them, some were identified as members of proteobacteria (e.g., Plesiocystis pacifica), Planctomycetota (Stieleria neptunia, Bremerella cremea, Bremerella volcania, Polystyrenella longa, Symmachiella macrocystis, Gimesia maris), and members of Chloroflexi (e.g., Litorilinea aerophila).
Carbapenem synthesis potency was indicated by the detection of two marker genes, encoding for ProB glutamate 5-kinase [EC:2.7.2.11: K00931] and ProA glutamate-5-semialdehyde dehydrogenase [EC:1.2.1.41: K00147]. These genes were detected in 1512 bacterial ASVs; among them, some were identified as members of Firmicutes (e.g., Alkalihalobacillus okhensis, Bacillus aerius) and Proteobacteria (Hyphomonas pacifica, Marisedimentalea aggregate, Paragaliceacola johnsnii, Kordiimonas lacus, Kordiimonas gwangyar, Kordiimonas aquimaris, Kordiimonas lacus).

3.5. Digestive Enzyme Production

The present study also identified marker genes which corresponded to digestive enzyme productions from many bacterial ASVs, indicated by lipid metabolism for lipase production, carbohydrate metabolism for amylase production, and amino acid metabolism for protease activity (Figure 5). For instance, there were marker genes which encoded for 58 KO ids, identified as being responsible for starch (carbohydrate source) digestion. The digestion of starch begins with the action of amylase enzymes, which convert it to maltotriose, maltose, dextrins (AMY, amyA, malS; alpha-amylase [EC:3.2.1.1]), and some glucose. A few other enzymes involved in starch digestion were identified, including SGA1; glucoamylase [EC:3.2.1.3], IMA, malL; oligo-1,6-glucosidase [EC:3.2.1.10]; UDPglucose 6-dehydrogenase [EC:1.1.1.22]; UTP-glucose-1-phosphate uridylyltransferase [EC:2.7.7.9]; glucose-6-phosphate isomerase [EC:5.3.1.9], etc. These enzymes were detected from 1787 bacterial ASVs. Among them, some were identified as a member of Proteobacteria (e.g., Thioprofundum lithotrophicum, Thalassotalea euphylliae, Thalassotalea fusca, Thalassotalea loyana, Thalassotalea eurytherma, Vibrio rotiferianus), Actinobacteria (e.g., Actinomarinicola tropica), Firmicutes (e.g., Bacillus inaquosorum, Blautia hominis, Clostridium saudiense), Bacteriodota (e.g., Salinirepens amamiensis), and Armatimonadetes (e.g., Chthonomonas calidirosea).
In addition, there were marker genes encoding four important digestive enzymes in the samples which indicated the lipase activities. These enzymes were phospholipase A1/A2 [EC:3.1.1.32 3.1.1.4], phospholipase C [EC:3.1.4.3], phospholipase D1/2 [EC:3.1.4.4], and alkyldihydroxyacetonephosphate synthase [EC:2.5.1.26]. These enzymes were detected from 434 bacterial ASVs, and among them, some were identified as members of Firmicutes (e.g., Bacillus tropicus, Paraclostridium benzoelyticum, Paeniclostridium sordellii, Eubacterium tenue, Romboutsia sedimentorum, Terrisporobacter petrolearius) and Proteobacteria (e.g., Chondromyces apiculatus, Sandaracinus amylolyticus, Thalassotalea insulae, Moritella marina, Acinetobacter junii, Marinomonas communis, Marinobacter salsuginis).

4. Discussion

Wild lobster is an opportunistic feeder, feeding on a wide range of zooplanktons which are available in their wild habitats [11]. In addition, lobster has complex life cycles involving the occupation of a wide range of habitats, from insular shelves during the reproduction stage and planktonic larvae floating in oceanic regions [14] to sedentary organisms at the juvenile stage onward in shallow bays [15]. Acknowledging these facts, lobsters may have diverse microbiomes harbored in their GI tracts, which may have the potential capacity to produce certain bioactive natural products, such as antimicrobial, antioxidant, or digestive enzymes [16]. The development of culture-independent techniques, such as next-generation sequencing and the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis, has enabled us to explore the structure and metabolic activity of more uncultured microorganisms in wild environments [9]. These advancements may contribute significantly to the screening processes of novel probiotic candidates in the future.
The present study reported the community structure and predicted metagenomic functional contents of microbiomes in the gastrointestinal tract of two wild spiny lobster species (Panulirus ornatus and Panulirus homarus) at two different life stages (post-puerulus and juvenile stages). The results showed that, in general, the five phyla appeared to be grouped based on life stages instead of species. The five most dominant phyla inhabiting both lobster species at the post-puerulus stage were Proteobacteria, Firmicutes, Bacteriodota, Patescibacteria, and Verrucomicrobiota. While at the juvenile stage, GI tracts of ornate and scalloped spiny lobsters were predominated by Proteobacteria, Bacteroidota, Acidobacteriota, Verrucomicrobiota, and Desulfobacterota. GI tracts of scalloped spiny lobster at the juvenile stage were predominated by Proteobacteria, Bacteroidota, Firmicutes, Cyanobacteriota, and Patescibacteria. The same result has been previously reported by Chen et al. [17], in which Proteobacteria is the most dominant bacteria in the GI tract of scalloped spiny lobsters. Similarly, the four main bacterial phyla were Tenericutes, Proteobacteria, Bacteroidetes, and Actinobacteria [18,19,20,21]. These results might suggest that Proteobacteria, Bacteriodetes, and Actinobacteria are autochthonous bacteria, and thus can be considered normal microbiomes in the GI tracts of lobsters. Some phyla, such as Actinobacteria and Tenericutes, appeared to fluctuate, and therefore can be categorized as allochthonous groups, in which their presence was highly influenced by environment and diets [22].
At the genus level, a total of 49 bacterial genera were identified in the GI tracts of ornate spiny lobsters, and 89 genera in the GI tracts of scalloped spiny lobsters at the post-puerulus stage. The microbial community genera in both species were very similar, as indicated by similar dominant genera, including Photobacterium, Candidatus Bacilliopora, Vibrio, and Catenococcus, which might be due to the similar habitat. While at the juvenile stage, 49 genera were identified from the GI tracts of ornate spiny lobsters, and 68 genera were identified from the GI tracts of scalloped spiny lobsters. The five dominant genera in the GI tracts of ornate spiny lobsters were Vibrio, Catenococcus, Acanthopleuribacter, Acinetobacter, and Pseudoalteromonas. The five most dominant genera in the GI tracts of scalloped spiny lobsters were Catenococcus, Vibrio, Pseudoalteromonas, Grimontia, and Photobacterium. The domination of Vibrio and Photobacterium has previously been reported from several lobster species, such as the European lobster (Homarus gammarus) [23], juvenile Caribbean spiny lobsters (Panulirus argus) [24], and the Japanese spiny lobster [25]. However, other dominant genera found in the present study were also different from the previous studies. Such discrepancies may be explained by the differences in host species, environmental conditions, diets, and temperature [18]. Furthermore, the differences in sampling times may also have significant effects on the composition and population of gut microbiota. The results may suggest that the microbial community in lobster may be determined by life stage and environment conditions.
Further analyses indicated that the microbial communities identified in the GI tract of the lobster samples had the potential capacity to synthesize several important secondary metabolites, indicated by the detection of marker genes corresponding to the synthesis of 10 antimicrobial compounds (streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, and neomycin), and antioxidants (flavonoid and carotenoids). Previously, antimicrobial-producing bacteria have been reported by Dopazo et al. [26], and the produced antimicrobials have been used to inhibit many fish bacterial pathogens, including Vibrio anguillarum, Aeromonas hydrophyla, Edwardsiella tarda, and Yersinia ruckeri. The ability of bacteria to produce antimicrobials has increased their usage as biocontrol agents in various sectors, including aquaculture and agriculture [27]. The interest in natural antimicrobial production has been increasing lately as a method to replace the indiscriminative uses of synthetic antibiotics in aquaculture industries to promote sustainable aquaculture production [2]. Some of the antimicrobial-producing bacteria were identified as members of Firmicutes, including Bacillus tropicus, Blautia hominis, Kineothrix alysoides, Caldicellulosiruptor bescii, Alkalihalobacillus okhensis, and Bacillus aerius. Among the identified species, Blautia hominis has been described to have a potential characteristic of being probiotic due to its antimicrobial activity [28]. One mechanism of this bacterial species to inhibit pathogen growth was by inhibiting nuclear factor kappa beta (NF-kB) activity in CaCo2 cells [29].
The other bacterial species detected in the present study which has been commonly reported as probiotics in fish is Bacillus aerius. Meidong et at. [30], for instance, reported that B. aerius was isolated from healthy hybrid catfish, and showed antimicrobial activity against Aeromonas hydrophila and Streptococcus agalactiae. Another study by Dutta et al. [31] added that B. aerius could kill pathogenic Aeromonas sp., could resist diluted bile juice, and is able to grow in intestinal mucus, as well as being non-pathogenic, which are very important characteristics for selecting probiotics for aquaculture purposes [2]. These results suggest that the GI tracts of wild aquatic species have a bacterial repertoire of many important secondary metabolites, which can be used to improve aquaculture production.
The present study also identified 434 bacterial ASVs, possessing the potential capacity to synthesize important digestive enzymes such as lipase, amylase, and protease. These enzymes play pivotal roles in feed digestion, which later contributes to nutrient adsorption, assimilation, and growth of cultured animals [32,33]. Similarly, many studies have reported the ability of certain intestinal microbiomes to synthesize digestive enzymes, including Bacillus amyloliquefaciens subsp. plantarum from the GI tract of hybrid abalone; Haliotis rubra x Haliotis laevigata [34], Bacillus sp., and Brevibacterium sp. from the GI tract of a silkworm; Bombyx mori [35], as well as Bacillus aerius, and Bacillus sonorensis for producing amylase from mrigal carp; and Cirrhinus mrigala [36]. Among them, some were identified as members of Firmicutes, including Bacillus tropicus, Paraclostridium benzoelyticum, Bacillus inaquosorum, and Blautia hominis. Of these identified species, Romboutsia sedimentorum has been previously reported to be isolated from marine sediment, and has the capacity to synthesize amylase [37]. This species has been described as spore-forming, gram-stain-positive, obligately anaerobic, straight or spiral rod-shaped, and able to utilize glucose, fructose, maltose, trehalose, and sorbitol as the sole carbon source. B. inaquosorum has also been reported to produce amylase in mrigal carp, Cirrhinus mrigala [36], while Bacillus sonorensis was reported to be significantly capable of inhibiting PirAB toxin-producing Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (AHPND) in shrimp aquaculture [38]. These results suggest that there were many potential bacterial probionts for increasing the yield of aquaculture industries.
The present study discovers many potential probionts from the GI tracts of wild spiny lobsters, which can contribute to the development of aquaculture industries. Many bacterial strains show the potential capacity to synthesize antimicrobial compounds (streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, and neomycin), antioxidants (e.g., flavonoids and carotenoids), and important digestive enzymes (e.g., lipase, protease, and amylase). Acknowledging the present study results, there were several possibilities which can be completed while regarding aquaculture purposes. The first possibility is to isolate those specific bacterial strains. Since the bacterial species have been identified, it will give critical information for preparing culture media [39]. For instance, B. inaquosorum is a spore-forming bacterium which can be isolated by bacillus-selective media such as polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate (PLET) agar, chromogenic agar (ChrA) [40], Mannitol Egg Yolk Polymyxin (MEYP) Agar, or Polymyxin Egg Yolk Mannitol Bromothymol Blue (PEMBA) Agar [41]. The second possibility to proceed with the present results is by cloning, especially those uncultured bacteria [7]. A basic principle of this approach is by constructing a metagenomic library in a suitable host by cloning a targeted fragmented DNA into expression systems, followed by the introduction of these metagenomic clones into a specific heterologous host [42] and re-function-based screening [43]. Through this approach, the chance of finding novel strains of probiont-producing natural products for use in aquaculture industries might be higher.

5. Conclusions

The bacterial community associated with the GI tract of wild spiny lobster was determined by life stage, instead of lobster species. The most dominant phyla were Proteobacteria, Firmicutes, Bacteriodota, Patescibacteria, and Verrucomicrobiota (post-puerulus stage), and Proteobacteria, Bacteroidota, Acidobacteriota, Verrucomicrobiota, Desulfobacterota, Firmicutes, Cyanobacteriota, and Patescibacteria (juvenile stage). At the genus level, the GI tract was mostly dominated by Photobacterium, Candidatus Bacillopora, Vibrio, and Catenococcus at the post-peurulus stage, and Vibrio, Catenococcus, Acanthopleuribacter, Acinetobacter, Pseudoalteromonas, Grimontia, and Photobacterium at the juvenile stage. Furthermore, the GI tract microbiomes of wild spiny lobsters showed a potential probiotic capacity, indicated by the detection of marker genes, which corresponded to the synthesis of antimicrobial compounds (streptomycin, vancomycin, carbapenem, tetracycline, novobiocin, penicillin, cephalosporin, ansamycines, butirosin, and neomycin), antioxidants (e.g., flavonoids and carotenoids), and important digestive enzymes (e.g., lipase, protease, and amylase).

Author Contributions

Conceptualization and experimental design, M.A. (Muhamad Amin), H.T., M.A. (Muhamad Ali), and T.A.; data collection, L.M., A.A., H.T., M.A. (Muhamad Ali), S.A, T.A. and M.A. (Muhamad Amin); data analysis, M.A. (Muhamad Amin), H.T., S.A., M.A. (Muhamad Ali), L.M. and A.A.; writing—manuscript draft, M.A. (Muhamad Amin), M.A. (Muhamad Ali), L.M., S.A. and A.A.; review and editing, H.T., M.A. (Muhamad Ali), and T.A.; funding acquisition, M.A. (Muhamad Amin), H.T. and T.A. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the Indonesian Ministry of Education, Culture, Research and Technology (Grant number: 1804/B/UN3.LPPM/PT.01.03/2024), and the Universiti Brunei Darussalam under the Faculty/Institute/Center Research Grant (No. UBD/RSCH/1.4/FICBF(b)/2020/029), (No. UBD/RSCH/1.4/FICBF(b)/2021/037), (No. UBD/RSCH/1.4/FICBF(b)/2022/051), (No. UBD/RSCH/1.4/FICBF(b)/2023/057) and (No. UBD/RSCH/1.4/FICBF(b)/2023/060).

Institutional Review Board Statement

Research procedures followed and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Universitas Airlangga, Indonesia (approval code 040/E5/PG.02.00.PL/2024).

Data Availability Statement

The datasets generated and/or analyzed during the current study are available in the GenBank with submission number SUB13731118 (Prokaryotic 16S rRNA/Microbiome of Spiny lobster).

Conflicts of Interest

The authors declare no conflicts of interest.

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Fishes 09 00264 g001
Figure 1. The numbers of raw and filtered reads of the bacterial 16s rRNA gene obtained from the gastrointestinal tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus and juvenile stages, collected from Prigi Bay, East Java Indonesia. PP_OSL is post-puerulus ornate spiny lobster, PP_SSL is post-puerulus scalloped spiny lobster, J_OSL is juvenile ornate spiny lobster, and J_SSL is juvenile scalloped spiny lobster.
Figure 1. The numbers of raw and filtered reads of the bacterial 16s rRNA gene obtained from the gastrointestinal tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus and juvenile stages, collected from Prigi Bay, East Java Indonesia. PP_OSL is post-puerulus ornate spiny lobster, PP_SSL is post-puerulus scalloped spiny lobster, J_OSL is juvenile ornate spiny lobster, and J_SSL is juvenile scalloped spiny lobster.
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Figure 2. Taxonomic composition at the phylum level of bacteria in the gastrointestinal tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus and juvenile stages. PP_OSL is the post-puerulus stage of an ornate spiny lobster, PP_SSL is the post-puerulus stage of a scalloped spiny lobster, J_OSL is the juvenile stage of an ornate spiny lobster and J_SSL is the juvenile stage of a scalloped spiny lobster. NA means unknown.
Figure 2. Taxonomic composition at the phylum level of bacteria in the gastrointestinal tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus and juvenile stages. PP_OSL is the post-puerulus stage of an ornate spiny lobster, PP_SSL is the post-puerulus stage of a scalloped spiny lobster, J_OSL is the juvenile stage of an ornate spiny lobster and J_SSL is the juvenile stage of a scalloped spiny lobster. NA means unknown.
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Figure 3. Taxonomic composition at the genus level of bacteria in the GI tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus stage. PP_OSL is the post-puerulus stage of ornate spiny lobster, PP_SSL is the post-puerulus stage of scalloped spiny lobster.
Figure 3. Taxonomic composition at the genus level of bacteria in the GI tract of ornate spiny lobster and scalloped spiny lobster at the post-puerulus stage. PP_OSL is the post-puerulus stage of ornate spiny lobster, PP_SSL is the post-puerulus stage of scalloped spiny lobster.
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Figure 4. Taxonomic composition at the genus level of bacteria in the gastrointestinal tract of ornate spiny lobster (a) and scalloped spiny lobster (b) at juvenile stages. J_OSL is a juvenile of an ornate spiny lobster, and J_SSL is a juvenile of a scalloped spiny lobster.
Figure 4. Taxonomic composition at the genus level of bacteria in the gastrointestinal tract of ornate spiny lobster (a) and scalloped spiny lobster (b) at juvenile stages. J_OSL is a juvenile of an ornate spiny lobster, and J_SSL is a juvenile of a scalloped spiny lobster.
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Figure 5. Histogram of predicted function abundance among the four GI tract sample groups. J_OSL is a juvenile of an ornate spiny lobster, and J_SSL is a juvenile of a scalloped spiny lobster. PP_OSL is the post-puerulus of ornate spiny lobster, and PP_SSL is the post-puerulus of scalloped spiny lobster.
Figure 5. Histogram of predicted function abundance among the four GI tract sample groups. J_OSL is a juvenile of an ornate spiny lobster, and J_SSL is a juvenile of a scalloped spiny lobster. PP_OSL is the post-puerulus of ornate spiny lobster, and PP_SSL is the post-puerulus of scalloped spiny lobster.
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Table 1. ORFs associated with streptomycin synthesis genes in the metagenome of wild spiny lobster GI tract microbiomes.
Table 1. ORFs associated with streptomycin synthesis genes in the metagenome of wild spiny lobster GI tract microbiomes.
PathwaysPP_OSLPP_SSLJ_OSLJ_SSLDescriptions
K01092118,581.5190,599.340,773.16124,961.4Myo-inositol-1(or 4)-monophosphatase [EC:3.1.3.25]
K01710125,918198,92033,84787,774E4.2.1.46, rfbB, rffG; dTDP-glucose 4,6-dehydratase [EC:4.2.1.46]
K00973124,573197,98230,68885,974E2.7.7.24, rfbA, rffH; glucose-1-phosphate thymidylyltransferase [EC:2.7.7.24]
K00067126,865170,77024,79385,249rfbD, rmlD; dTDP-4-dehydrorhamnose reductase [EC:1.1.1.133]
K01835110,010185,14326,73566,997pgm; phosphoglucomutase [EC:5.4.2.2]
K0179085,732156,62822,42762,023rfbC, rmlC; dTDP-4-dehydrorhamnose 3,5-epimerase [EC:5.1.3.13]
K0084557,86741,06521,73852,923glk; glucokinase [EC:2.7.1.2]
K1577825,650.810,507.679245.343,916.6phosphomannomutase/phosphoglucomutase [EC:5.4.2.8; 5.4.2.2]
K0001060926712488711,708iolG; myo-inositol 2-dehydrogenase/D-chiro-inositol 1-dehydrogenase [EC:1.1.1.18 1.1.1.369]
K018587851061972INO1, ISYNA1; myo-inositol-1-phosphate synthase [EC:5.5.1.4]
K106735131333352strA; streptomycin 3″-kinase [EC:2.7.1.87]
K0434320022313331067strB; streptomycin 6-kinase [EC:2.7.1.72]
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Amin, M.; Taha, H.; Musdalifah, L.; Ali, M.; Alimuddin, A.; Alim, S.; Arai, T. Structure and Diversity of Microbiomes Associated with the Gastrointestinal Tracts of Wild Spiny Lobsters and Profiling Their Potential Probiotic Properties Using eDNA Metabarcoding. Fishes 2024, 9, 264. https://doi.org/10.3390/fishes9070264

AMA Style

Amin M, Taha H, Musdalifah L, Ali M, Alimuddin A, Alim S, Arai T. Structure and Diversity of Microbiomes Associated with the Gastrointestinal Tracts of Wild Spiny Lobsters and Profiling Their Potential Probiotic Properties Using eDNA Metabarcoding. Fishes. 2024; 9(7):264. https://doi.org/10.3390/fishes9070264

Chicago/Turabian Style

Amin, Muhamad, Hussein Taha, Laila Musdalifah, Muhamad Ali, Alimuddin Alimuddin, Sahrul Alim, and Takaomi Arai. 2024. "Structure and Diversity of Microbiomes Associated with the Gastrointestinal Tracts of Wild Spiny Lobsters and Profiling Their Potential Probiotic Properties Using eDNA Metabarcoding" Fishes 9, no. 7: 264. https://doi.org/10.3390/fishes9070264

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