Abstract

The present study aimed to explore and compare the gut microbiome of Labeo rohita (rohu) from wild and aquaculture setting through 16S rDNA sequencing. The amplification of the library of 16S rDNA V3-V4 hypervariable regions of the gut microbiome was carried out followed by sequencing using Illumina MiSeq. The analysis of sequencing data was carried out through the Quantitative Insights into Microbial Ecology (QIIME2) pipeline. Proteobacteria, Firmicutes, Cyanobacteria and Actinobacteria represented the bacterial phyla with large abundance. The abundance of Bacillus spp. and Clostridium spp. was high in cultured forms. Vagococcus spp. and Carnobacterium spp. were found to be abundant in wild forms. Cyanobium spp., and Pseudomonas spp. were abundant in both wild and cultured forms. In the present study, the gut of cultured forms of rohu shows a diverse microbiome than that of wild forms. The harbouring gut microbes may offer several benefits to the host fish such as carbohydrate digestion, antimicrobial activity, xenobiotic degradation and faster growth rate. The functional profiles from the 16s rRNA gene amplicon confirm that the cultured rohu hosts the bacteria that have a role in metabolism and compromised disease resistance. Absence of Bacillus spp. in wild form, and low abundance of Lactococcus spp. indicates the need of finding alternatives for probiotics. The information from the present study and its understanding can be used for exploring the role of the gut microbiome with regard to the growth, immunity and other physiological functions of the fish.

1. Introduction

Aquatic foods are considered to be crucial as they offer values such as food and nutrition. The percentage of total food fish supplied by aquaculture is expected to rise up to 50% by 2030 ¹. The consumption and trade related to the industry are also expected to climb, albeit at a slower rate ². This reaffirms that aquaculture is and is going to remain the world's fastest-growing food-producing industry. India tops the list of the top 25 countries with respect to inland water capture production contributing to 16% of the total world capture production ².

In India, carp culture is practised as a polyculture of Indian Major Carps (IMC- Labeo rohita, Catla catla and Cirrhinus mrigala). Three exotic carps namely silver carp, grass carp and common carp are also cultured together with them. India leads in the production of Rohu, Catla and Mrigal with Bangladesh as another major producer ^{3, 4, 5}. Cyprinus carpio was introduced in India for aquaculture about six to seven decades ago. But now it has invaded the main river Ganga while contributing enormously to the fishery in Uttar Pradesh state ⁶. Indian major carps dominate the total inland fish landing in India as presented in Figure 1.

Several investigations have been carried out to ascertain the differences between fish inhabiting the wild and aquaculture settings. The gut microbial community is affected more by the host-habitat than its taxonomy and trophic level ⁷. The fish inhabiting wild habitats and aquaculture settings show morphological variations. This could be the result of variations in environmental factors. It can also be an outcome of genetic differences also together with environmental factors ^{8, 9}. The change in habitat may also affect reproductive health such as that of better sperm characteristics in males from wild habitats ¹⁰. The productivity in carp polyculture through wild-sourced seeds is more as compared to that via seeds from a hatchery. This can be linked to the more genetic variety of the population in its wild habitat, disease resistance and higher survival rate ¹¹. The protein levels in the muscles of cultured fish are higher as they are raised on an artificial diet ¹². The fat content of fish from wild habitats and aquaculture settings may also vary. Farmed fish are better sources of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) as compared to fish from wild habitats ^{13, 14}. Farmed fish have been reported to be more nutritious than the wild population ¹⁵.

The physical, chemical, and biological characteristics of all surface waters, including ponds, lakes, rivers, and oceans, vary. The bacterial diversity and evenness vary considerably with habitat ¹⁶. Carnivorous species are shown to have substantially higher levels of trypsin activity and bacteria that produce lipase and protease than herbivorous species which supports the significance of microbiota in host digestion ¹⁷. The gut microbiome, therefore, has always been an area of investigation for several workers.

Studies till early 2000 concentrated mainly on culture-based techniques that involved culturing and identification of bacteria ^{18, 19, 20, 21, 22, 23}. These studies though significant might have provided biased assessment because culturing gut bacteria in vitro is a big challenge ²⁴. In view of the difficulties related to isolation and maintenance, genomic sequencing of bacteria is a preferred option. The use of Next Generation Sequencing (NGS) and metagenomics have resulted in quick and unbiased evidence of gut microflora in fish ^{25, 26, 27, 28, 29, 30, 31, 32}.

The present study investigates the diversity of gut microbiome in Labeo rohita from wild and aquaculture settings using NGS to evaluate the variations in the core microbiome and the possible impact of change in the habitat on the gut microbiome.

2. Materials and Methods

In the present study, the main sampling area was from the Paithan Taluka of Aurangabad district, Maharashtra, where gut from Labeo rohita was sampled. The gut samples were also obtained from the fish procured from the wild habitat, Jayakwadi dam (19°29'0.31"N, 75°22'24.63"E) and aquaculture setting, Paithan Fish seed farm (19°28'37.02"N, 75°22'6.07"E). The gut samples were collected in sterile 1.5 mL Eppendorf tubes ensuring that no bacterial contamination resulted. During the transportation, the samples were kept in cool and dark. Samples were stored at -20°C until DNA from wild and farmed gut samples were extracted.

DNA was extracted using the NucleoSpin® DNA stool isolation kit (gut) (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) coupled with bead-beating. A bead tube and extraction buffer provided with the kit were used to collect the dissected intestine. Post isolation, DNA was dissolved in 50 μL of Elution buffer provided with the kit. The DNA extracts were stored at -80°C, till further processing.

The DNA extracts were used as templates in PCR to amplify the variable regions V3–V4 of the 16S rRNA gene for 25 cycles.

The gene-specific sequences used, target the 16S V3 and V4 regions as per earlier investigations ³³. Illumina adapter overhang nucleotide sequences are added to the gene-specific sequences. The full-length primer sequences targeting this region are:

F5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3’

R5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3’

The overhang adapter sequences added to the locus-specific primer for the region to be targeted were:

Forward overhang:

5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[locus specific sequence]

Reverse overhang:

5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[locus specific sequence]

Amplicons were visualised on a 1% agarose gel. For library preparation, two-stage PCR was carried out. PCR clean-up using magnetic bead technology (Ampure XP Beads) was done after each PCR. This was followed by library quantification (fluorometric quantification using dsDNA binding dyes) and normalization. The library was pooled. It was denatured using NaOH followed by dilution with a hybridization buffer. After heat denaturation, it was sent for Illumina MiSeq sequencing (MiSeq v3 reagent kit).

Sequence analysis was performed using the software package Quantitative Insights Into Microbial Ecology, QIIME2 ³⁴. Quality filtering of raw sequences was done and they were assigned to the samples according to their barcodes. After removing the primer sequences, chimeric sequences identified by de novo (abundance-based) and reference-based chimera detection with UCHIME were filtered out ³⁵. 16s metagenomic analysis was done using SILVA reference databases ³⁶.

Proportions of microbes found in each sample community (relative abundance data per sample) were analysed using the following script:

The level specified at specific taxonomic ranks can be specified by -L parameters for the script (1 for kingdom, 2 for phylum, 3 for class, 4 for order, 5 for family, 6 for genus, 7 for species).

To create visualized plots following script was used:

plot_taxa_summary.py -i <output/out_table_w_tax.txt> -l <taxonomic rank> -c pie,bar,area -o < taxsCharts >

The functional prediction was carried out using MicFunPred ³⁷ which predicts functional profiles from 16S rRNA gene amplicon sequence data. STAMP v2.1.3 ³⁸ was used for the visualization of predicted functional features.

Predictions for metabolism (carbohydrate, energy, lipid, nucleotide, amino acid, cofactors and vitamins), infectious diseases (bacterial, viral and parasitic) and antibacterial drug resistance were evaluated.

3. Results

For the present study, the gut samples of wild and cultured L. rohita were collected. All samples were collected in triplicate using the surface sterile environment technique.

The total read count obtained was 4082 whereas the average read count accounts for 2041 (Table 1).

From the substantial quality filtered reads, OTUs were assigned to 8 phyla, 9 classes, 16 orders, 17 families, and 18 genera. Among the classified reads, Phylum Cyanobacteria has maximum representation (42%), followed by Firmicutes (35%) and Proteobacteria (17%).

Phylum Cyanobacteria have shown the highest representation in the gut of the wild Labeo (42.5%) followed by Firmicutes (35%) whereas Planktomycetes represent a minimum (2.4%). Bacteroidetes and Chlamydiae were absent.

Similarly, in the gut of the Labeo from aquaculture settings, Cyanobacteria was most dominating (27.3%) followed by Firmicutes (26.3%) and Bacteroidetes has the least representation (0.6%) (Figure 2).

In the gut of wild-caught Labeo, Bacilli were found to be 59.5% followed by Alphaproteobacteria (17.2%), Gammaproteobacteria (12.6%), Actinomecetia (6.2%) and Planktomycetia (4.5%).

The metagenomic communities from the gut samples of the cultured Labeo have shown the dominancy of class Alphaproteobacteria (28.6%) followed by Clostridia (18.5%), Bacilli and Gammaproteobacteria (15.6% each) and Planktomycetia (13.8%). The lowest representation was observed for Chlamidiia (3.9%), Verrucomicrobiae (2.4%) and Acidimicrobiia (1.6%) (Figure 3).

From the gut of wild Labeo, out of the total 7 orders, 2 orders (Synechococcales, and Lactobacillales) contributed 76% of the flora and the remaining 5 orders make up 24% of the total. In cultured Labeo, gut diversity belonging to 14 orders was observed of which only 2 orders (Synechococcales and Eubacteriales) cover up to 47% microflora while the other 12 orders contribute 53% microbiome. These orders include Hyphomicrobiales, Rickettsiales, Sphingomonadales, Alteromonadales, Oceanospirillales Pseudomonadales, Pirellulales, Planctomycetales, Verrucomicrobiales, Bacillales Lactobacillales and Chroococcales (Figure 4).

In wild gut samples, Prochlorococcaceae (54.8 %), Enterococcaceae (16.4%), and Carnobacteriaceae (10.7 %) were dominant and share almost 82% of the total microbiome while other 6 families share the remaining 18 % of the total microbiome.

Out of 12 families obtained in cultured Labeo gut samples, the most dominant families were Prochlorococcaceae (54.8 %), Clostridiaceae (15.6%), and Bacillaceae (11.1%). They share almost 85% of the total microbiome and the remaining 9 families make up 15% of the total microbiome (Figure 5).

In wild gut samples, Cyanobium (54.8 %) and Vagococcus (16.4%) were dominant and share almost 72% of the total microbiome while the other 9 genera share the remaining 28 % of the total microbiome.

Out of 10 genera obtained in cultured Labeo gut samples, the most dominant were Cyanobium (50.3 %), Bacillus (15.2%) and Clostridium (9.8%). They share almost 75% of the total microbiome and the remaining 7 genera make up 55% of the total microbiome (Figure 6).

The abundance tree at the genus level using SILVA ribosomal RNA gene database project (Figure 7) clearly reflects that Cyanobium is the most abundant genus in both wild and cultured forms of L. rohita. The abundance of Bacillus and Clostridium in cultured forms in more as compared to the wild form. Vagococcus and Carnobacterium, which are abundant in wild form did not represent the cultured forms.

It was found that out of the total 18 genera reported from wild and cultured varieties of Labeo rohita in the present study, 8 were unique to the wild type and 7 were unique to the fish from aquaculture settings. 4 genera were common in both varieties (Figure 8).

The studies on functional prediction show a considerable variation in the functional potential of microbial communities from taxonomic profiles at the genus level. The wild forms show better carbohydrate, lipid and nucleotide metabolism (P<0.05). It also appears that the cultured forms are more susceptible to viral and parasitic infections (Figure 9).

4. Discussion

Multicellular eukaryotes, their existence and interaction with microbial communities existed for billions of years ³⁹. The microorganisms that live in environments including the stomach, skin, and vagina differ significantly among even healthy persons, according to studies on the human microbiome. Even though nutrition, environment, host genetics, and early microbial exposure have all been linked to this diversity, a large part of it is still unknown ⁴⁰. On the basis of the findings of the human microbiome research and the analogy so derived, it can be argued that the community structure of the fish microbiome can have a huge impact on fish health and growth. The expansion of the aquaculture industry calls for the manipulation of gut microbiomes to support the health of hosts. A baseline data on microbial diversity is required to understand the role of the gut microbiome in fish species. There are only a few studies on the gut microbiome of different fish species from varied geographical locations and habitats such as wild and aquaculture settings.

The present study elaborated on the composition of gut microbiota of the Indian major carp, Labeo rohita, from wild and aquaculture settings.

Fish have a simpler intestinal microbiota as compared to mammals. Proteobacteria, Cyanobacteria, Firmicutes, Actinobacteria, Chloroflexi, Verrucomicrobia and Bacteroidetes have been reported as the main microbiota in recent studies ^{41, 42, 43}. In the present study, Cyanobacteria was found to be the most abundant phylum in both wild and cultured rohu. This might be due to the fact that Cyanobacteria are the major food source. Similar observations have been reported by other workers ⁴⁴. Another abundant phylum Proteobacteria, a group of Gram-negative bacteria, might have a role in various processes such as Carbon, Nitrogen and Sulphur cycling as has been reported earlier from sludge and municipal wastes ^{42, 45}. Our study also highlights the presence of Sphingomonas (2.8%) in the cultured rohu which has been reported to be associated with xenobiotics degradation ^{46, 47}. The Proteobacteria, Pseudomonas, was found to be equally abundant in the wild (6.6%) and cultured form (8.2%), in the present study. Pseudomonas spp. has been reported to offer multiple benefits to the host such as microplastic degradation ⁴⁸, and antimicrobial activity ⁴⁹, Firmicutes were found to represent wild and cultured forms in high abundance. Firmicutes have been reported to be crucial for carbohydrate metabolism in man ⁵⁰. A high abundance of Firmicutes and very less presence of Bacteroidetes in cultured form can be correlated with the role of Firmicutes in a faster growth rate ⁵¹. Bacillus was not observed in the gut microbiome of wild rohu; however, they were abundant in the gut microbiome of the cultured counterpart (15.2%). Bacilli are known for cellulase-producing capacity ⁵², probiotic potential and fish pathogen antagonists ⁵³. The abundance of Bacillus in the cultured Rohu, therefore, must be offering several health benefits. A Firmicute, Clostridium (9.8%) represented only in the cultured form. Commonly associated with soil, Clostridium has been reported from the gut of other animals such as man, rainbow trout, and carp. Clostridium can ferment carbon sources, especially cellulose. This may play a crucial role in digesting food from plant sources ⁵⁴. Their role as mutualistic symbionts in marine herbivores has been reported by some workers ⁵⁵. Clostridium dominates the gut of herbivores and omnivores more than carnivores. Though, Halomonas normally dominate carnivorous fish, they have been reported from the gut of omnivorous fish as well. Halomonas are protease-producing bacteria. Clostridium contributed to nutrition by producing digestive enzymes to degrade cellulose ⁵¹. In the present study, the presence of Halomonas in the cultured rohu can be attributed to its role in protein digestion. In common carp, Clostridium butyricum has been reported to alter the composition of intestinal flora and negatively impact the multiplication of pathogens ⁵⁶. The gut microbiota of cultured rohu showed high dominance of phylum Verrucomicrobia (10.1%). Their abundance is generally less as compared to Proteobacteria and Bacteroidetes. Verrucomicrobia, rich in glycoside hydrolase genes are involved in polysaccharide hydrolysis ⁵⁷. Hence, their high abundance in the cultured Rohu can be attributed to polysaccharide hydrolysis. Other variations in the microbiome can be correlated with the change in habitat, diet and several other factors.

We investigated the functional profiles from 16S rRNA gene amplicon sequence data and concluded that the pathways for Nucleotide metabolism, metabolism of cofactors and vitamins, carbohydrate metabolism, amino acid metabolism, energy metabolism, lipid metabolism and antimicrobial drug resistance were abundant (>2%). The carbohydrate metabolism was higher (P<0.05) in the wild rohu. The carbohydrate metabolism in cultured forms can be correlated with the higher abundance of Bacillus and Clostridium. Firmicutes mainly dominated by Bacilli are known to play important role in the fermentation of dietary carbohydrates ⁵⁸. The abundance of nucleotide metabolism was higher in the wild rohu (P<0.05) and the functional profile for viral infectious diseases was significantly more abundant in the cultured form.

5. Conclusion

Our study furnishes an in-depth analysis of the gut microbiome of the Indian major carp Labeo rohita from wild habitat and aquaculture setting. Our findings give a clear overview of how changes in the habitat and environmental factors influence gut microbiome composition. Our results emphasize that the gut microbiome of the fish from wild and aquaculture settings varies to a great extent. Further, the core microbiome persisted in both environments and excluded other bacteria. The present study shows that the present aquaculture practices suit the carp culture in India as they offer gut microbiome providing several health benefits to the cultured fish at par with the wild variety. However, it appears that cultured fish are more susceptible to disease in the crowded, relatively unnatural environment of fish farms. The enrichment of microbial community functions in cultured forms confirms this finding. Our findings will aid the manipulation of the gut microbiome for enhancing aquaculture production.

References