Next Article in Journal
An Evaluation of Suitable Habitats for Amur Tigers (Panthera tigris altaica) in Northeastern China Based on the Random Forest Model
Previous Article in Journal
Novel Covalent Modifier-Induced Local Conformational Changes within the Intrinsically Disordered Region of the Androgen Receptor
Previous Article in Special Issue
Dietary Canthaxanthin Supplementation Promotes the Laying Rate and Follicular Development of Huaixiang Hens
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biology
Volume 12
Issue 11
10.3390/biology12111443
biology-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus)

by
Su-Jeong Lee
1,†,
So Hee Kim
2,†,
Da-In Noh
1,
Young-Sun Lee
1,
Tae-Rim Kim
1,
Md Tawheed Hasan
3,
Eun-Woo Lee
1,4 and
Won Je Jang
1,4,*
1
Biopharmaceutical Engineering Major, Dong-Eui University, Busan 47340, Republic of Korea
2
Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science, Tongyeong 53085, Republic of Korea
3
Department of Aquaculture, Sylhet Agricultural University, Sylhet 3100, Bangladesh
4
Core-Facility Center for Tissue Regeneration, Dong-Eui University, Busan 47340, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2023, 12(11), 1443; https://doi.org/10.3390/biology12111443
Submission received: 13 October 2023 / Revised: 15 November 2023 / Accepted: 15 November 2023 / Published: 17 November 2023
(This article belongs to the Special Issue Application and Mechanism of Marine Bioactive Substances in Animal Nutrition)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Global concerns have been raised about the negative effects of antibiotic usage on aquaculture. Synthetic antibiotics have biological alternatives, namely, probiotics, which have the potential to improve fish growth, immunity, intestinal microbial population, and disease resistance. Host-associated probiotics are more adaptable and functional in the known intestinal environment, and two novel strains of Rummeliibacillus sp. and Microbacterium sp. were identified from the flounder (Paralichthys olivaceus) intestine. It is scientifically established that the effect of multiprobiotics was better than that of individual probiotics; as such, these two strains were administered in 1 × 108 CFU/g feed at a 50:50 ratio. At the end of the feeding experiment, the growth and feed utilization of the probiotic group (Pro) were higher than those of the control fish. Although serum biochemical parameters did not change, the immune parameter myeloperoxidase increased in the combined probiotic-fed group. These two strains also increased the beneficial microbial abundance in the intestine of the Pro group compared with that in the control fish. Therefore, the identification of the two novel probiotic strains from the flounder intestine has provided a basis for conducting further research, and their combination can be supplemented in commercial flounder cultures in future applications.

Abstract

Two novel strains of Rummeliibacillus sp. and Microbacterium sp. were identified from the intestine of olive flounder (Paralichthys olivaceus) and characterized in vitro as potential probiotics. Feeds without probiotic and with a 50:50 mixture of these two strains (1 × 108 CFU/g feed) were denoted as the control and Pro diets, respectively. Three randomly selected tanks (20 flounders/tank, ~11.4 g each) were used for each diet replication. After 8 weeks of feeding, the growth and feed utilization of the flounder in the Pro group improved (p < 0.05) compared to the control. Among four immune parameters, only myeloperoxidase activity was elevated in the Pro group. Serum biochemistry, intestinal microbial richness (Chao1), and diversity (Shannon index) remained unchanged (p ≥ 0.05), but phylogenetic diversity was enriched in the Pro fish intestine. Significantly lower Firmicutes and higher Proteobacteria were found in the Pro diet; the genus abundance in the control and Pro was as follows: Staphylococcus > Lactobacillus > Corynebacterium and Lactobacillus > Staphylococcus > Corynebacterium, respectively. Microbial linear discriminant scores and a cladogram analysis showed significant modulation. Therefore, the combination of two host-associated probiotics improved the growth and intestinal microbial population of flounder and could be supplemented in the Korean flounder industry.

1. Introduction

The olive flounder (Paralichthys olivaceus) aquaculture industry has intensified in the Republic of Korea, and it needs a considerable amount of feed supply, high stocking density, and frequent water exchange. According to the KOSIS [1], in 2021, the production of this fish was 41,776 metric tons, which was 46.7% of the total marine fish production of 89,423 metric tons. Intensive aquaculture has also resulted in environmental degradation, and changes in the physical, chemical, and biological parameters of culture environment have made this fish susceptible to infectious pathogens and other opportunistic bacteria that cause outbreaks of infectious diseases. Synthetic antibiotics and chemical chemotherapeutics are used to control these diseases, but this approach results in the accumulation of antibiotic residues in the fish body; consequently, these residues can be transmitted to humans, produce antibiotic-resistant pathogens, and cause a mass killing of harmful and beneficial bacteria [2,3]. Prevention is better than cure; aquaculture scientists worldwide have expressed their concern about the indiscriminate antibiotic usage and advised the application of probiotics as an alternative to antibiotics to increase immunity and thus prevent infectious diseases.
Probiotics are live microorganisms that confer a health benefit on the host when they are administered in adequate amounts [4]. They secrete antagonistic compounds to kill pathogens, exclude infectious bacteria by competing for space in the intestine, improve digestive enzyme activities, increase innate immune parameters, serve as a source of different nutrients, and improve disease protection [5]. Some intestinal commensal bacteria produce biodegradable antibiotics, namely, nisin and bacteriocin, to eliminate external pathogenic bacterial threats; they are also strong candidates for host-associated probiotic identification and characterization. Moreover, in light of aquaculture ecological parameters, water temperature, and salinity, as well as to ensure higher viability and functionality in a harsh intestinal environment, host-associated probiotics are more suitable than commercial probiotics. Probiotics modulate the intestinal microbial population and decrease the load of pathogenic bacteria; a well-balanced intestinal microbial population helps maintain the immune physiology and well-being of aquaculture species [6,7].
Two novel bacterial strains, Rummeliibacillus sp. and Microbacterium sp., were isolated and identified from healthy flounder intestine. These two potential host-associated probiotic bacteria successfully passed different in vitro characterization tests, namely, antibiotic susceptibility, hemolysis activity, HT-29 cell line cytotoxicity, gastric juice and bile acid viability, antagonistic nature, different enzyme secretion, and prebiotic utilization (data not published). In flounder research, apart from flounder host-associated probiotics, Hasan et al. [8] provided a dietary supplement with Bacillus SJ-10 from jeotgal, Cha et al. [9] used commercial B. subtilis or B. licheniformis for feeding, and Kim et al. [10] administered Lactococcus lactis BFE920 from sprouts to increase production, innate immunity, and protection against infectious diseases. Few studies have focused on flounder host-associated probiotic supplementation and its effect on this fish species [11,12]. The metabolites of one probiotic influence the growth, survival, and functionality of others; therefore, multiprobiotics instead of individual probiotics are increasingly used day by day in aquaculture ventures. However, studies have yet to quantify the combined effects of two flounder intestinal bacteria, Rummeliibacillus sp. and Microbacterium sp., after they are used as a dietary supplement for this species culture.
Therefore, the objective of this research was fixed as the dietary application of a 50:50 combination of these two potential flounder intestinal probiotics (Rummeliibacillus sp. and Microbacterium sp.) to quantify their combined effects on P. olivaceus growth, feed utilization, and immune responses. Moreover, any changes in the serum biochemical parameters and intestinal microbial population were also estimated.

2. Materials and Methods

2.1. Fish Diet Preparation

Fish diet composition, preparation, and preservation were performed in accordance with the methods of Hasan et al. [13]. Briefly, feed ingredients were weighed; then, fish oil and distilled water (300 mL/kg) were added and mixed thoroughly. Potential intestinal probiotic strains of Rummeliibacillus sp. and Microbacterium sp. were suspended in 300 mL of distilled water (1.67 × 108 CFU/mL/strain) to obtain stable 1 × 108 CFU of total 2 probiotics per gram of feed and formulate a probiotic-inoculated “Pro” diet. Diet without any probiotic inoculation was known as control feed. The prepared diets were subsequently air-dried at room temperature and stored in sealed plastic bags at 4 °C.

2.2. Fish Collection, Husbandry, and Feeding Trial

Juvenile P. olivaceus were obtained from a private hatchery; before the feeding trial was initiated, all the fish were fed with the control diet for 2 weeks to acclimatize to the experimental environment. At the beginning, ~11.4 g/flounder was stocked into six tanks; each tank contained 20 fish and had a volume of 60 L. Three tanks were randomly assigned as replicates for each of the control and Pro diets. Fish were fed to apparent satiation twice a day (10:00 and 16:00) for 8 weeks. Water quality was regularly monitored, and stable environmental parameters were maintained as temperature, 20.0 ± 0.5 °C; salinity, 31 ± 1 ppt; dissolved oxygen, 6.0 ± 0.5 mg/L; pH, 7.3 ± 0.3; and water flow, 1.2 L/min.

2.3. Sample Collection and Analysis

At the end of 8 weeks, the fish were starved for 24 h, and all surviving fish in the tanks were captured and weighed. Afterward, nine fish per diet group were anesthetized with 500 μL/L of 2-phenoxyethanol (Sigma-Aldrich, St. Louis, MO, USA), and blood was collected using nonheparinized and heparinized syringes for serum preparation (clotted blood centrifugation at 16,000× g for 10 min) and respiratory burst (RB) analysis, respectively.

2.4. Quantification of Growth, Feed Utilization, Innate Immunity, and Serum Biochemical Parameters

After the feeding experiment was completed, growth parameters (final body weight [FBW], weight gain [WG%], and specific growth rate [SGR%]) and feed utilization parameters (feed conversion ratio [FCR] and protein efficiency ratio [PER]) were calculated using the recognized scientific equations.
For innate immune parameters, superoxide dismutase (SOD) activity in the serum was quantified using a SOD kit (K335-100, BioVision, Milpitas, CA, USA) in accordance with the manufacturer’s instructions. Briefly, 20 μL serum was used to inhibit the superoxide anion produced by xanthine and xanthine oxidase on a water-soluble tetrazolium dye. The reaction endpoint was read at 450 nm after 20 min reaction at 37 °C, and percentage inhibitions were calculated following the equations mentioned in the kit.
Serum myeloperoxidase (MPO) activity was estimated as described by Quade and Roth [14] with minor modifications. Initially, in a 96-well plate, 20 μL serum was diluted in 80 μL of Hanks’ balanced solution and then 35 μL of 20 mM 3,3′,5,5′-tetramethylbenzidine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 35 μL of 5 mM H2O2 were added to each well. After 2 min of incubation, 35 μL of 4 M H2SO4 was added to stop the color change and the absorbance read at 450 nm.
The generation of oxidative radicals by neutrophils involved in phagocytic activity during RB in flounder blood was measured using the nitroblue tetrazolium assay, as described by Anderson and Siwicki [15]. Serum antiprotease (AP) levels were estimated using the method outlined by Heo et al. [11].
Total glucose (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were quantified using specific kits in BS-390 chemistry analyzer (Mindray Bio-Medical Electronics, Shenzhen, China) at the Core-Facility Center for Tissue Regeneration, Dong-Eui University (Busan, Republic of Korea).

2.5. Intestinal Microbiome Analysis

In accordance with the described method of Jang et al. [16], flounder intestinal microbiome analysis was performed using the whole intestine of nine fish per group. Briefly, total DNA was extracted using a PowerSoil DNA isolation kit (MoBio, Carlsbad, CA, USA), and its quality and quantity were measured with a NanoDrop spectrophotometer. MiSeq sequencing (Illumina, San Diego, CA, USA) and library construction were performed by the company TheMOAGEN Co., Ltd. (Daejeon, Republic of Korea). Primers were designed to target the V3 to V4 regions of the 16S rRNA sequence. Raw sequences were filtered for quality, denoised, and merged; chimeras were removed using the q2-dada2 plugin [17]. The differential abundance of microbial taxa in the intestine because of dietary difference was estimated via linear discriminant analysis effect size (LEfSe) analysis. A cladogram was prepared to illustrate the abundant taxa between Pro and control group fish.

2.6. Statistical Analysis

The normality and homogeneity of variance of the dataset were assessed using Shapiro–Wilk and Levene tests, respectively. Data were then subjected to one-way ANOVA using the IBM SPSS software (SPSS Inc., version 17.0, Chicago, IL, USA) following Student’s t-test. Results with p < 0.05 were considered statistically significant, and means are presented as mean ± standard deviation.

3. Results

3.1. Effects of the Combined Probiotic Strains on Growth, Feed Utilization, Innate Immunity, and Serum Biochemical Parameters

After 8 weeks of feeding, the diets inoculated with novel Rummeliibacillus sp. and Microbacterium sp. strains (Pro diet) positively influenced the growth parameters of olive flounder by significantly improving FBW, WG, and SGR compared with those of the control (Table 1).
Similarly, feed utilization parameters, such as FCR and PER, were modulated (p < 0.05) in the Pro group compared with those in the nonprobiotic diet-fed group.
No changes (p ≥ 0.05) were detected in serum biochemical parameters, such as ALT, AST, TG, and TC, between the Pro and control groups. Similarly, innate immune parameters, such as SOD, RB, and AP, remained unchanged after 8 weeks of feeding with the Pro diet. However, the MPO significantly increased in the Pro group compared with that in the control group (Table 2).

3.2. Effects of Combined Probiotics on the Alpha and Beta Diversity of the Intestinal Microbiota

After 8 weeks of feeding with the Pro diet, the observed species richness (Chao1 index) and diversity (Shannon index) remained unchanged (p ≥ 0.05) relative to the control. However, phylogenetic diversity (PD) was significantly higher in the combined host-associated potential probiotic-supplemented fish intestine than in the control intestine (Figure 1C).
Principal coordinate analysis (PCoA) based on unweighted unifrac metrics was used to analyze beta diversity (Figure 1E,F). The Pro and control intestinal microbiota differed, but the microorganisms in each group were closely associated.

3.3. Modulation of the Relative Abundance of Microbiota at Phylum and Genus Levels

At the phylum level, Firmicutes was the most abundant in the intestine of the control flounder, followed by Proteobacteria and Actinobacteria (Figure 2A). However, the abundance of Firmicutes was significantly lower and the abundance of Proteobacteria was higher in the intestine of the Pro group than in the intestine of the control group.
The relative abundance of the genus in the control group exhibited the following order: Staphylococcus > Lactobacillus > Corynebacterium; in the Pro group, the order was Lactobacillus > Staphylococcus > Corynebacterium. The abundance of Lactobacillus was significantly higher, and the abundance of Staphylococcus was significantly lower in the intestine of the fish fed with combined probiotics than in the intestine of the control fish (Figure 2B).
The LDA scores of Lactobacillaceae, Proteobacteria, and Lactobacillus were > 4.8 in the Pro group; by comparison, Firmicutes, Staphylococcaceae, Staphylococcales, and Staphylococcus scores were > −4.8, which indicated the difference in the abundance of microbiota at the taxon level (Figure 3A). The cladogram (Figure 3B) demonstrated significant variations; that is, Firmicutes and Bacilli were dominant in the intestine of the control group, whereas Actinobacteria, Actinobacteriota, Proteobacteria, and Planctomyetes were dominant in the Pro group.

4. Discussion

Because of low pH and presence of different digestive and metabolic enzymes, the fish intestinal environment is challenging for maintaining the viability and functionality of supplemented probiotics. Thus far, different probiotics are provided as dietary supplements in commercially cultured species, and they are efficient in modulating growth, immunity, and intestinal microbial population. The combination of two or more probiotics is supplemented in aquaculture to achieve a synergistic effect because microbial metabolites produced by one probiotic may influence the performance of the other; as a result, the effect of combined probiotics is stronger than that of individual probiotics [18]. Nayak [19] mentioned that the survival and activity of the sustained microbial community of the gastrointestinal tract of fish fed with multispecies probiotics are higher than those of fish fed with single-species probiotics.
In this research, equal concentrations of the Pro diet inoculated with host-associated Rummeliibacillus sp. and Microbacterium sp. improved the growth and feed utilization of olive flounder. These types of positive parameter modulations were previously detected after dietary administration with multiprobiotics composed of Bacillus subtilis and B. licheniformis in Nile tilapia (Oreochromis niloticus) [20]; Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp., and Pediococcus acidilactici in rainbow trout (Oncorhynchus mykiss) [21]; and Enterococcus faecium QAUEF01 and Geotrichum candidum QAUGC01 in rohu fish (Labeo rohita) [22]. However, feeding a mixture of B. licheniformis SK3927, B. amyloliquefaciens SK4079, B. subtilis SK4082, L. brevis SK1751, L. plantarum SK3494, and Saccharomyces cerevisiae SK3587 has failed improve the growth and feed utilization of flounder [23]. Probiotics can produce different essential enzymes that can increase digestive enzyme activities in the intestine to accelerate diet digestion and acclimation, resulting in increased feed utilization and growth performance [24,25]. In this present study, the actual underlying mechanism of growth and feed utilization modulation by Rummeliibacillus sp. and Microbacterium sp. was unknown, and digestive enzymes were not quantified; however, both these novel strains produced six different enzymes in vitro (API test), which might increase the intestinal enzyme actions for the enhanced feed utilization, thereby improving growth performance.
ALT and AST are stress enzymes secreted from the liver to the blood stream when fish are exposed to toxicants and subjected to cellular degradation [26]. Under stress conditions, fish require energy that can be provided by glucose production; as a stress indicator, the presence of increased glucose levels in the blood indicates that fish is exposed to stress [27]. In this study, serum biochemical parameters, specifically ALT, AST, and TG, had no significant differences after the fish were fed with the combination of the two novel probiotic strains; therefore, these two potential probiotics had no toxic or harmful effects on the flounder culture. In fish, the innate immune system is composed of cellular components, humoral parameters, and epithelial or mucosal barrier [28]. Cytotoxic cells, macrophages, monocytes, natural killer cells, and neutrophils are considered phagocytic cells [29], and humoral innate immunity involves bacteriolytic or hemolytic enzymes [19] that nonspecifically kill or destroy invading pathogens. In this innate immune system, phagocytic cells become activated during RB, and consumed oxygen is converted to superoxide (O2−), and MPO transforms O2− to hypochlorous acid (HClO) [30]. O2− and HClO are detrimental to invading infectious pathogens and involved in their elimination from the fish body [31]. In the present study, MPO in the Pro diet-fed flounder was significantly higher than that in the control fish, indicating that the immunological status of the Pro-fed fish was better than that of the nonprobiotic-fed group. The dietary application of probiotics improves immunological status [8,23,32,33] and does not elicit harmful effects [13,34,35,36] on different commercially cultured species, including flounder, thus supporting the current findings.
Pérez-Sánchez et al. [5] indicated that the intestinal microbial community of fish plays a vital role in host nutrient digestion, absorption, and assimilation, as well as metabolism, physiological status, pathological defense, and innate immune system activation. The fish intestinal microbiome can be modulated by temperature, environment, diet composition and dietary supplements, and different developmental stages [37]. In the present study, the 50:50 combination of two potential probiotics was unable to change the intestinal microbial diversity, richness, and observation, but phylogenetic diversity in the Pro group was improved. Differences between the Pro and control diets were observed through using PCoA, and the microbiota of the individual feeding group were closely associated. Beta diversity normally estimates the community similarity or distance between/among samples; in the present study, closeness in the Pro diet group may be an indicator of microbial implantation through Rummeliibacillus sp. and Microbacterium sp. supplementation. Moreover, the significant decrease in Firmicutes and the significant increase in Proteobacteria at the phylum level of the Pro fish intestine validated the phylogenetic diversity estimated by using the alpha diversity analysis. Firmicutes and Proteobacteria are the most abundant phyla in cultured and wild fine flounder (P. adspersus), respectively [38]. Dietary application with Bacillus ap. SJ-10 probiotic increases the Proteobacteria concentration compared with that in the control P. olivaceus [39]. Beneficial Lactobacillus concentrations increased in Pro-fed fish by decreasing the concentration of harmful Staphylococcus compared with those in the nonprobiotic-supplemented fish. Staphylococcus may cause soft skin infections, such as abscesses, furuncles, and cellulites, and S. aureus causes infection in the blood [40]. In the present study, the abundance of microbial taxa (LDA score) and the presence of Actinobacteria and Proteobacteria (cladogram observation) were higher in the treatment. Actinobacteria are known to produce different secondary metabolites [41,42]. The increased proportions of natural antibiotics (nisin and bacteriocin) producing Lactobacillus in the intestine of probiotic-fed fish were consistent with these phylum-level findings. Additionally, it is not confirmed, but the combination of Rummeliibacillus sp. and Microbacterium sp. might make the intestinal environment favorable to colonizing different microbes, thereby increasing taxon abundance in Pro-fed fish intestine. The application of probiotics through diet positively improves the beneficial bacteria in the intestinal microbial community of commercially cultured fish species [32,43,44,45], and this observation is consistent with the present findings.

5. Conclusions

This research demonstrates that the combination of two flounder host-associated probiotics positively affected the different aspects of this species. The increase in feed utilization with these novel strains means less feed application for increased production, resulting in farmers’ profitability. Changes in some innate immune parameters are likely to improve disease prevention, ultimately eliminating antibiotics from the flounder culture. Moreover, these host-associated strains can increase the beneficial bacteria in the intestine by decreasing pathogenic concentrations. In the future, these two probiotics may be supplemented in the Korean aquaculture industry to resolve the current problems. However, a small-scale earthen pond experiment may be needed to observe variations in laboratory and field experiments before these probiotics can be applied in large aquaculture ventures.

Author Contributions

S.-J.L.: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, and Writing—original draft. S.H.K.: Conceptualization, Methodology, Project administration, and Writing—review and editing. D.-I.N.: Conceptualization, Methodology, Investigation, Formal analysis, and Data curation. Y.-S.L.: Conceptualization, Methodology, Investigation, Formal analysis, and Data curation. T.-R.K.: Methodology, Investigation, and Formal analysis. M.T.H.: Data curation and Writing—original draft. E.-W.L.: Conceptualization, Methodology, Validation, and Writing—review and editing. W.J.J.: Conceptualization, Methodology, Project administration, and Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from the National Institute of Fisheries Science (R2023046) and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A1A01049238).

Institutional Review Board Statement

All experimental activities were performed in accordance with the rules and protocols of the Animal Ethics Committee (Approval No. R2022-031) at Dong-Eui University, Korea.

Informed Consent Statement

No applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Korean Statistical Information Service (KOSIS). Survey on the Status of Fish Culture. 2021. Available online: http://kosis.kr/ (accessed on 24 March 2023).
  2. Lee, W.; Ahn, G.; Oh, J.Y.; Kim, S.M.; Kang, N.; Kim, E.A.; Kim, K.N.; Jeong, J.B.; Jeon, Y.J. A Prebiotic Effect of Ecklonia cava on the Growth and Mortality of Olive Flounder Infected with Pathogenic Bacteria. Fish Shellfish. Immunol. 2016, 51, 313–320. [Google Scholar] [CrossRef] [PubMed]
  3. Beck, B.R.; Kim, D.; Jeon, J.; Lee, S.M.; Kim, H.K.; Kim, O.J.; Lee, J.I.; Suh, B.S.; Do, H.K.; Lee, K.H.; et al. The Effects of Combined Dietary Probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on Innate Immunity and Disease Resistance in Olive Flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2015, 42, 177–183. [Google Scholar] [CrossRef] [PubMed]
  4. Food and Agricultural Organization; World Health Organization. Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria. In A Joint FAO/WHO Expert Consultation Report; Food and Agricultural Organization; World Health Organization: Cordoba, Argentina, 2001; pp. 1–34. [Google Scholar]
  5. Pérez-Sánchez, T.; Ruiz-Zarzuela, I.; de Blas, I.; Balcázar, J.L. Probiotics in Aquaculture: A Current Assessment. Rev. Aquac. 2014, 6, 133–146. [Google Scholar] [CrossRef]
  6. Lee, C.; Cha, J.H.; Kim, M.G.; Shin, J.; Woo, S.H.; Kim, S.H.; Kim, J.W.; Ji, S.C.; Lee, K.J. The Effects of Dietary Bacillus subtilis on Immune Response, Hematological Parameters, Growth Performance, and Resistance of Juvenile Olive Flounder (Paralichthys olivaceus) Against Streptococcus iniae. J. World Aquac. Soc. 2020, 51, 551–562. [Google Scholar]
  7. Kim, H.N.; Yun, Y.; Ryu, S.; Chang, Y.; Kwon, M.J.; Cho, J.; Shin, H.; Kim, H.L. Correlation between Gut Microbiota and Personality in Adults: A Cross-Sectional Study. Brain Behav. Immun. 2018, 69, 374–385. [Google Scholar]
  8. Hasan, M.T.; Jang, W.J.; Kim, H.; Lee, B.J.; Kim, K.W.; Hur, S.W.; Lim, S.G.; Bai, S.C.; Kong, I.S. Synergistic Effects of Dietary Bacillus sp. SJ-10 Plus β-glucooligosaccharides as a Synbiotic on Growth Performance, Innate Immunity and Streptococcosis Resistance in Olive Flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2018, 82, 544–553. [Google Scholar] [CrossRef]
  9. Cha, J.H.; Rahimnejad, S.; Yang, S.Y.; Kim, K.W.; Lee, K.J. Evaluations of Bacillus spp. as Dietary Additives on Growth Performance, Innate Immunity and Disease Resistance of Olive Flounder (Paralichthys olivaceus) Against Streptococcus iniae and as Water Additives. Aquaculture 2013, 402, 50–57. [Google Scholar] [CrossRef]
  10. Kim, D.; Beck, B.R.; Heo, S.B.; Kim, J.; Kim, H.D.; Lee, S.M.; Kim, Y.; Oh, S.Y.; Lee, K.; Do, H.; et al. Lactococcus lactis BFE920 Activates the Innate Immune System of Olive Flounder (Paralichthys olivaceus), Resulting in Protection Against Streptococcus iniae Infection and Enhancing Feed Efficiency and Weight Gain in Large-scale Field Studies. Fish Shellfish Immunol. 2013, 35, 1585–1590. [Google Scholar] [CrossRef]
  11. Heo, W.S.; Kim, Y.R.; Kim, E.Y.; Bai, S.C.; Kong, I.S. Effects of Dietary Probiotic, Lactococcus lactis subsp. lactis I2, Supplementation on the Growth and Immune Response of Olive Flounder (Paralichthys olivaceus). Aquaculture 2013, 376, 20–24. [Google Scholar] [CrossRef]
  12. Niu, K.M.; Khosravi, S.; Kothari, D.; Lee, W.D.; Lee, B.J.; Lim, S.G.; Hur, S.W.; Lee, S.M.; Kim, S.K. Potential of Indigenous Bacillus spp. as Probiotic Feed Supplements in an Extruded Low-Fish-Meal Diet for Juvenile Olive Flounder, Paralichthys olivaceus. J. World Aquac. Soc. 2021, 52, 244–261. [Google Scholar] [CrossRef]
  13. Hasan, M.T.; Jang, W.J.; Lee, B.J.; Hur, S.W.; Lim, S.G.; Kim, K.W.; Han, H.S.; Lee, E.W.; Bai, S.C.; Kong, I.S. Dietary Supplementation of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 Combinations Enhance Growth and Cellular and Humoral Immunity in Olive Flounder (Paralichthys olivaceus). Probiotics Antimicrob. 2021, 13, 1277–1291. [Google Scholar] [CrossRef] [PubMed]
  14. Quade, M.J.; Roth, J.A. A Rapid, Direct Assay to Measure Degranulation of Bovine Neutrophil Primary Granules. Vet. Immunol. 1997, 58, 239–248. [Google Scholar] [CrossRef] [PubMed]
  15. Anderson, D.P.; Siwicki, A.K. Basic hematology and serology for fish health programs. In Diseases in Asian aquaculture II; Shariff, M., Arthur, J.R., Subasinghe, R.P., Eds.; Philippines Fish Health Section, Asian Fisheries Society: Manila, Philippines, 1995; pp. 185–202. [Google Scholar]
  16. Jang, W.J.; Hasan, M.T.; Lee, B.J.; Hur, S.W.; Lee, S.; Kim, K.W.; Lee, E.W.; Kong, I.S. Effect of Dietary Differences on Changes of Intestinal Microbiota and Immune-related Gene Expression in Juvenile Olive Flounder (Paralichthys olivaceus). Aquaculture 2020, 527, 735442. [Google Scholar]
  17. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-resolution Sample Inference from Illumina Amplicon Data. Nat. Methods 2016, 13, 581–583. [Google Scholar] [CrossRef] [PubMed]
  18. Salinas, I.; Abelli, L.; Bertoni, F.; Picchietti, S.; Roque, A.; Furones, D.; Cuesta, A.; Meseguer, J.; Esteban, M.Á. Monospecies and Multispecies Probiotic Formulations Produce Different Systemic and Local Immunostimulatory Effects in the Gilthead Seabream (Sparus aurata L.). Fish Shellfish Immunol. 2008, 25, 114–123. [Google Scholar] [CrossRef] [PubMed]
  19. Nayak, S.K. Probiotics and Immunity: A Fish Perspective. Fish Shellfish Immunol. 2010, 29, 2–14. [Google Scholar] [CrossRef]
  20. Abarike, E.D.; Jian, J.; Tang, J.; Cai, J.; Yu, H.; Lihua, C.; Jun, L. Influence of Traditional Chinese Medicine and Bacillus species (TCMBS) on Growth, Immune Response and Disease Resistance in Nile Tilapia, Oreochromis niloticus. Aquac. Res. 2018, 49, 2366–2375. [Google Scholar] [CrossRef]
  21. Pérez-Sánchez, T.; Balcázar, J.L.; Merrifield, D.L.; Carnevali, O.; Gioacchini, G.; de Blas, I.; Ruiz-Zarzuela, I. Expression of Immune-related Genes in Rainbow Trout (Oncorhynchus mykiss) Induced by Probiotic Bacteria During Lactococcus garvieae Infection. Fish Shellfish Immunol. 2011, 31, 196–201. [Google Scholar] [CrossRef]
  22. Ghori, I.; Tabassum, M.; Ahmad, T.; Zuberi, A.; Imran, M. Geotrichum candidum Enhanced the Enterococcus faecium Impact in Improving Physiology and Health of Labeo rohita (Hamilton, 1822) by Modulating Gut Microbiome Under Mimic Aquaculture Conditions. Turkish J. Fish. Aquat. Sci. 2018, 18, 1255–1267. [Google Scholar]
  23. Harikrishnan, R.; Kim, M.C.; Kim, J.S.; Balasundaram, C.; Heo, M.S. Probiotics and Herbal Mixtures Enhance the Growth, Blood Constituents and Nonspecific Immune Response in Paralichthys olivaceus against Streptococcus parauberis. Fish Shellfish Immunol. 2011, 31, 310–317. [Google Scholar]
  24. Hasan, M.T.; Kim, H.J.; Hur, S.W.; Jeong, S.M.; Kim, K.W.; Lee, S. Dietary Exogenous α-Amylase Modulates the Nutrient Digestibility, Digestive Enzyme Activity, Growth-Related Gene Expression and Diet Degradation Rate of Olive Flounder (Paralichthys olivaceus). J. Microbiol. Biotechnol. 2023, 33, 1390. [Google Scholar]
  25. Akbari Nargesi, E.; Falahatkar, B.; Sajjadi, M.M. Dietary Supplementation of Probiotics and Influence on Feed efficiency, Growth Parameters and Reproductive Performance in Female Rainbow Trout (Oncorhynchus mykiss) Broodstock. Aquac. Nutr. 2020, 26, 98–108. [Google Scholar] [CrossRef]
  26. Rahimnejad, S.; Guardiola, F.A.; Leclercq, E.; Esteban, M.Á.; Castex, M.; Sotoudeh, E.; Lee, S.M. Effects of Dietary Supplementation with Pediococcus acidilactici MA18/5M, Galactooligosaccharide and Their Synbiotic on Growth, Innate Immunity and Disease Resistance of Rockfish (Sebastes schlegeli). Aquaculture 2018, 482, 36–44. [Google Scholar] [CrossRef]
  27. Fira, M.D.; Santanumurti, M.B.; Jamal, M.T.; Muttaqin, A.; Subekti, S.; Sari, P.D.W. Blood Glucose Profile as a Rapid Method for Observing Koi Carp (Cyprinus carpio) Health Status-Case Study of Ectoparasites in Blitar, Indonesia. Rev. Bras. DE Parasitol. Vet. 2023, 32, e014622. [Google Scholar]
  28. Akhter, N.; Wu, B.; Memon, A.M.; Mohsin, M. Probiotics and Prebiotics Associated with Aquaculture: A Review. Fish Shellfish Immunol. 2015, 45, 733–741. [Google Scholar] [PubMed]
  29. Dobaño, C.; Moncunill, G. Naturally Acquired Immunity (NAI). In Encyclopedia of Malaria; Springer: New York, NY, USA, 2018; pp. 1–15. [Google Scholar]
  30. Klebanoff, S.J. Myeloperoxidase: Friend and Foe. J. Leukoc. Biol. 2005, 77, 598–625. [Google Scholar]
  31. Vallejos-Vidal, E.; Reyes-López, F.; Teles, M.; MacKenzie, S. The Response of Fish to Immunostimulant Diets. Fish Shellfish Immunol. 2016, 56, 34–69. [Google Scholar]
  32. Tan, H.Y.; Chen, S.W.; Hu, S.Y. Improvements in the Growth Performance, Immunity, Disease Resistance, and Gut Microbiota by the Probiotic Rummeliibacillus stabekisii in Nile Tilapia (Oreochromis niloticus). Fish Shellfish Immunol. 2019, 92, 265–275. [Google Scholar] [CrossRef]
  33. Li, Y.; Hu, S.; Gong, L.; Pan, L.; Li, D.; Cao, L.; Khan, T.A.; Yang, Y.; Peng, Y.; Ding, X.; et al. Isolating a New Streptomyces amritsarensis N1-32 Against Fish Pathogens and Determining Its Effects on Disease Resistance of Grass Carp. Fish Shellfish Immunol. 2020, 98, 632–640. [Google Scholar] [CrossRef]
  34. Won, S.; Hamidoghli, A.; Choi, W.; Park, Y.; Jang, W.J.; Kong, I.S.; Bai, S.C. Effects of Bacillus subtilis WB60 and Lactococcus lactis on Growth, Immune Responses, Histology and Gene Expression in Nile Tilapia, Oreochromis niloticus. Microorganisms 2020, 8, 67. [Google Scholar] [CrossRef]
  35. Ahmadifar, E.; Sadegh, T.H.; Dawood, M.A.; Dadar, M.; Sheikhzadeh, N. The Effects of Dietary Pediococcus pentosaceus on Growth Performance, Hemato-immunological Parameters and Digestive Enzyme Activities of Common Carp (Cyprinus carpio). Aquaculture 2020, 516, 734656. [Google Scholar] [CrossRef]
  36. Park, Y.; Lee, S.; Hong, J.; Kim, D.; Moniruzzaman, M.; Bai, S.C. Use of Probiotics to Enhance Growth, Stimulate Immunity and Confer Disease Resistance to Aeromonas salmonicida in Rainbow Trout (Oncorhynchus mykiss). Aquac. Res. 2017, 48, 2672–2682. [Google Scholar] [CrossRef]
  37. Qiao, G.; Xu, C.; Sun, Q.; Xu, D.H.; Zhang, M.; Chen, P.; Li, Q. Effects of Dietary poly-β-hydroxybutyrate Supplementation on the Growth, Immune Response and Intestinal Microbiota of Soiny Mullet (Liza haematocheila). Fish Shellfish Immunol. 2019, 91, 251–263. [Google Scholar] [PubMed]
  38. Ramírez, C.; Romero, J. Fine Flounder (Paralichthys adspersus) Microbiome Showed Important Differences Between Wild and Reared Specimens. Front. Microbiol. 2017, 8, 271. [Google Scholar] [CrossRef] [PubMed]
  39. Jang, W.J.; Lee, J.M.; Hasan, M.T.; Lee, B.J.; Lim, S.G.; Kong, I.S. Effects of Probiotic Supplementation of a Plant-based Protein Diet on Intestinal Microbial Diversity, Digestive Enzyme Activity, Intestinal Structure, and Immunity in Olive Flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2019, 92, 719–727. [Google Scholar]
  40. Rasigade, J.P.; Vandenesch, F. Staphylococcus aureus: A Pathogen with Still Unresolved Issues. Infect. Genet. Evol. 2014, 21, 510–514. [Google Scholar] [CrossRef]
  41. Wu, S.; Wang, G.; Angert, E.R.; Wang, W.; Li, W.; Zou, H. Composition, Diversity, and Origin of the Bacterial Community in Grass Carp Intestine. PLoS ONE 2012, 7, e30440. [Google Scholar] [CrossRef]
  42. Ventura, M.; Canchaya, C.; Tauch, A.; Chandra, G.; Fitzgerald, G.F.; Chater, K.F.; Van Sinderen, D. Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum. Microbiol. Mol. Biol. Rev. 2007, 71, 495–548. [Google Scholar]
  43. Niu, K.M.; Khosravi, S.; Kothari, D.; Lee, W.D.; Lim, J.M.; Lee, B.J.; Kim, K.W.; Lim, S.G.; Lee, S.M.; Kim, S.K. Effects of Dietary Multi-strain Probiotics Supplementation in a Low Fishmeal Diet on Growth Performance, Nutrient Utilization, Proximate Composition, Immune Parameters, and Gut Microbiota of Juvenile Olive Flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2019, 93, 258–268. [Google Scholar]
  44. Sahandi, J.; Jafaryan, H.; Soltani, M.; Ebrahimi, P. The Use of Two Bifidobacterium Strains Enhanced Growth Performance and Nutrient Utilization of Rainbow Trout (Oncorhynchus mykiss) Fry. Probiotics Antimicrob. 2019, 11, 966–972. [Google Scholar] [CrossRef]
  45. Merrifield, D.L.; Dimitroglou, A.; Bradley, G.; Baker, R.T.M.; Davies, S.J. Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) II. Effects on Growth Performance, Feed Utilization, Intestinal Microbiota and Related Health Criteria. Aquac. Nutr. 2010, 16, 504–510. [Google Scholar] [CrossRef]
Biology 12 01443 g001
Figure 1. Alpha (A–D) and beta (E,F) diversity of the intestinal microbiota of olive flounder fed with the control and probiotic-inoculated diets for 8 weeks. Beta diversity as a principal coordinate analysis (PCoA) was performed on the basis of unweighted unifrac metrics. * p < 0.05.
Figure 1. Alpha (A–D) and beta (E,F) diversity of the intestinal microbiota of olive flounder fed with the control and probiotic-inoculated diets for 8 weeks. Beta diversity as a principal coordinate analysis (PCoA) was performed on the basis of unweighted unifrac metrics. * p < 0.05.
Biology 12 01443 g001
Biology 12 01443 g002
Figure 2. Relative abundance of the intestinal bacterial communities of olive flounder at the phylum (A) and genus (B) levels after supplementation with the control and probiotic-inoculated diets for 8 weeks.
Figure 2. Relative abundance of the intestinal bacterial communities of olive flounder at the phylum (A) and genus (B) levels after supplementation with the control and probiotic-inoculated diets for 8 weeks.
Biology 12 01443 g002
Biology 12 01443 g003
Figure 3. Linear discriminant analysis effect size (LEfSe) analysis of the differential abundance of taxa within the olive flounder intestinal microbiota following random sampling from each group. (A) Linear discriminant analysis (LDA) score of the abundance of taxa; (B) cladogram showing differentially abundant taxa of phylum to genus between the two groups.
Figure 3. Linear discriminant analysis effect size (LEfSe) analysis of the differential abundance of taxa within the olive flounder intestinal microbiota following random sampling from each group. (A) Linear discriminant analysis (LDA) score of the abundance of taxa; (B) cladogram showing differentially abundant taxa of phylum to genus between the two groups.
Biology 12 01443 g003
Table 1. Growth performance and feed utilization of olive flounder supplemented with experimental feed additives.
Table 1. Growth performance and feed utilization of olive flounder supplemented with experimental feed additives.
GroupsGrowth Performance, Feed Utilization, and Organosomatic Parameters
FBW (g)WG (%)SGR (% day−1)FCRPER
Con31.55 ± 0.50 a192.95 ± 6.79 a1.92 ± 0.04 a1.23 ± 0.04 b1.60 ± 0.08 a
Pro35.98 ± 1.05 b233.31 ± 6.80 b2.15 ± 0.04 b1.02 ± 0.04 a1.93 ± 0.11 b
Values are mean ± SD of three replicates. Values with different superscript letters within the same column in the table are significantly different (p < 0.05). FBW, Final body weight = Final weight of total fish in tank/Fish number; WG, Weight gain = [(Final weight − Initial weight)/Initial weight] × 100; SGR, Specific growth rate = [(ln final weight − ln initial weight)/days] × 100; FCR, Feed conversion ratio = Dry feed intake/Wet body weight gain; PER, Protein efficiency ratio =Wet weight gain/Protein fed.
Table 2. Serum biochemical and nonspecific immune parameters of olive flounder supplemented with experimental feed additives.
Table 2. Serum biochemical and nonspecific immune parameters of olive flounder supplemented with experimental feed additives.
GroupsSerum Biochemical Parameters
ALTASTTGTC
Con108.33 ± 14.5746.33 ± 5.6916.67 ± 2.08147.33 ± 12.22
Pro102.33 ± 16.2645.33 ± 3.2117.00 ± 3.61138.67 ± 9.71
GroupsNonspecific immune parameters
SODRBMPOAP
Con37.11 ± 5.380.52 ± 0.030.46 ± 0.07 a72.33 ± 6.81
Pro41.68 ± 4.830.68 ± 0.111.19 ± 0.22 b81.67 ± 9.07
Values are mean ± SD of three replicates. Values with different superscript letters within the same column in the table are significantly different (p < 0.05). No superscript letter indicates no significant difference (p > 0.05). ALT, Alanine aminotransferase (U/L); AST, Aspartate aminotransferase (U/L); TG, Total glucose (mg/dL); TC, Total cholesterol (mg/dL); SOD, Superoxide dismutase (% superoxide inhibition); RB, Respiratory burst (absorbance at 540 nm); MPO, Myeloperoxidase activity (absorbance at 450 nm); AP, Antiprotease activity (% of trypsin inhibition).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Lee, S.-J.; Kim, S.H.; Noh, D.-I.; Lee, Y.-S.; Kim, T.-R.; Hasan, M.T.; Lee, E.-W.; Jang, W.J. Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus). Biology 2023, 12, 1443. https://doi.org/10.3390/biology12111443

AMA Style

Lee S-J, Kim SH, Noh D-I, Lee Y-S, Kim T-R, Hasan MT, Lee E-W, Jang WJ. Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus). Biology. 2023; 12(11):1443. https://doi.org/10.3390/biology12111443

Chicago/Turabian Style

Lee, Su-Jeong, So Hee Kim, Da-In Noh, Young-Sun Lee, Tae-Rim Kim, Md Tawheed Hasan, Eun-Woo Lee, and Won Je Jang. 2023. "Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus)" Biology 12, no. 11: 1443. https://doi.org/10.3390/biology12111443

APA Style

Lee, S. -J., Kim, S. H., Noh, D. -I., Lee, Y. -S., Kim, T. -R., Hasan, M. T., Lee, E. -W., & Jang, W. J. (2023). Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus). Biology, 12(11), 1443. https://doi.org/10.3390/biology12111443

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop