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. 2024 May 17;19(5):e0302522. doi: 10.1371/journal.pone.0302522

Microbiota in different digestive tract of paddlefish (Polyodon spathula) are related to their functions

Chengxing Long 1,*, Jieqi Wu 2, Jialin Liu 1
Editor: Nafiu Bala Sanda3
PMCID: PMC11101114  PMID: 38758940

Abstract

Paddlefish has high economic and ecological value. In this study, microbial diversity and community structure in intestine, stomach, and mouth of paddlefish were detected using high-throughput sequencing. The results showed that the diversity and richness indices decreased along the digestive tract, and significantly lower proportion of those were observed in intestine. Firmicutes, Bacteroidetes and Proteobacteria were the dominant phyla. In top 10 phyla, there was no significant difference in mouth and stomach. But compared with intestine, there were significant differences in 8 of the 10 phyla, and Firmicutes and Bacteroidetes increased significantly, while Proteobacteria decreased significantly. There was no dominant genus in mouth and stomach, but Clostridium_sensu_stricto_1 and uncultured_bacterium_o_Bacteroidales was predominant in intestine. In conclusion, the species and abundance of microbiota in the mouth and stomach of paddlefish were mostly the same, but significantly different from those in intestine. Moreover, there was enrichment of the dominant bacteria in intestine.

Introduction

The digestive tract is a very important and specialized organ system, mainly involved in the digestion and absorption of substances, and is also the largest immune organ of body [1]. There is a diverse and variety of microorganisms inhabited in the cavity of the digestive tract, which is responsible for maintain the physiological homeostasis of the host [2]. Therefore, understanding the characteristics of gastrointestinal microbiota is vital for host health [3]. In recent years, microbiota, especially in intestine, has become a research hotspot. Increasingly, research has revealed the relationship between different intestinal microbiota and body diseases, and predict or determine the severity of diseases based on the species and abundance in intestinal microbiota [4,5].

The microbial composition in the digestive tract is mainly affected by many factors, such as dietary, environmental and physiological factors of the host, etc. Different sections of the digestive tract exhibit various environmental conditions [6–8]. Environmental conditions can impact the microbial composition in the digestive tract. In turn, the study on the characteristics of microbiota in the digestive tract can reflect the living environment [9]. Therefore, a balanced microbiota is essential for the maintenance of overall health in fish [10]. During the past decade, a large number of studies have been reported on gastrointestinal microorganisms of aquatic animals [3,11]. Studies have shown that the stability of gastrointestinal microorganisms in aquatic animals depends on the number and abundance of species, as well as complex interactions within the community [12].

Paddlefish, Polyodon spathula, is a large freshwater fish native to the Mississippi River basin in North America, belonging to the phylum vertebrata, class bony fish, order sturgeons, family sturgeons [13]. As an ancient freshwater economic fish, Polyodon spathula has strong adaptability, rapid growth, short food chain, and mainly feeds on crustacean zooplankton, and is the only species that filters and feeds zooplankton in paddlefish [14]. Paddlefish eggs can be made into caviar, is a rare tonic, meat is rich in a variety of amino acids. The content of cartilage is high, which contains high crude protein and sodium, potassium, calcium and other trace elements, has a high edible and economic value. In addition, its unique long-snout shape makes it has high ornamental value [15,16]. Therefore, paddlefish breeding has very high economic value. Furthermore, paddlefish has the physiological function of optimizing water quality because of its dense and slender gill rake [17]. In this respect, paddlefish has very good ecological value again.

The paddlefish is an important filter feeding fish in mixed pond in China. At present, many scholars have reported the intestinal microbial composition of paddlefish [18–20], but have mainly focused on breeding and reproductive technology [21–23], digestive enzymes and nutritional meat quality evaluation [14,16,24,25], probiotics [26], and involving different parts of the digestive tract [1]. In this study, hybrid aquaculture pond samples from a regional fishery science research institute were selected, and the characteristics and differences of the microbiota in the paddlefish mouth, stomach and intestine were analyzed by using second-generation high-throughput sequencing technology and bioinformatics methods. This study aims to analyze the characteristics and differences of microbiota in different parts of the paddlefish digestive tract, and to reveal their correlations with functions. The results will provide a microbiological basis for promoting the healthy development of aquaculture.

Materials and methods

Experimental fish and farming patterns

The paddlefish used in the experiment were collected in the same pond at the Fisheries Research Institute, Loudi, Hunan Province, China. It was the same batch of breeding fish, healthy and disease-free, with body weight 1967.2–2471.4 g. The breeding time of the fish was from Janunary 4, 2020 to Janunary 19, 2021, a total of 380 days, and the stocking specification was 350–400 g. During the whole experiment, the fish have been raised in natural water without feeding under the following water conditions: depth 2.0 m, temperature 17.8 ℃, dissolved oxygen > 4.35 mg/L, pH 8.10–8.56.

Sample collection and processing

The sampling time was 8:00 a.m on January 19, 2021. From 21 fish caught in a trawling net, five healthy individuals with similar body weight (length 75.4–80.2 cm) were randomly selected and transported back to the laboratory in a special fish bucket, while the rest were returned to the pond.

After measuring the body length and mass of the sample fish in the laboratory, the paddlefish was deeply anesthetized in water containing overdose of MS222, the surface of the paddlefish was rinsed with sterile water and 75% ethanol [27]. Five mouth samples (YZ1-YZ5), five stomach samples (YW1-YW5) and five intestine samples (YC1-YC5) were collected aseptically with tweezers, and stored with sterilized centrifuge tubes and refrigerated at -80 ℃ for later use.

16S rRNA gene sequencing

MN NucleoSpin 96 Soi kit was used extract DNA from the collected digesta samples. The V3-V4 variable region of 16S rRNA was amplified with 338F and 806R primers and followed by high-throughput sequencing using the Illumina HiSeq 2500 platform. The amplification primers, reaction system and amplification conditions are as follows. The primers were synthesized by Beijing Biomarker Technologies Co., Ltd (Beijing, China).

The sequence for the forward primer was 5′-ACTCCTACGGGAGGCAGCA-3′, and the reverse primer sequence was 5′-GGACTACHVGGGTWTCTAAT-3′. The amplification reaction was performed as follows: 5 μL KOD FX Neo Buffer, 0.3 μL (10 μM) of forward primer, 0.3 μL (10 μM) of reverse primer, 2 μL (2mM) dNTP, 0.2 μL KOD FX Neo, 50 ng of DNA template. The amplification conditions were as follows: initial denaturation at 95°C for 5 min, followed by 25 cycles consisting of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 40 s, and a final extension of 7 min at 72°C.

Analysis of microbial composition

Splicing the original data (FLASH, version1.2.11) [28], high-quality Tags sequence can be obtained by quality filtering (Trimmomatic, version 0.33) [29] and removal of chimera (UNHIME, version8.1) [30]. The sequences were clustered at the 97% similarity level (USEARCH, Version10.0) [31], and 0.005% of the sequence numbers was used as the threshold to filter out [32]. Mothur software (Version V.1.30, http://www.mothur.org/) was used to calculate the alpha diversity (including Chao1 richness estimator, Ace richness estimator, Shannon diversity index and Simpson Diversity index) and Beta diversity (including principal component analysis, principal coordinate analysis and non-metric multidimensional scale) in samples, respectively, so as to comprehensively evaluate the overall diversity and reveal differences among samples. LefSe analysis (http://huttenhower.sph.harvard.edu/lefse/) was used to find species with significant differences among all groups. Based on the four distance matrices obtained from beta diversity analysis, the samples were hierarchically clustered using the unweighted paired average method (UPGMA) with R to determine the similarity of species composition among the samples.

Statistical analysis

SPSS24.0 statistical software (IBM Corp., Armonk, NY, USA) was used for data statistics. Measurement data were expressed as the means ± standard deviations (x ± s), and independent sample T test was used to compare the pairwise means. A one-way analysis of variance was used for differences among normal distribution data groups, and Mann-Whitney U test was used for non-normal distribution data. P < 0.05 was regarded as a significant difference. All raw data obtained in this study have been submitted to the NCBI sequence read archive (accession number is PRJNA753213 https://www.ncbi.nlm.nih.gov/).

Ethics statement

The animal study was reviewed and approved by The Institutional Animal Care and Use Committed of Hunan University of Chinese Medicine (SYXK (Xiang) 2019–0009).

Results

Sequencing characteristics of 16S rRNA gene

After quality control, a total of 1,189,396 high-quality sequences were obtained from 15 samples in mouth (YZ), stomach (YW) and intestine (YC) of paddlefish, and the proportion of effective sequences in each sample was between 0.9153 and 0.9770. The average Coverage index of each sample was 0.9978, between 0.9970 and 0.9982, which could reflect the real situation of species in the community (Table 1).

Table 1. Coverage and diversity indices of bacterial species in paddlefish (Polyodon spathula).

group Chao1 Ace Simpson Shannon Coverage
YC 675.1619±88.1881 695.4459±88.4495 0.6992±0.0668 2.7510±0.2822 0.9979±0.0001
YW 996.6823±62.4031** 1023.4613±83.1413** 0.9723±0.0250** 7.5987±0.8204** 0.9977±0.0002
YZ 1049.0519±82.2196** 1079.5702±154.4841** 0.9758±0.0379** 7.9248±0.9886** 0.9977±0.0005
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Note: YC stands for Paddlefish intestine, YW stands for paddlefish stomach; YZ stands for Paddlefish mouth; Compared with Paddlefish intestine

*stands for p<0.05

**stands for p<0.01.

Chao1 index, Ace index, Shannon index and Simpson index were calculated to illustrate the diversity and richness of each sample. According to the calculation results of diversity and richness index (Table 1), Chao1 index, Ace index, Simpson index and Shannon index in YC presented the lowest value, all of which were significantly different from those in YZ and YW (P < 0.01). However, Chao1, Ace, Simpson and Shannon diversity index were similar between YZ and YW, with no significant difference. These results indicated that the lowest richness and diversity of bacteria were found in YC and the highest one in YZ, the bacteria in YZ and YW were relatively stable.

Overall microbiota structures

A total of 35 phyla, 733 genera and 821 species were identified in all the samples from the digestive tract of the paddlefish. Among them, 25, 34 and 35 phyla, 462, 659 and 644 genera and 512, 737 and 721 species were identified in YC, YW and YZ, respectively. Among the identified phyla, bacteria from Firmicutes, Bacteroidetes and Proteobacteria took dominant. The relative abundance of these three phyla accounted for 74%, 68.45% and 92.96% of YZ, YW and YC, respectively (Fig 1).

Fig 1. The bacterial community in all samples at phylum level.

Fig 1

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Note: YC stand for Paddlefish intestine, YW stand for paddlefish stomach; YZ stand for Paddlefish mouth.

Characteristics of bacterial community composition

Characteristics of bacterial community composition at the phylum level

The microbiota compositions in YC, YW and YZ were detected at the phylum level. There were 35 phyla detected in 15 samples of the three groups, including 25 in YC sample, 34 in YW sample and 35 in YZ sample. Proteobacteria (35.21%, 27.87% and 8.79% respectively), Firmicutes (27.94%, 31.71% and 46.88% respectively), Bacteroidetes (10.84%, 8.87% and 37.29% respectively), Acidobacteria (7.20%, 5.21% and 0.24% respectively), Actinobacteria (6.96%, 7.30% and 0.46% respectively) and Cyanobacteria (2.49%, 11.58% and 0.55% respectively) were the dominant phylum in YC, YW and YZ of paddlefish. However, in the top 10 phyla, the proportion of Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes and Verrucomicrobia in YW and YZ were significantly different from YC (P < 0.01 or P < 0.05), but there was no significant difference in the abundance of these phyla in YW and YZ (Fig 2, Table 2). In addition, except for the relative abundance of Firmicutes, Bacteroidetes and Fusobacteria increased in YC, the other seven phyla all decreased in differenct degrees.

Fig 2. Relative abundance of bacteria in each digestive tract on top 10 phyla.

Fig 2

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Note: YC stand for Paddlefish intestine, YW stand for paddlefish stomach; YZ stand for Paddlefish mouth.

Table 2. Relative abundance of bacteria in each digestive tract of paddlefish (Polyodon spathula) on top 10 phyla.
Phylum YC YW YZ
Firmicutes 0.4710±0.2315 0.3148±0.1099 0.2796±0.0231
Proteobacteria 0.0882±0.0889 0.2787±0.0404** 0.3557±0.0824**
Bacteroidetes 0.3705±0.1965 0.0879±0.0281* 0.1070±0.0401*
Actinobacteria 0.0046±0.0055 0.0725±0.0265** 0.0691±0.0210**
Cyanobacteria 0.0055±0.0061 0.1198±0.1047* 0.0249±0.0055**
Acidobacteria 0.0024±0.0030 0.0521±0.0148** 0.0713±0.0420**
Fusobacteria 0.0554±0.0489 0.0152±0.0110 0.0089±0.0034
Chloroflexi 0.0004±0.0003 0.0168±0.0066** 0.0138±0.0093*
Gemmatimonadetes 0.0002±0.0002 0.0090±0.0069* 0.0197±0.0113*
Verrucomicrobia 0.0008±0.0003 0.0071±0.0022** 0.0075±0.0045**
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Note: YC stands for Paddlefish intestine, YW stands for paddlefish stomach; YZ stands for Paddlefish mouth; Compared with Paddlefish intestine

*stands for p<0.05

**stands for p<0.01.

Characteristics of bacterial community composition at genus level

There were 733 genera detected in 15 samples of the three groups, including 242 uncultured bacteria. Of the 733 identified genera, 462 genera were detected in YC, 659 genera were detected in YW and 644 genera were detected in YZ of paddlefish. Furthermore, in the top 10 genera, uncultured bacteria accounted for 5. Clostridium_sensu_stricto_1 (34.83%) and uncultured_bacterium_o_Bacteroidales (33.31%) were predominant in YC, but their relative abundance was very low in YW and YZ, accounted for 9.57%, 0.16% and 0.29%, 0.14% respectively. Interestingly, there was no dominant genus in YW and YZ, and the relative abundance of the top 10 genera accounted for less than 30%. Overall, the species in YW and YZ were much richer than those in YC, but there was no dominant genus. Furthmore, there was no significant difference in the top 10 genera. These observations suggest that the microbial composition between YW and YZ was similar, and there were no significant differences (Fig 3, Table 3). Furthermore, we can get the same conclusion in UPGMA analysis (Fig 4).

Fig 3. Relative abundance of bacteria in each digestive tract on top 10 genus.

Fig 3

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Note: YC stand for Paddlefish intestine, YW stand for paddlefish stomach; YZ stand for Paddlefish mouth.

Table 3. Relative abundance of bacteria in each digestive tract of paddlefish (Polyodon spathula) on top 10 genus.

genus YC YW YZ
Clostridium_sensu_stricto_1 0.3497±0.1527 0.0959±0.1501* 0.0016±0.0011**
 o_Bacteroidales 0.3306±0.2047 0.0029±0.0026* 0.0014±0.0014*
 f_Enterobacteriaceae 0.0037±0.0039 0.0407±0.0152** 0.1089±0.1001*
 Lactobacillus 0.0013±0.0017 0.0289±0.0174** 0.0508±0.0498**
 Cetobacterium 0.0022±0.0027 0.0410±0.0526* 0.0215±0.0072**
 o_Chloroplast 0.0549±0.0491 0.0072±0.0114 0.0023±0.0017
 Bacteroides 0.0317±0.0637 0.0134±0.0084 0.0167±0.0061
 c_Subgroup_6 0.0017±0.0024 0.0236±0.0059** 0.0301±0.0158**
 Aeromonas 0.0473±0.1053 0.0010±0.0006 0.0017±0.0013
 f_Muribaculaceae 0.0011±0.0018 0.0216±0.0125* 0.0193±0.0067**
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Note: YC stands for Paddlefish intestine, YW stands for paddlefish stomach; YZ stands for Paddlefish mouth; Compared with Paddlefish intestine

*stands for p<0.05

**stands for p<0.01.

Fig 4. Uphma analysis of bacteria in each digestive tract of Polyodon spathula.

Fig 4

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Note: YC stand for Paddlefish intestine, YW stand for paddlefish stomach; YZ stand for Paddlefish mouth.

Discussion

The microbial community is a complicated ecosystem, which is kept in dynamic balance by the interactions between species [33]. The balance of intestinal bacteria in healthy fish plays an important role in immune regulation and nutrient metabolism [34,35]. In the aquaculture water environment, Firmicutes, Bacteroidetes and Proteobacteria are the main microbiota [36,37]. Sustainable development of aquaculture requires full consideration of the interaction between the environment and aquatic organisms [38–40]. Therefore, good feedback can be obtained on the microbial community of the aquaculture water by analyzing the characteristics of the gastrointestinal microbial community of its colonized aquatic organisms. In this study, we investigated the characteristics of microbial community in YC, YW and YZ of paddlefish using Miseq sequencing technology and bioinformatics, and analyzed the differences among them.

From the results of Alpha diversity, the microbial diversity in YC was significantly lower than that in YZ and YW, which was consistent with the highest microbial diversity in the stomach and the lowest microbial diversity in the intestine (Yang et al., 2020). It was also consistent with the fact that the microbial diversity in the aquaculture water was significantly higher than that in the intestine [4]. Paddlefish are filter feeders, their mouths are in direct contact with the aquatic environment, and their intestines passing through the selection barrier of the stomach, thus reducing diversity [41]. These results indicate that microorganisms maintain the relative stability of community through interspecies interactions to adapt to different physicochemical conditions and functions in different organs of the body.

At the phylum level, we identified 35 phyla. The dominant phyla in YZ and YW samples were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Acidobacteria. Whereas YC was rich in Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria. Specially, the relative abundance of Firmicutes and Bacteroidetes was absolutely predominant in YC. There was no significant difference between YZ and YW in the top 10 phyla. However, compared with YC, there were significant differences in 8 of the 10 phyla, among which Firmicutes and Bacteroidetes increased significantly, while Proteobacteria decreased significantly. In general, in human intestinal microbes, there is a significant correlation between the proportion of Bacteroidetes and Firmicutes, and the ratio of Firmicutes to Bacteroidetes can reflect the lipid situation of the body [42]. It has been reported that increased members of Firmicutes can increase the amount of lipids [43]. Bacteroides is a common bacteria in the intestinal tract of aquatic fish [44,45]. The significant increase in the relative abundance of bacteroides in the intestine can promote the digestion of carbohydrates, which is closely related to the digestive and absorption function of the intestine.

At the genus level, we identified 733 genera, including 462 in YC sample, 659 and 644 in YW and YZ samples, respectively. Of these, Clostridium_sensu_stricto_1 and uncultured_bacterium_o_Bacteroidales were in absolutely predominant in YC, but low in YW and YZ. More interestingly, of the top 10 genera, there was no dominant genus in YW and YZ. Clostridium_sensu_stricto_1 is the dominant genus in Clostridiales and belongs to Firmicutes. Clostridium can antagonize a variety of pathogenic fungi and also promote nitrogen accumulation [46], which indicates that the paddlefish has the potential function of optimizing water quality. Bacteroidales are the main representative of intestinal anaerobic microorganisms and an important microorganism that affects animal metabolism [47–50]. They play a crucial role in nutrient absorption, immune response, fat accumulation and intestinal microbiota balance, which directly or indirectly maintain the health of the host [50]. These effects directly or indirectly indicate that the composition and function of microbiota in the intestine are significantly different from those in the mouth and stomach, and these differences are related to the intestinal function.

In summary, our work confirmed the similarity of microbial species in YW and YZ from the community characteristics in YZ, YW and YC of paddlefish. This study showed that the characteristics of the microbiota community in YC were different from those in YW and YZ, and there was a phenomenon of enrichment of dominant bacterial species. These findings will further support the correlation between the colonization of specific microbiota and their functions in paddlefish.

Data Availability

The relevant data of this article has been submitted to the NCBI sequence read archive (accession number is PRJNA802701 https://www.ncbi.nlm.nih.gov/).

Funding Statement

The research was supported by the Natural Science Foundation of Hunan Province (2022JJ30316) to CX. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Nafiu Bala Sanda

14 Jan 2024

PONE-D-23-38263Microbiota in different compartments of the digestive tract of paddlefish (Polyodon spathula) are related to their functionsPLOS ONE

Dear Dr. Long,

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PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #3: Yes

**********

5. Review Comments to the Author

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Reviewer #1: This study used high-throughput sequencing to analyze the microbial communities in different digestive tract compartments (mouth, stomach, and intestine) of paddlefish (Polyodon spathula). The aim is to reveal their characteristics and differences, exploring their associations with host functions. The research, focusing on the economically and ecologically valuable freshwater fish, paddlefish, holds significance for the sustainable development of aquaculture.

Reviewer's Comments:

1. Provide more detailed descriptions of the high-throughput sequencing technology and bioinformatics analysis methods in the article, including steps involved in data analysis and specifics about the software used, ensuring the study's replicability.

2. Offer a more specific explanation of the steps involved in denoising and deduplication of sequencing data to ensure the accuracy and reliability of research results.

3. Provide a more thorough explanation, elucidating the ecological significance of diversity and abundance indices such as Chao1, Ace, Shannon, and Simpson. This will help readers better understand how these indices reflect the characteristics of microbial communities.

4. Provide detailed explanations of the statistical analysis methods used to characterize the microbial communities in different parts, including which statistical tests were employed and whether multiple comparison corrections were applied.

5. Include graphical representations of the results from Mothur software for diversity analysis to better illustrate the outcomes of diversity analysis.

6. The article mentions changes in microbial diversity and community structure without delving into the relationship between these changes and the physiological functions and health status of paddlefish. Further exploration through functional analysis is recommended to investigate the correlation between microbial composition and physiological functions such as nutrient absorption and the immune system in paddlefish.

7. The ariticle does not address whether the study considered temporal variations. Microbial communities may undergo changes over time. Conducting a time-series study would provide better insights into the dynamic changes of microbial communities and their relationship with seasonal or other time-related factors.

8. Analyze the metabolic functions of the microbial community to gain a deeper understanding of their roles in different parts of paddlefish. This includes studying functions such as enzyme production and acid production, which can help explain the microbial involvement in the digestive process of paddlefish.

9. Consider the microbial differences among individual paddlefish, taking into account factors such as age and gender. This in-depth analysis can provide insights into the individual variations in microbial communities among paddlefish.

Reviewer #2: The authors conducted a preliminary exploration of the microbiome in different segments of the digestive tract of paddlefish (Polyodon spathula). It is noteworthy that the experimental structure and design closely resemble the approach outlined by Yang G. et al., as published in 2020 (https://doi.org/10.1016/j.aqrep.2020.100402). While the study offers some insights, the novelty is somewhat lacking, and the microbiome analysis appears restricted to composition and abundance. Moreover, the content presented in tables and figures seems repetitive.

To enhance the manuscript, the authors should consider providing a more in-depth discussion of the findings, emphasizing unique aspects that distinguish their work from Yang G. et al. Additionally, addressing the following points will contribute to the overall clarity and rigor of the study:

Materials and Methods:

Line 100: Clarify the rationale for using fish within the weight range of 1967.2-2471.4g for the study.

Lines 112-115: Specify which part of the organ (e.g., saliva, mouth tissue, digesta, or intestine tissue) was utilized for microbiome analysis.

Line 148: Reconsider the use of T-test for comparing three groups of data. A more suitable approach, such as Kruskal-Wallis, followed by post hoc tests like Dunn’s test, should be employed.

Reviewer #3: This study investigated the microbial diversity and community structure in intestine, stomach, and mouth of paddlefish using high-throughput sequencing, which provide a microbiological basis for the development of aquaculture. However, there are still some problems worth further discussion and revision. Please refer to the attachment for details.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Attachment

Submitted filename: Comments.doc

pone.0302522.s001.doc (24.5KB, doc)
PLoS One. 2024 May 17;19(5):e0302522. doi: 10.1371/journal.pone.0302522.r002

Author response to Decision Letter 0

7 Feb 2024

Dear PhD Nafiu Bala Sanda

Thank you for your letter and reviewers' constructive comments on our manuscript entitled "Microbiota in different digestive tract of paddlefish (Polyodon spathula) are related to their functions". Those comments are all valuable and helpful for revising and improving our paper. We have studied the comments carefully. According to the reviewers' detailed suggestions, we have made extensive revision on the original manuscript and below we present a point-by-point response to the comments.

With best wishes,

Sincerely yours

Chengxing Long

Replies to comments from the Editors and Reviewers:

******************

Comments

******************

This study investigated the microbial diversity and community structure in intestine, stomach, and mouth of paddlefish using high-throughput sequencing, which provide a microbiological basis for the development of aquaculture. However, there are still some problems worth further discussion and revision. Details are given below.

（1）The title should be revised, since the article only conducted microbial sequencing and did not measure its function.

Response: Thanks for the reasonable advice. I made a few revisions to the title. In this study, we mainly elaborate the relationship between different digestive tract microbial characteristics and their functions of the paddlefish. At the same time, in the discussion section, we added some materials explaining the relationship between different digestive tract microbes and their functions. Look forward to your further good advice.

（2）I don't quite understand: Line 100 “...with body weight 1967.2-2471.4 g... ” , but Line 102 “...the stocking specification was 350-400 g.”, what does it refer to?

Response: Thanks for the reasonable advice. 350-400 g is the weight of the paddlefish when it was first released into the pond. 1967.2-2471.4 g is the weight of the paddlefish when it was caught after 380 days of feeding.

（3）The sampling time is only one, why does it take one year for breeding? Where is the meaning?

Response: Thanks for the reasonable advice. In order to better analyze the correlation between the microbiota in different parts of the digestive tract and the function of each digestive tract of paddlefish. We select the same batch of fish, feed the same pond, sample at the same time.

（4）There are only 5 samples, not enough.

Response: Thanks for the reasonable advice. Indeed, the larger the sample size, the more convincing it can be. However, due to the limitations of funds and conditions, we finally selected 5 healthy individuals of the same size from the captured fish for the experiment.

（5）The standard deviations of the table 2 and 3 data are very large, why?

Response: Thanks for the reasonable advice. The standard deviation of some species is indeed a bit large, but it is acceptable for species with higher abundance, and these results may be related to individual differences, or the low abundance of these species themselves.

（6）Line 202: “.644 genera were detected in YC of paddlefish. ”, should be “...644 genera were detected in YZ of paddlefish.”

Response: Thanks for the reasonable advice. Line 202: should be “...644 genera were detected in YZ of paddlefish.”

（7）Line 248-249: “These research results were the best evidence for the high body fat content in the paddlefish.” this sentence is inappropriate, so suggested to delete it.

Response: Thanks for the reasonable advice. The sentence has been deleted.

（8）Line 255-257: “Clostridium can antagonize a variety of pathogenic fungi and also promote nitrogen accumulation (Qian et al., 2018), which will provide evidence for the water quality optimization function of paddlefish.” this sentence is also not very accurate, it is recommended to modify it.

Response: Thanks for the reasonable advice. The sentence has been revised.

******************

Journal Requirements:

******************

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: Thanks for the reasonable advice. It has been modified as required.

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

Response: Thanks for the reasonable advice. In order to reduce the pain of the fish as much as possible, the paddlefish was deeply anesthetized in water containing overdose of MS222. After wiping the outer surface of the paddlefish with 75% ethanol, the contents of the mouth, stomach and intestine of the paddlefish were collected by sterile tools.

3. Thank you for stating the following financial disclosure:

"the Natural Science Foundation of Hunan Province (2022JJ30316)."

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Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: Thanks for the reasonable advice. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, and added in the cover letter.

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"This work was supported by the Natural Science Foundation of Hunan Province (2022JJ30316)"

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"the Natural Science Foundation of Hunan Province (2022JJ30316)."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Thanks for the reasonable advice. The fund information was removed from the Acknowledgments section.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Response: Thanks for the reasonable advice. The ORCID iD has been provided.

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Response: Thanks for the reasonable advice. Figures and tables in the manuscript have been removed.

******************

Reviewer #1:

******************

1. Provide more detailed descriptions of the high-throughput sequencing technology and bioinformatics analysis methods in the article, including steps involved in data analysis and specifics about the software used, ensuring the study's replicability.

Response: Thanks for the reasonable advice. We have listed detailed references for the methods and software involved in the article, which can ensure the repeatability of the research.

2. Offer a more specific explanation of the steps involved in denoising and deduplication of sequencing data to ensure the accuracy and reliability of research results.

Response: Thanks for the reasonable advice. In order to ensure the accuracy and reliability of the research results, the steps involved in de-noising and de-duplication of sequencing data were explained in detail, and detailed references were noted. (p132-p137)

3. Provide a more thorough explanation, elucidating the ecological significance of diversity and abundance indices such as Chao1, Ace, Shannon, and Simpson. This will help readers better understand how these indices reflect the characteristics of microbial communities.

Response: Thanks for the reasonable advice. The ecological significance of the diversity and abundance indices such as Chao1, Ace, Shannon and Simpson has been well studied, so I will not elaborate in detail here, and I will add it if necessary.

4. Provide detailed explanations of the statistical analysis methods used to characterize the microbial communities in different parts, including which statistical tests were employed and whether multiple comparison corrections were applied.

Response: Thanks for the reasonable advice. Statistical methods have been supplemented.

5. Include graphical representations of the results from Mothur software for diversity analysis to better illustrate the outcomes of diversity analysis.

Response: Thanks for the reasonable advice. I consider that the data in the table can illustrate the change in diversity more clearly than the graph, so the results of the diversity analysis are not represented by the graph. We look forward to your approval.

6. The article mentions changes in microbial diversity and community structure without delving into the relationship between these changes and the physiological functions and health status of paddlefish. Further exploration through functional analysis is recommended to investigate the correlation between microbial composition and physiological functions such as nutrient absorption and the immune system in paddlefish.

Response: Thanks for the reasonable advice. Exploring the correlation between microbial composition and physiological functions such as nutrient absorption and the immune system in paddlefish is the main content of my next research.

7. The ariticle does not address whether the study considered temporal variations. Microbial communities may undergo changes over time. Conducting a time-series study would provide better insights into the dynamic changes of microbial communities and their relationship with seasonal or other time-related factors.

Response: Thanks for the reasonable advice. You have provided me with a good research idea. In the following research, I will consider the relationship between dynamic changes of microbial communities and seasonal or other time-related factors.

8. Analyze the metabolic functions of the microbial community to gain a deeper understanding of their roles in different parts of paddlefish. This includes studying functions such as enzyme production and acid production, which can help explain the microbial involvement in the digestive process of paddlefish.

Response: Thanks for the reasonable advice. Analyze the metabolic functions of the microbial community to gain a deeper understanding of their roles in different parts of paddlefish. We will focus on this in the following research.

9. Consider the microbial differences among individual paddlefish, taking into account factors such as age and gender. This in-depth analysis can provide insights into the individual variations in microbial communities among paddlefish.

Response: Thanks for the reasonable advice. There are many factors affecting the differences in the microbial communities of paddlefish. In this study, we only considered the relationship between the digestive tracts of paddlefish and the water environment

******************

Reviewer #2:

******************

Materials and Methods:

Line 100: Clarify the rationale for using fish within the weight range of 1967.2-2471.4g for the study.

Response: Thanks for the reasonable advice. The weight of the paddlefish is not controlled by us, and we only want to analyze the microbiota characteristics of different digestive tracts of the paddlefish after one year of feeding. The results of this study aim to show that the microbial characteristics are related to their function.

Lines 112-115: Specify which part of the organ (e.g., saliva, mouth tissue, digesta, or intestine tissue) was utilized for microbiome analysis.

Response: Thanks for the reasonable advice. The digestive system was analyzed in this study

Line 148: Reconsider the use of T-test for comparing three groups of data. A more suitable approach, such as Kruskal-Wallis, followed by post hoc tests like Dunn’s test, should be employed.

Response: Thanks for the reasonable advice. We have modified and supplemented the statistical methods.

******************

Reviewer #3:

******************

This study investigated the microbial diversity and community structure in intestine, stomach, and mouth of paddlefish using high-throughput sequencing, which provide a microbiological basis for the development of aquaculture. However, there are still some problems worth further discussion and revision. Please refer to the attachment for details.

Response: Thanks for the reasonable advice .We have made effective revised to the article in accordance with the requirements of reviewers and editors, and hope that you will continue to give valuable comments.

Attachment

Submitted filename: Response to the Editors and Reviewers.doc

pone.0302522.s002.doc (54KB, doc)

Decision Letter 1

Nafiu Bala Sanda

8 Apr 2024

Microbiota in different digestive tract of paddlefish (Polyodon spathula) are related to their functions.

PONE-D-23-38263R1

Dear Dr. Long Chengxing,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewers' comments:

Acceptance letter

Nafiu Bala Sanda

8 May 2024

PONE-D-23-38263R1

PLOS ONE

Dear Dr. Long,

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* All references, tables, and figures are properly cited

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Kind regards,

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on behalf of

Dr. Nafiu Bala Sanda

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Comments.doc

    pone.0302522.s001.doc (24.5KB, doc)
    Attachment

    Submitted filename: Response to the Editors and Reviewers.doc

    pone.0302522.s002.doc (54KB, doc)

    Data Availability Statement

    The relevant data of this article has been submitted to the NCBI sequence read archive (accession number is PRJNA802701 https://www.ncbi.nlm.nih.gov/).

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