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. 2023 Apr 29;13(5):153. doi: 10.1007/s13205-023-03561-8

Configuration of gut bacterial community profile and their potential functionality in the digestive tract of the wild and cultivated Indonesian shortfin elver-phase eels (Anguilla bicolor bicolor McClelland, 1844)

Diah Kusumawaty 1,✉, Stella Melbournita Noor Augustine 1, Any Aryani 1, Yunus Effendi 2, Talha Bin Emran 3, Trina Ekawati Tallei 4,✉
PMCID: PMC10148933  PMID: 37131968

Abstract

This study aimed to explore the bacteria present in the digestive tracts of wild and cultivated Indonesian shortfin eel during the elver phase. The eel has high export potential due to its vitamin and micronutrient content, but slow growth and vulnerability to collapse in farm conditions hinder its cultivation. The microbiota in the eel's digestive tract is crucial for its health, particularly during the elver phase. This study used Next Generation Sequencing to analyze the community structure and diversity of bacteria in the eels' digestive tracts, focusing on the V3–V4 regions of the 16S rRNA gene. Mothur software was used for data analysis and PAST v.3.26 was used to calculate alpha diversity. The results showed that Proteobacteria (64.18%) and Firmicutes (33.55%) were the predominant phyla in the digestive tract of cultivated eels, while Bacteroidetes (54.16%), Firmicutes (14.71%), and Fusobacteria (10.56%) were predominant in wild eels. The most prevalent genera in cultivated and wild elver were Plesiomonas and Cetobacterium, respectively. The microbiota in the digestive tract of cultivated eels was diverse despite uneven distribution. The KEGG database analysis revealed that the primary function of the microbiome was to facilitate the eel's absorption of nutrients by contributing significantly to the metabolism of carbohydrates and amino acids. This study's findings can aid in assessing eel health and improving eel farming conditions.

Keywords: Indonesian shortfin eel, Elver phase, Wild, Cultivation, Microbiome, Metagenomic, Digestive tracts, 16S rRNA, PICRUSt, KEGG

Introduction

Eel (Anguilla sp.) is a highly valuable fish in domestic and international markets. Indonesia is home to at least six species of eel, including Anguilla bicolor bicolor, A. b. pacifica, A. marmorata, A. celebensis, A, ancentralis, and A. borneensis (Affandi 2005). According to the IUCN threat criteria, A. bicolor and A. celebensis are categorized as Near Threatened (Nijman 2015) due to the difficulty of cultivating eels in an artificial environment, which is necessary as the eel breeds exclusively in the natural environment. The practice of catching eels in their glass eel stage and rearing them in an artificial environment poses a significant mortality risk to the eel population (Carda-Diéguez et al. 2014). All eel species are facultative catadromous, living primarily in fresh, brackish, and coastal waters and migrating to pelagic oceanic waters to breed (Tsukamoto 1992). Catadromous eels begin their lives in the sea and then migrate to the shore as larvae (leptocephali) before maturing into juveniles (referred to as glass eels). Then they migrate to the river (where they are known as elvers) and turn into pigmentation (Henkel et al. 2012). They then spend several years as yellow eels in freshwater (Arai 2020).

This fish is rich in vitamins, micronutrients, and minerals, such as vitamin A (4.700 IU), eicosapentaenoic acid (EPA) (1.337 mg/100 g), docosahexaenoic acid (748 mg/100 g) (Bae et al. 2010), protein (61.78%), fat (15.55%), arginine, lysine (Herawati et al. 2020), leucine, glutamic acid, palmitate, oleic acid (Widyasari et al. 2013), Mg, Zn, and Fe (Wijayanti et al. 2018). Due to its high nutritional content, eel is in high demand and has the potential to be developed as a functional food (Febrianta and Rawendra 2019).

Japan is the largest consumer of eels. The amount of eel consumption per year in the country is 150,000, out of the 250,000 needed worldwide (Wahjuningrum et al. 2018). This is because eel production in Japan is declining, and they must import their needs from other countries. The Japanese have a strong preference for Indonesian shortfin eel. Indonesia exported 4.1 million kilograms of live eel to Japan during the first 10 months of 2012 (Nijman 2015).

Behind the high market demand, there are several cultivation obstacles related to the eel's survival. Constraints that frequently occur when rearing eels include disease-infected seeds and cannibalism (Affandi et al. 2013). In the critical phase of the nursery, keeping the eel seed in the glass eel phase until it develops into an elver and then transitioning it to a fingerling pose a challenge. Additionally, poor water quality and low digestibility of feed by the eels are two issues that eel cultivators frequently face (Alika et al. 2021).

Eels' health is influenced by several factors, one of which is the balance of the microbiota living in their digestive tract. These microbial communities are critical for the fish's development, physiology, and overall well-being (Talwar et al. 2018; Butt and Volkoff 2019). The microbes contribute to the host's health by aiding in digestion, nutrient absorption, and immunity (Gajardo et al. 2016; Butt and Volkoff 2019). The composition of the microbiota in the digestive tracts of fish depends highly on the bacterial population. The naturally occurring bacteria present in the digestive tract of wild elver eel are largely unknown. Understanding the composition and function of the digestive tract microbiome in wild and cultivated eels would be extremely beneficial for the eel-rearing process. A rapid method for determining bacterial populations’ composition and diversity without culture is to use a metagenomic approach (Forbes et al. 2017). The purpose of this study was to use this approach to characterize the structure and diversity of the bacterial community in the digestive tract of Indonesian shortfin eels during the elver phase in the wild and in captivity. Additionally, PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) (Langille et al. 2013) was used to infer the functions of the microbiome in the digestive tract of the eels based on the 16S rRNA gene.

Materials and methods

Sample collection and digestive tract retrieval

A total of 50 elver-phase eel specimens were collected from an eel farm in Giri Mekar Village, Bandung (West Java, Indonesia). The eels were fed solely on sludge worms (Tubifex sp.). Figure 1 shows a representative sample. The eels were dissected using a scalpel, and then the digestive tracts (from the esophagus to the tip of the anus) were removed to obtain a total of 100 mg of digestive tracts.

Fig. 1.

Fig. 1

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A representative sample of the Indonesian shortfin elver phase eel

Total DNA genomic extraction

Total DNA containing the bacterial DNA was extracted from the digestive tracts of cultivated Indonesian shortfin elver-phase eel using the Sambrook (1989) protocol. The DNA quality and purity were analyzed by electrophoresis on a 0.8% agarose and NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). The pure extracted DNA was used for sequencing according to the protocol by Kusumawaty et al. (2020).

Amplicons generation

Amplification of the V3–V4 regions of 16S rRNA was carried out in an Agilent SureCycler 8800 Thermal Cycler using the Phusion® High-Fidelity PCR Master Mix (New England Biolabs) under the following reaction conditions: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 52 °C for 30 s, and extension at 72 °C for 45 s. This was followed by a final extension at 72 °C for 3 min (Lombogia et al. 2020). The PCR products were then diluted in the same volume of 1 × loading buffer and electrophoresed on a 2% agarose gel. The presence of bright and sharp bands measuring 400–450 bp indicated the successful amplification, which was intended for subsequent use.

Amplicons purification and library preparation

The Qiagen Gel Extraction Kit (Qiagen, Germany) was used to purify the amplicons. The DNA library was prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina, and its quantification was done via Qubit and Q-PCR. Subsequently, the Illumina platform was used to analyze the DNA library.

Bioinformatics analysis

Sequencing data processing

Sequencing data in fastq files were analyzed using mothur v.1.35.1 (Schloss et al. 2009). The two sets of readings (forward and reverse) were merged using the make.contigs command and the contig results were saved in FASTA format. The merged sequences were then processed using the screen.seqs command, followed by the count.seqs command, to determine the quality of the resulting sequences. Sequences that were duplicated of each other were sorted using the unique.seqs command to get unique sequences. The sequences were aligned to the SILVA alignment database (SILVA SSU v.132) to create a cluster and a distance matrix (Pruesse et al. 2007; Quast et al. 2013). The align.seqs command was used for alignment, which compares and detects similarities between sequences. The aligned sequences were then filtered using the filter.seqs command, followed by another use of the unique.seqs to obtain unique sequences.

The next step was pre-clustering to reduce sequence error and the number of unique sequences. Chimeric sequences were detected and removed using the chimera.uchime command, followed by the remove.seqs command. The sequences were classified using Bayesian clustering through the classify.seqs command. Quality control was implemented to remove sequences other than bacterial communities (archaeal, chloroplast, mitochondrial, eukaryotic, and unknown sequences) using the remove.lineage command. Clustering was then performed to obtain the values of OTUs (operational taxonomic units). The OTUs were classified based on their taxonomy using Microsoft Excel. Finally, a rarefaction curve was generated using the rarefaction.single command.

Alpha diversity analysis

Analysis of alpha diversity was conducted using PAST v.3.26 (Hammer-Muntz 2001). The analysis included dominance (D), Simpson (1 − D), Shannon–Wiener (H′), evenness (eH/S), Margalef (Dmg), and equitability (J) indices (Fatimawali et al. 2020).

Functional prediction analysis

PICRUSt was employed to predict the functional properties of the microbiome found in the digestive tracts of eels. The 16S rRNA copy numbers were normalized, and the microbiome potential functionality were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database (Kanehisa et al. 2012) as a reference. All graphical profiles were visualized using R software.

Results and discussion

Bacterial composition in the digestive tract of elver-phase eels

The diversity of microbial communities inhabiting the digestive tract of the fish plays a crucial role in modulating fish physiology, which ultimately affects their health (Butt and Volkoff 2019). The structure and composition of the digestive tract bacteria in cultivated elver-phase eel was determined using a representative sequence from the OTUs. An OTU is defined to delineate bacteria based on 97% 16S rRNA sequence similarity threshold (Nguyen et al. 2016), according to the SILVA database.

As illustrated in Fig. 2, the composition of the predominant phylum varies significantly between wild and cultivated elvers. Proteobacteria (64.18%) were more prevalent in cultivated elver, while Bacteroidetes (54.16%) predominated in wild elver. Similar findings were previously reported in cultivated European eel (A. anguilla), where bacteria belonging to the phyla Proteobacteria and Fusobacteria dominated the intestinal microbiota (Huang et al. 2018). These phyla also dominated the adult phase of silver and yellow eels. Additionally, they were also found in the wild elver phase of A. bicolor bicolor (Kusumawaty et al. 2020). Proteobacteria have facultative anaerobic characteristics, which means they can live and grow in an oxygen-free environment, allowing them to survive in the digestive tract. They can also withstand a variety of toxic environments and grow on a variety of organic compounds, including proteins, carbohydrates, and lipids.

Fig. 2.

Fig. 2

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Bacterial composition at phylum level. The predominant phylum in cultivated elvers is Proteobacteria, making up 64.18% of the population. In contrast, wild elvers are predominantly made up of Bacteroidetes, which make up 54.16% of the population

The second most abundant phylum in both elvers was Firmicutes, accounting for 33.55% in the cultivated elver and 14.71% in the wild elver, as illustrated in Fig. 2. The majority of bacteria in this phylum have probiotic properties that aid in the suppression of pathogenic bacteria (Thomas and Versalovic 2010). Furthermore, these bacteria assist with the absorption of micronutrients (Barkhidarian et al. 2021). Probiotics are commonly added to fish feed because they can improve nutrient utilization efficiency and aid in fish growth (Martínez Cruz et al. 2012). Probiotic-enriched feeds have been reported to increase the growth of eel seeds (Muchlisin et al. 2020).

The finding of this study highlights the significant role of evaluating elver health in creating functional feeds and probiotic formulations that can support their growth, development, and disease resistance. The findings suggest that the data obtained from such evaluations can aid in developing feeds that match the dietary requirements of elvers at different stages of growth, identifying efficient probiotics, and recognizing feed additives that enhance nutritional value and health benefits. The data can also assist in preventing and managing diseases that affect elvers by including feed additives or probiotics in their diets.

Fish feed that contains high fiber and carbohydrates can increase the number of Firmicutes bacteria. Several bacterial species in the phylum Firmicutes can degrade insoluble complex carbohydrates into simple carbohydrates, which are then used as an energy source for the host and other digestive tract microbial communities (Qin et al. 2010). Mekuchi et al. (2018) discovered that when fish were fasting, Proteobacteria predominated, while Firmicutes predominated when they were fed, and that it took 12 h to exchange the dominant bacteria.

Fusobacteria were discovered in 10.56% of wild elver and 1.00% of cultivated elver. These findings also corroborate those of Chen et al. (2019), who discovered that Firmicutes, Fusobacteria, Proteobacteria, Bacteroidetes, and Actinobacteria were the five most abundant phyla in the digestive tract of swamp eels (Monopterus albus). Proteobacteria were found to be the most prevalent bacteria in the anterior intestine of Anguilla japonica, with Bacteroidetes gradually replacing them in the posterior intestine region (Zhu et al. 2021). Proteobacteria, Fusobacteria, and Firmicutes are frequently found in the digestive tracts of marine and freshwater fish. These three phyla are reported to dominate the digestive tract microbiome of most marine (Hennersdorf et al. 2016) and freshwater fish species (Larsen et al. 2014; Eichmiller et al. 2016). Each phylum and its members have distinct characteristics and roles in the digestive tract, particularly in aiding in the digestion of food substances so that fish can obtain nutrients, which are then converted into energy.

The classes with the highest abundance are shown in Fig. 3. The cultivated elver had a high abundance of Gammaproteobacteria (63.10%), while the wild elver had a high abundance of Bacteroidia (54.20%). Gammaproteobacteria is a class that often appears in the metagenomic identification of aquatic animals. Clostridia were detected in 32.90% of cultivated elver and 14.4% of wild elver. Fusobacteria were found in up to 10% of wild elver and only 1% of cultivated elver. Gammaproteobacteria and Fusobacteria are the two major bacterial classes found in the intestinal tracts of many aquatic animals (Sun and Xu 2021). Clostridia play a crucial role in digestion and act as beneficial bacteria, which can enhance eel's health. Moreover, due to their unique biological activities, Clostridia have been shown to effectively reduce inflammation and allergic diseases. Its probiotic properties are attributed to metabolites, such as butyrate, secondary bile acids, and indolepropionic acid, which have been shown to strengthen the intestinal epithelial cell barrier and interact with the immune system (Guo et al. 2020).

Fig. 3.

Fig. 3

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Bacterial composition at class level. Gammaproteobacteria was more abundant in cultivated elvers, while Bacteroidia was more abundant in wild elvers. Clostridia were found in both, but in a higher proportion in cultivated elvers. Fusobacteria were found in higher proportions in wild elvers compared to cultivated elvers

Figure 4 displays the most abundant orders in cultivated elver, which were Enterobacterales (38.19%) and Clostridiales (32.94%), whereas Bacteroidales (54.15%) was the most abundant in wild elver. In addition, Clostridiales (14.41%), Fusobacteriales (10.56%), and Verrucomicrobiales (7.77%) were prevalent in wild elver. Aeromonadales (17.29%) and Pseudomonadales (7.07%) were also significantly observed in cultivated elvers. The results indicate a difference in order between the two samples.

Fig. 4.

Fig. 4

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Bacterial composition at order level. The most abundant orders in cultivated elver were Enterobacterales and Clostridiales, while Bacteroidales was the most abundant in wild elver. Wild elver also had a higher prevalence of Clostridiales, Fusobacteriales, and Verrucomicrobiales. Cultivated elver had significant observations of Aeromonadales and Pseudomonadales

As seen in Fig. 5, Enterobacteriaceae (38.19%) was the most abundant family in cultivated elver, followed by Clostridiaceae (31.02%), Aeromonadaceae (17.29%), and Moraxellaceae (6.89%). In contrast, Porphyromonadaceae (49.8%) appeared to be quite prevalent in wild elver. Several significant bacterial families, including Clostridiaceae (11.93%), Fusobacteriaceae (10.55%), Verrucomicrobiaceae (7.77%), and Bacteroidaceae (4.28%), were detected in wild elver. Previous studies have discovered Enterobacteriaceae on the gills, skin, and digestive tract of European eels in Latvian lakes (Strazdina et al. 2015). Enterobacteriaceae are commonly found in the gastrointestinal tracts of fish, and their presence in fish farming can pose serious health risks to humans (Oliveira et al. 2017). Aeromonadaceae and Moraxellaceae are two well-known families found in aquatic animals' digestive tracts (Wang et al. 2019). Members of Aeromonadaceae are known to be pathogenic and capable of causing diseases in fish (Adriana Dos Santos Silva 2020). Ruminococcaceae is a family that ranks among the top five most abundant families, encompassing 25 genera. The bacteria in this family are capable of degrading complex plant materials, such as cellulose, hemicellulose, and lignin (Biddle et al. 2013). They are a group of bacteria that are strictly anaerobic found in the colonic mucosal biofilms of healthy individuals (Gu et al. 2021). However, this family was found in very insignificant numbers in both elver samples.

Fig. 5.

Fig. 5

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Bacterial composition at family level. Enterobacteriaceae was the most abundant family in cultivated elver, followed by Clostridiaceae, Aeromonadaceae, and Moraxellaceae. However, Porphyromonadaceae was prevalent in wild elver. Significant bacterial families, including Clostridiaceae, Fusobacteriaceae, Verrucomicrobiaceae, and Bacteroidaceae, were also detected in wild elver

The intestinal microbiome of bighead carp (Aristichthys nobilis) fed with natural food, such as zooplankton, cladocera, copepods, and rotifers revealed a predominance of the families Porphyromonadaceae (40.2%), Fusobacteriaceae (29.7%), and Peptostreptococcaceae (27.4%) (Li et al. 2018). This indicates that these three families dominate in fish fed with natural feed or living in the wild. Peptostreptococcaceae were found in 0.34% and 0.65% of wild and cultivated elvers, respectively.

At least 164 genera (representing 78.35% of detectable genera) were found in the digestive tract of cultivated elver. In contrast, 64 genera (representing 52.06% of detectable genera) were identified in the digestive tract of the wild elver. This indicates that the bacteria in the digestive tracts of cultivated elver, fed solely with Tubifex, were more diversified than those of wild elver. Plesiomonas (35.71%) and Clostridium sensu stricto 1 (30.95%) were the most abundant bacteria in cultivated elver, followed by Aeromonas (17.29%) and Acinetobacter (6.89%). Meanwhile, the digestive tract of wild elver is dominated by five genera: Cetobacterium (10.55%), Clostridium sensu stricto 1 (8.94%), Akkermansia (7.77%), Odoribacter (4.45%), and Desulfovibrio (3.87) (Fig. 6). Except for Clostridium sensu stricto 1, which was prevalent in both elvers, there was a considerable variation in bacterial genera between the two elvers. Clostridium was detected with an abundance of 12%. The members of this genus are generally considered to be pathogens and are interpreted as indicators of an unhealthy microbiota (Lakshminarayanan et al. 2013; Yang et al. 2019). However, not all Clostridium bacteria are pathogenic, as some are beneficial for the host's growth and development. Clostridium has been reported as a producer of volatile fatty acids and vitamins, as well as helping in the intestinal digestion of fish through the production of digestive enzymes (Ray et al. 2012). The addition of the probiotic bacteria C. butyricum to tilapia feed increased the fish's weight gain (Poolsawat et al. 2020).

Fig. 6.

Fig. 6

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Bacterial composition at genus level. Plesiomonas and Clostridium sensu stricto 1 were the most abundant bacteria in cultivated elver, followed by Aeromonas and Acinetobacter. On the other hand, Cetobacterium, Clostridium sensu stricto 1, Akkermansia, Odoribacter, and Desulfovibrio were the dominant genera in wild elver. Except for Clostridium sensu stricto 1, there was considerable variation in bacterial genera between the two elvers

Studies found that, along with Fusobacterium and Aeromonas, the genus Plesiomonas was a major component of the bacterial microbiota of the intestinal tracts of a variety of freshwater fish (Larsen et al. 2014; Duarte et al. 2015). Members of the Fusobacterium can synthesize a variety of vitamins, elicit a proinflammatory response in the host, and exhibit virulence characteristics that enhance their adhesion to and ability to invade host cells (Kostic et al. 2012). Fish cultured for commercial purposes appears to have a strong association with the presence of Plesiomonas (Pakingking et al. 2015). However, unchecked populations of Plesiomonas bacteria can cause infection and death in elver-phase eels. The presence of P. shigelloides was known to cause digestive tract diseases, such as diarrhea.

Aeromonas was found to be quite abundant in cultivated elver. It may be possible that the remains of feed that accumulated at the bottom of the pond had the potential to fertilize Aeromonas, as the water turns acidic due to fermentation. However, Kusumawaty et al. (2020) reported that this bacterium was also detected in as much as 17% in wild fingerling-phase eels. This bacterium is pathogenic to most organisms and poses a risk of disease in elver. Elver infected with Aeromonas hydrophila exhibits clinical symptoms, such as passive and weak movement, a non-slippery body, bleeding on the chest, abdomen, and base of the fins, loss of body balance, decreased appetite, and broken and cracked dorsal, pectoral, and tail fins.

Acinetobacter is known to be pathogenic in various fish species (Xie et al. 2020; Ellison et al. 2021). This bacterium was also present in wild fingerling-phase eels (7%) (Kusumawaty et al. 2020), along with Cetobacterium and Clostridium, which were the most abundant bacteria. Acinetobacter was also detected in the digestive tract of farmed rainbow trout (Michl et al. 2017; Parshukov et al. 2019). Cetobacterium was found to predominate (23%) of the healthy intestine of Yunlong grouper (Ma et al. 2019).

In cultivated elver eels, Plesiomonas, Aeromonas, Clostridium, and Acinetobacter are considered part of the normal microflora of the digestive tract, and their potential beneficial effects can be enhanced by supplementing fish feed with probiotics. Although certain pathogenic bacteria can occasionally hinder the fish's development and growth, the presence of beneficial bacteria in the digestive tract can counteract their negative effect.

Lactic acid bacteria are beneficial bacteria found in the digestive tracts of fish. These bacteria are part of the normal flora found in the digestive tracts of terrestrial and aquatic animals. In cultivated elver-phase and wild fingerling-phase eels, Lactobacillus, Bacillus, Bifidobacterium, Lactococcus, and Streptococcus were found in a very significant number (Kusumawaty et al. 2020). Alika et al. (2021) found that shortfin eel seeds fed with fermented feed and probiotic supplementation performed better in terms of survival and growth than those fed with this supplementation. In a study by Omenwa et al. (2015), fish fed a feed containing the probiotic Lactobacillus had a 96.22% survival rate of fry and fingerlings of Clarias gariepinus. Lactobacillus supplementation has been reported to improve the immune system of Anguilla marmorata (Pinoke et al. 2015). Dietary supplementation of Nile tilapia fingerlings with Bifidobacterium longum, B. thermophilum, Bacillus subtilis, and L. acidophilus improved growth performance and fish health (Khalafalla et al. 2020). It is evident that diet type affects the fish’s gut microbiota and immune response (Ingerslev et al. 2014).

Bacterial diversity indices in the digestive tract of cultivated elver-phase eels

Alpha diversity indices summarize community structure in terms of richness (number of taxonomic groups), evenness (distribution of group abundance), or both (Willis 2019). The alpha diversity indices of bacterial genera in the digestive tracts of wild and cultivated elver-phase eels are presented in Table 1. The bacterial communities in this study have very low to moderate alpha diversity indices. The dominance index (D) is used to determine how much one taxon dominates another. The greater the value of the dominance index, the greater the presence of the dominant species. The communities in this study were not dominated by one or more taxa, as indicated by the dominance (D) value of 0.27 and 0.14 for wild and cultivated elver, respectively. The dominance index ranges from 0 to 1, with 0 representing infinite diversity (Lemos et al. 2011).

Table 1.

Alpha diversity indices of bacterial genera in cultivated elver-phase eels' digestive tracts

Alpha diversity Wild ever Cultivated elver
Indices Value Description Value Description
Dominance (D) 0.27 Low dominance 0.14 Low dominance
Simpson (1-D) 0.73 High diversity 0.86 High diversity
Shannon–Wiener (H’) 2.89 Moderate diversity 2.61 Moderate diversity
Evenness (e^H/S) 0.11 Low evenness 0.07 Low evenness
Margalef richness (R) 13.69 24.24 High richness
Chao1 358.04 329.40
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The Simpson (1 − D) value of 0.73 for wild elver and 0.86 for cultivated elver indicated that the bacterial genera in the digestive tract of both elvers were very diverse. However, the bacterial community in the cultivated elver were more diverse. The index indicates the likelihood that two randomly selected individuals from a sample will belong to different species (Moore 2013). The Shannon–Wiener (H´) index typically ranges between 1.5 and 3.5 and rarely exceeds 4.5 (Magurran 2021). With an H' index of 2.89 and 2.61 for wild and cultivated elver, respectively, bacterial diversity in both elvers was moderate. A high value can be obtained as the number of OTUs increases and the distribution of individuals among the taxa becomes more evenly distributed.

The evenness index is a measure of how similar one species is to another in terms of the relative abundance of the species (Fatimawali et al. 2020). A value of 0.07 indicates that the bacterial communities exhibit a low degree of evenness. The abundance distribution of each taxon becomes more uneven with an increase in the diversity of taxa (Tallei and Saroyo 2018). The evenness index value ranges between 0 and 1, with a lower value indicating a more uneven distribution that favors certain phyla or lower taxa (Koneri et al. 2017).

The Margalef (R) index is the simplest biodiversity metric to measure bacterial community richness (Magurran 2021). According to the index's criteria, taxa richness is low if R is less than 2.5, moderate if R is greater than 2.5, and high if R is greater than 4. A value greater than 4 indicates a rich taxa diversity. The bacterial community in both wild and cultivated elvers was extremely rich, with a value of 13.69 and 24.24, respectively. The Chao1 index is a non-parametric method used to estimate richness by measuring the expected OTUs in samples based on all of the bacterial species/genera found in the samples (Chao et al. 2006). The genus richness in the digestive tracts of cultivated elver-phase eels was very high, consistent with their R indices, with a value of 358.04 in wild elver and 329.40 in cultivated elver.

The diversity of bacteria in the digestive tract of elver eels will be influenced by the quality of their food intake. Huang et al. (2018) found that the gut microbiota of elver-phase eels had a greater diversity of bacteria compared to adult-phase eels. These differential microbial functional capacities are likely related to the physiological functions of the host and the interactions between the host and microbes. This is supported by the distinct differences in potential functional capacities across different gut sections (Chen et al. 2019).

KEGG-based PICRUSt analysis

Prediction of the functional potential of the microbial community

In KEGG-based PICRUSt, the predicted functional gene content of a microbial community is assigned to KEGG orthologs (KOs), which are then mapped to KEGG pathways. The relative abundance of each KEGG pathway can then be calculated based on the predicted KO content of the community. The results of KEGG-based PICRUSt analysis on the relative abundance of functional gene content performed by the microbial community in the digestive tract of elver-phase eels are displayed in Fig. 7. Several differences exist between the types of gene contents in the two elvers, indicating different metabolic processes. These differences are most likely due to variations in bacterial composition between the samples. The prominent functional potential in cultivated elver is related to cellobiose phosphotransferase system (PTS) EIIC component, iron complex transport system permease protein, cold shock protein, ATP-binding cassette subfamily C (ABCC), major type 1 subunit fimbrin (pilin), outer membrane usher protein, and LacI family transcriptional regulator. On the other hand, the most important functional potential in wild elver is linked to putative transposase and peptide/nickel transport system permease protein.

Fig. 7.

Fig. 7

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Relative abundance of the predicted functional gene content of microbial communities in the digestive tract of wild and cultivated elvers using KEGG-based PICRUSt analysis. There were differences in the types of genes present in the two elvers, indicating different metabolic processes, likely due to variations in bacterial composition. The functional potential in cultivated elvers was related to various proteins, while the most important functional potential in wild elvers was linked to putative transposase and peptide/nickel transport system permease protein

The EIIC component of the PTS is responsible for selectively transferring sugar molecules across the inner bacterial cells (McCoy et al. 2014). The PTS regulatory network not only regulates carbohydrate absorption and metabolism but also affects the use of nitrogen and phosphorus and the pathogenicity of some diseases (Deutscher et al. 2006). The iron complex transport system permease protein facilitates transmembrane ion transport (Askwith and Kaplan 1997). Iron, which is primarily absorbed in the intestine, is vital for intestinal microbiota, and its availability influences this microbial system (Seyoum et al. 2021). Many bacteria produce small cold shock protein (Csp) in response to a rapid decrease in temperature (cold shock) (Keto-Timonen et al. 2016). ABCC genes encode proteins involved in phase III metabolism that translocate xenobiotics outside of cells (Encarnación-Medina et al. 2017). Fimbrin is a member of the actin-binding protein family. This protein may be required for efficient bacterial entry into host cells, such as Sip A, which is encoded by Salmonella (Zhou et al. 1999).

The outer membrane protein usher is an integral protein that serves as the site from which the fimbriae emerge. Fimbriae are long, projecting proteins that enable bacteria, particularly members of the family Enterobacteriaceae, to adhere to certain receptor sites and colonize specific surfaces (Stubenrauch et al. 2017). LacI family transcriptional regulators recognize sugar effectors and regulate the expression of genes involved in carbohydrate utilization (Tsevelkhoroloo et al. 2021). Transposons are mobile genetic components that have been linked to bacterial genome plasticity and host adaptability (Vigil-Stenman et al. 2017).

Microbial metabolism prediction

Figure 8 provides statistics on the relative abundance of metabolic pathways within each of the KEGG categories. A high abundance of the bacterial metagenome, primarily associated with cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems, was observed among the most represented Gene Ontology (GO) categories of the intestinal microbiota. These categories were also found in the functional composition of the intestinal microbiota of hadal amphipods (Chan et al. 2021), Atlantic salmon (Salmo salar L.) (Dehler et al. 2017), and omnivorous fishes (Bi et al. 2021).

Fig. 8.

Fig. 8

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The statistical data regarding the relative abundance of metabolic pathways present within each KEGG category. There was a significant proportion of the bacterial metagenome, which is primarily associated with cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems, highly represented among the Gene Ontology (GO) categories of the intestinal microbiota

The statistical analysis of a one-way t-test with a confidence level of 0.01 revealed that the average gene abundance in the cultivated elver was greater than that of the wild elver, except for the membrane transport in the environmental information processing, which was greater in the wild elver. Based on the 10 most abundant genes, metabolic genes predominate in both cultivated and wild elvers. This is further supported by genomic data demonstrating a high gene expression process (transcription and translation).

Cell motility was the most prominent function in the category of cellular process. Membrane transport played a critical in environmental information processing. Replication and repair were identified as the most active processes in the category of genetic information processing. Carbohydrate and amino acid metabolism were the most prominent activities in the metabolic process. At KEGG level 1, metabolic processing took precedence, followed by environmental and genetic information processing. At KEGG level 2, other functions were observed, followed by membrane transport, carbohydrate, and amino acid metabolism, then replication and repair. Other functions also were observed at KEGG level 2, followed by transporters and general function prediction. Previous studies have reported that the healthy intestines of rainbow trout and Yunlong grouper had more sequences related to the metabolism of sugar, protein, and amino acid. Additionally, it was hypothesized that the intestinal microflora has an effect on the host's nutritional metabolism (Lyons et al. 2017; Ma et al. 2019).

Multiple studies have examined the gene expression profiles of fish raised in aquaculture environments, and research indicates that fish reared in farms exhibit altered gene expression patterns in comparison to their wild counterparts. Carnivorous fish species, such as Atlantic salmon, rainbow trout, European sea bass, and gilthead sea bream, commonly exhibit upregulated genes involved in stress response, immune function, and metabolism. Stressors, such as overcrowding, poor water quality, and fluctuations in temperature, light, and feeding schedules that occur in aquaculture settings, are likely to activate various physiological responses in fish, leading to changes in gene expression (Beemelmanns et al. 2021; Bermejo-Nogales et al. 2014; Cheyadmi et al. 2022; Momoda et al. 2007).

Conclusion

This study presented the first report on the investigation of the composition, diversity, and abundance of intestinal bacteria in the digestive tract of the wild and cultivated elver phases of Indonesian shortfin eels. Our findings demonstrate that the diversity of bacteria in the digestive tracts of cultivated elver eels, from the genus to the phylum level, decreased in comparison to wild elver eels. The predominant phyla in wild elver were Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Verrucomicrobacteria. In contrast, in cultivated elver stage eels the predominating phyla were Proteobacteria, and Firmicutes. The genera Plesiomonas, Aeromonas, Clostridium, and Acinetobacter were found to be in abundance in the samples of cultured eels, suppressing the development of other genera found in wild elver eels. Although several lactic acid bacteria were detected, only  a small number found. However, due to their native presence in the digestive tracts of elver phase eels, these bacteria have the potential to be developed into a probiotic supplement. The bacterial communities may have uneven distribution, but they are highly diverse and abundant, suggesting a well-balanced ecosystem. The presence of microbiota in the digestive tracts of cultivated as well as wild Indonesian shortfin elver-phase eels indicates that they contribute significantly to carbohydrate and amino acid metabolism, thereby facilitating nutrient absorption from these eels.

Funding

This research was funded by the Ministry of Education, Culture, Research, and Technology, Republic of Indonesia, through the Excellent Basic Research for Higher Education scheme with grant number 3572/E4/AK.04/2021 dan agreement/contract number 10/E1/KP.PTNBH/2021 and 265/UN40.LP/PT.01.03/2021.

Data availability

All data are included within this article.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Contributor Information

Diah Kusumawaty, Email: diah.kusumawaty@upi.edu.

Stella Melbournita Noor Augustine, Email: stellamelbournita@student.upi.edu.

Any Aryani, Email: any_aryani@upi.edu.

Yunus Effendi, Email: effendiy@uai.ac.id.

Talha Bin Emran, Email: talhabmb@bgctub.ac.bd.

Trina Ekawati Tallei, Email: trina_tallei@unsrat.ac.id.

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