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Paenibacillus polymyxa improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus
Journal Pre-proof Paenibacillus polymyxa, improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus. Kwaku Amoah, Qin-cheng Huang, Xiao-hui Dong, Bei-ping Tan, Shuang Zhang, Shuyan Chi, Qi-hui Yang, Hong-yu Liu, Yuan-zhi Yang PII:
S0044-8486(19)30903-2
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734563
Reference:
AQUA 734563
To appear in:
Aquaculture
Received Date: 16 April 2019 Revised Date:
1 September 2019
Accepted Date: 1 October 2019
Please cite this article as: Amoah, K., Huang, Q.-c., Dong, X.-h., Tan, B.-p., Zhang, S., Chi, S.-y., Yang, Q.-h., Liu, H.-y., Yang, Y.-z., Paenibacillus polymyxa, improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus., Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734563. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Paenibacillus polymyxa, improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus.
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Kwaku Amoahab, Qin-cheng Huangab, Xiao-hui Dongabc*, Bei-ping Tanabc*, Shuang Zhangabc,
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Shu-yan Chiabc, Qi-hui Yangabc, Hong-yu Liuabc, Yuan-zhi Yanga
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a
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Zhanjiang, Guangdong 524088, PR China
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b
Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University,
Aquatic Animals Precision Nutrition and High-Efficiency Feed Engineering Research Centre of Guangdong
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Province, Zhanjiang, Guangdong 524088, PR China
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c
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Agriculture, Zhanjiang, Guangdong 524000, China
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* Correspondence: X-H Dong, Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries,
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Guangdong Ocean University, Zhanjiang, Guangdong 524088, China.
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E-mail address: [email protected]
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* Correspondence: B-P Tan, Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries,
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Guangdong Ocean University, Zhanjiang, Guangdong 524088, China.
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E-mail address: [email protected]
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Key Laboratory of Aquatic, Livestock and Poultry Feed Science and Technology in South China, Ministry of
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ABSTRACT
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This present study was performed to examine the effects of different levels of dietary
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Paenibacillus polymyxa ATCC 842 (PP) on the growth, immune response, antioxidant activities,
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intestinal morphology and microbiota, and defense against Vibrio parahaemolyticus (VP)
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infection in white leg shrimp, Litopeneaus vannamei. Shrimps (initial weight of 0.58 ± 0.001g)
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were fed diet containing 0 (control, PP0), 106 (PP1), 107 (PP2) and 108 (PP3) cfu g-1 P. polymyxa.
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After 8 weeks feeding trial, shrimps fed the PP treated diet displayed a synergistic effect which
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significantly enhanced (P < 0.05) the final body weight, survival rate, weight gain rate, specific
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growth rate, protein efficiency ratio, condition factor; total protein, albumin, globulin,
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triglyceride, lysozyme, total antioxidant capacity, superoxide dismutase, acid phosphatase
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activity in the serum; alkaline phosphatase activity in the hepatopancreas; glutathione,
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glutathione peroxidase activity in both the serum and hepatopancreas, with decreased feed
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conversion rate; aspartate aminotransferase, alanine aminotransferase, and malondialdehyde
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levels in both serum and hepatopancreas compared to the control group (PP0). Correspondingly,
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the probiotic-treated group experienced significantly improved (P < 0.05) mid-intestinal
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morphological structures such as villus height, villus width, muscle thickness and digestive
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enzyme activities including amylase, trypsin, and lipase than the untreated group with the PP3
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group obtaining the highest. Dietary PP supplementation in diets again was observed to change
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the shrimps’ intestinal microbial composition. Explicitly, the most dominant bacterial phyla,
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Proteobacteria, Bacteriodetes, and Planctomycetes observed in this study were significantly
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higher (P < 0.05) in the probiotic-enriched group compared to the control. At the genus level, the
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relative
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Tenacibaculum, and Shewanella) were significantly decreased (P < 0.05), whereas beneficial
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bacteria (Ruegeria and Pseudoalteromonas) were significantly enhanced (P < 0.05) in the
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probiotic-treated group than the untreated. Additionally, dietary supplementation of PP in L.
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vannamei’s diet significantly improved (P < 0.05) the protection against VP infections with PP3
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treatment group obtaining the highest relative percentage survival of 78.3 %. These results
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collectively suggest that dietary PP had a positive effect on the intestinal health of L. vannamei
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via the modulation of the microbial composition; thus, promoting the digestion and absorption of
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nutrients in boosting shrimps’ immunity. The optimal supplementation dosage in diets was found
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to be 108 cfu g-1 diet.
abundance
of
opportunistic
bacterial
pathogens
(Vibrio,
Photobacterium,
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KEYWORDS: Paenibacillus polymyxa ATCC 842, Litopenaeus vannamei, midgut, relative
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percentage survival, microbiota.
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1. Introduction
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Pacific white shrimps, Litopenaeus vannamei, owing to their higher benefits have been
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reported as the most cultured shrimp species (Chiu et al., 2007; FAO, 2012). Aquaculturists upon
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realizing the benefits these shrimp possess and also having the desire of meeting the increasing
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demand for shrimp food have shifted from the extensive system of culturing to the semi-
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intensive and intensive system causing challenges such as bacterial and viral disease infections
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as a result of the higher stocking densities (Bondad-Reantaso et al., 2005). Shrimps’
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susceptibility to bacterial, viral, fungal and parasitic infections are very wide. Bachère et al.
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(2004) in their work reported that shrimps lack adaptive immune features in removing pathogens
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hence are easily infected, causing poor growth and development, as well as high mortality. This
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has led researchers into finding lasting solutions to the disease menace to prevent the re-
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occurrence of the previously described disease outbreaks (Flegel, 2006; Lightner et al., 2012).
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Antibiotic usage in shrimp aquaculture, though proven to help combat these diseases by
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improving growth, feed utilization and disease resistance, have been critiqued because of their
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potential adverse effects on humans, the environment such as the development and spread of
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drug-resistant pathogens (Das et al., 2013), destroying of microbial populations in the
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aquaculture environment and the suppression of aquatic animal immune systems (Li et al., 2007;
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Zhou et al., 2009). Probiotics which have proven otherwise is being investigated due to the
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positive results it has concerning the immune response, antioxidant defense system, disease
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resistance and the gut microbiota (Spinler et al., 2008; Verschuere et al., 2000). Among the
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commercial probiotics being tested currently, Bacillus spp. have been noted as the fore of recent
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research in shrimp culture due to the colossal sum of beneficial properties they possess as
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compared to others (Hong et al., 2005; Verschuere et al., 2000). Paenibacillus polymyxa
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formerly recognized as Bacillus polymyxa is a Gram-positive, rod-shaped bacterium that
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distinctly swells its sporangium (Lal and Tabacchioni, 2009) with their spores able to resist harsh
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environmental conditions, including extreme pH, temperature, high pressure, UV irradiation,
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aridity and chemical infiltration (Huo et al., 2012). It is worth mentioning that their spores are
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known to produce a variety of beneficial bioactive substances including fusaricidins,
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antimicrobial polypeptides and polymyxin antibiotic compounds as a result of the presence of
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non-ribosomal peptide synthetase systems in them (Shaheen et al., 2011; Grady et al., 2016).
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Studies show P. polymyxa to be the source of dispase enzyme which is reported to be used in the
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isolation of tissues of animals (Ono et al., 1977; Stenn et al., 1989). The principal role of this
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wholesome strain P. polymyxa is described as the phytopathogen disease control in numerous
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crops and aquatic animals (Zhou et al., 2016; Gupta et al., 2016a). For example, P. polymyxa
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NSY50 was reported to effectively reduce Fusarium wilt (56.4%) by altering the soil
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physicochemical properties and all enzyme activities of the soil (Shi et al., 2017). It is
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noteworthy this strain is comparatively used as the last remedy for infections caused by multiple-
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drug-resistant, Pseudomonas aeruginosa (Velkov et al., 2013). Isolated surfactant from P.
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polymyxa has been reported to disrupt biofilms of Bacillus subtilis, Pseudomonas aeruginosa,
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Micrococcus luteus, Streptococcus bovis, and Staphylococcus aureus effectively (Quinn et al.,
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2012). P. polymyxa ATCC 842 bacterium has been noted to improve on the digestibility of
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nutrients and gut microflora in broiler chicken when added to fermented palm kernel cake
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(FPKC) (Alshelmani et al., 2016). Furthermore, reports of P. polymyxa has been noted as being
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able to improve the growth, enhance the immune and antioxidant activities of aquatic animals
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(Gupta et al., 2016a, 2014). However, to the best of our knowledge, no research has been
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conducted to investigate the effects of P. polymyxa on the gut microbiota and morphology
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changes, as well as the disease resistance using Vibrio parahaemolyticus on L. vannamei.
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Moreover, the efficacy during the application of probiotic research depends on many factors
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including species composition and viability, probiotic inclusion level, mode, and frequency of
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probiotic supplementation and environmental conditions (Gomez-Gil et al., 2000; Gupta et al.,
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2014; Lara-flores, 2011). Hence, here in this study, we report the impact of dietary Paenibacillus
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polymyxa ATCC 842 (PP) on the growth, immune response, digestive enzyme activity, intestinal
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morphology and microbiota, and defense against V. parahaemolyticus infection of Pacific white
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shrimp, L. vannamei.
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2. Materials and Methods
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2.1 Bacterial strain
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The potential probiotic strain, P. polymyxa ATCC 842 (PP) bacterium was acquired from the
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Guangdong Microbial Culture Center (GDMCC), Guangdong, China. Under sterile conditions,
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deionized water was added to the probiotic PP, streaked on Luria-Bertani (LB media, Sangon
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Biotech) agar plate and incubated at 37 °C for 48 h. In confirming the purity of the strain,
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colonies were identified by their morphological and biochemical characterization. Cell density
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was calculated from optical density (OD) at 600 nm and correlated with a colony-forming unit
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(cfu) counts using serial dilution and spread plating technique on LB agar. The quantified
120
bacteria were maintained at 4 °C in a suspended form with phosphate-buffered saline (PBS, pH
121
7.5). Cells were re-suspended in the same buffer before use.
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2.2 Experimental set-up and shrimp management
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Pacific white shrimp, L. vannamei, not displaying signs of disease (gross examination of the
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gills of individual samples, carapace, thoracic and abdominal segments, pods (uro, pere and
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pleo), without previous history of parasitic infections were collected from the shrimp farm of the
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College of Fisheries, Guangdong Ocean University (Zhanjiang, Guangdong province, China) and
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maintained in aerated cement pools (4.5 m (l) × 3.45 m (w) × 1.8 m (h)) for two weeks where
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they were hand-fed four times daily (07:00, 11:00, 17:00 and 21:00) with commercial diets
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(purchased from Zhanjiang Aohua Feed Co. Ltd., Guangdong, China). After adaptation, shrimps
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were starved for 24 h and a total of 480 shrimps of similar size (0.58 ± 0.001g) were randomly
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distributed into (40 shrimps per tank) 12 fiberglass (four treatments in triplicates) tanks (0.3 m3)
132
in an indoor facility of the Marine Biological Research Base of Guangdong Ocean University for
133
the experiment. The treatments consisted of three P. polymyxa concentrations at 106 (PP1), 107
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(PP2) and 108 (PP3) cfu g-1 feed and one control (PP0, without any probiotic). The selected strain
135
was grown in LB media in a shaking incubator (190 rpm) at 37 °C for 24 - 28 h and cells
136
harvested by centrifugation (4,300 × g at 4 °C for 10 min) to obtain the microbial pellet. The
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pellet was washed twice with PBS (pH 7.2) and re-suspended in the same buffer. The optical
138
density (OD) measured at 600 nm (Gupta et al., 2014) were found to be 1.021. Serial dilutions
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were prepared, and the concentration of microbial pellet adjusted to suit the required dose for the
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various feed preparation. The shrimps were fed the basal pelleted diets at 10 % of their body
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weight for an acclimatization period of one week, which shifted to their experimental diet
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feeding. The experiment lasted 56 days (under 12 h light per 12 h dark photoperiod regime) and
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siphoning done daily to remove uneaten food which was weighed and recorded. Single-air stones
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were used for aeration. Water quality was maintained daily by a renewal of 50 % of the water
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with a maintained temperature range of 28 – 30 °C, pH range of 7.8 – 8.1, dissolved oxygen
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concentration of ≥ 6 mg L-1, and a salinity range of 28– 32 ‰ (YSI 556 MPS, YSI, Inc., USA).
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2.3 Experimental diets
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Table 1 shows the nutritional composition and proximate analysis of the basal diet in the
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current study, which was also in conformity to our previous experiment formulation (Amoah et
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al., 2019). Fish meal, soybean meal, and corn gluten served as the main source of protein while
151
fish oil, soy lecithin oil, and soybean oil also served as the primary lipid sources. The proximate
152
composition analysis of the basal diet was assessed following the AOAC (2002, 1995) method.
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Crude protein (40.65) was measured by the Kjeldahl system (8400-Autoanalyzer, FOSS), crude
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lipid (7.7) was extracted with ether by the Soxhlet method, crude ash content (10.9) was
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determined by combustion of muffle oven which involves oven incineration at 550 °C for 5 hrs,
156
and moisture (10.31) content also identified through oven drying at 105 °C. With continuous
157
mixing of ingredients sieved with 80 meshes/Inch (0.2 mm side length/square hole) in the
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Hobart-type mixer, the bacterial suspensions made previously (see section 2.2) were slowly
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added to the dough to achieve final concentrations in the diet. Each diet was then made to pass
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through a mincer with its resulting pellets air-dried until moisture levels were around 10%.
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Pellets after drying were broken up, sieved into pellets of similar sizes and stored at -20 °C in
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sealed Ziploc bags until used. All the processes were done under sterile conditions. After
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assessing the survival of the bacteria in diets for 8 weeks following the methods of Irianto and
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Austin (2002) and Gupta et al. (2016b), fresh diets were prepared every week to ensure high
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probiotic level in the experimental diets.
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2.4 Sample collection
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2.4.1
Assessment of growth performance
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After the 56-day feeding trial, the shrimps were being fasted for 24 h before harvesting. Total
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remaining numbers of shrimps were counted, and their mean body weight measured. Based on
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the recording of their weight, growth parameters such as survival rate (SR), weight gain rate
171
(WGR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER),
172
and condition factor (CF) were calculated as described below;
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1. SR, % = 100 ×
174
2. WGR, % = 100 ×
175
3. SGR, % = 100 ×
176
4. FCR =
177
5. PER =
178
6. CF, % = 100 ×
179
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2.4.2 Serum, hepatopancreas and intestinal collection
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After being fasted for 24 h, shrimps per tank were collectively weighed. Hemolymph and
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hepatopancreas samples from five randomly sampled shrimps in each tank were collected. The
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hemolymph was collected at their ventral sinus with 1-mL sterile syringes into 1.5-mL
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Eppendorf tubes and stored at 4 °C overnight. The supernatant of stored hemolymph samples at
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–80 °C after centrifugation (1,252 × g for 10 min at 4 °C), were used for subsequent serum
185
biochemical analysis. In analyzing the intestinal microbiota, the same shrimp samples used for
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the collection of hemolymph and hepatopancreas were subsequently dissected under sterile
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conditions to obtain the intestines with their stool samples. All samples from shrimps fed the
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same diet were gathered into one sterile Eppendorf tube and immediately stored in liquid
189
nitrogen until usage. For histological evaluation, five shrimps from each tank were randomly
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selected to remove the intestinal samples. The shrimps’ midguts were then cut and kept in
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Bouin’s fluid for future analysis. Five shrimps from each tank were again selected randomly and
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kept wholly in sealed Ziploc bags and stored at -20 °C for whole body composition analysis.
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2.5 Whole body composition analysis
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The proximate analysis of the whole shrimp body was determined using methods proposed by
195
the AOAC (2002, 1995). Thus, crude protein, crude lipid, ash contents, and the moisture were
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determined as earlier described (see section 2.3).
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2.6 Digestive enzyme assays
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The digestive enzyme activity of the intestines was measured as previously described (Amoah
199
et al., 2019). Briefly, the shrimps’ intestinal samples after weighing were homogenized by
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adding sterile 0.9% saline solution in preparing 10 % (w:v) homogenates of which the
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homogenate was centrifuged at 4 °C for 10 min (489 × g) and the supernatant collected in a 1.5-
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mL Eppendorf tubes for the digestive enzyme activity analysis. The protein concentration used to
203
calculate the standard of the digestive enzyme activities was determined following the
204
manufacturer’s instructions using the Coomassie brilliant blue kit (provided by the Nanjing
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Jiancheng Biological Engineering Research Institute, P. R. China) by reading in a
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spectrophotometer (OD595nm). Trypsin (TRP), amylase (AMS), and lipase (LPS) activities were
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analyzed by using the colorimetric method with procured commercial kits (Nanjing Jiancheng
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Biological Engineering Research Institute, P. R. China) which was later read in a
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spectrophotometer at 253, 660 and 540 nm wavelengths respectively following the company’s
210
instructions.
211
2.7 Activity of immune-related enzymes
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Serum and hepatic immunological assays were measured separately for probiotic treated and
213
control shrimp. By using bead homogenizer, hepatic samples after weighing were homogenized
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in sterile 0.9 % saline (1:9 (w:v) ratio) and centrifuged (959 × g for 10 min at 4 °C). The
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supernatant was collected and stored for later hepatopancreas analysis. Serum total protein (TP)
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(OD540nm) and albumin (ALB) (OD628nm) were determined by applying the biuret method (Lowry
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et al., 1951) and bromocresol green calorimetric technique (Webster, 1974), respectively.
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Deducting of ALB from the TP led to the determination of globulin protein (GLO) content.
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Lysozyme (LYZ) activity was measured using the turbidimetric assay following Ellis (1990)
220
methodology with partial modification. Briefly, 100 µL of serum was added to 1 ml lyophilized
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Micrococcus lysodeikticus (cultured in LB media at 37 °C, centrifuged at 3,622 × g (4 °C for 5
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min), washed two times, and re-suspended in PBS) which was later adjusted to 0.243 mg mL-1
223
(c:v) concentration in PBS (pH 6.4). The OD was recorded at 530 nm at 1, and 20 min at 22 °C.
224
LYZ activity was expressed as units/mL, where a unit was defined as the amount of serum that
225
caused a reduction in absorbance of 0.001 units min-1. Serum triglyceride (TG) content was
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carried out using a commercial kit (Nanjing Jiancheng Biological Engineering Institute, China)
227
measured at an OD of 510 nm following the company’s protocol.
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Glutathione (GSH), glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), acid
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phosphatase (ACP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and
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malondialdehyde (MDA) were determined using commercial kits (Nanjing Jiancheng Biological
231
Engineering Institute, China) following the company’s protocol. Based on the reaction ability of
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ditho-dinitrobenzoic acid with sulfhydryl compounds at 405 nm absorption peak in producing a
233
relatively stable yellow color, GSH activity was measured. GSH-Px is preferably represented by
234
catalyzed GSH reaction rate by measuring absorbance at 412 nm. The SOD activity was
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determined according to Oyanagui (1984) with the reaction based on its inhibitory effect on the
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scale of superoxide anion generating by xanthine and xanthine oxidase reaction system. A SOD
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unit was measured as the amount of enzyme that led to a 50 % inhibition of the nitroblue
238
tetrazolium reduction rate measured at 550 nm using ELISA microplate reader. ACP activity
239
measurement was spectrophotometrically measured by the use of disodium phenyl phosphate
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being the substrate with an acid phosphate detection kit at an absorbance of 530 nm. AKP
241
catalyzes the hydrolysis of disodium phenyl phosphate and was measured at 520 nm absorbance
242
peak with its unit being KU g-1. One king unit is defined as one-milligram phenol generated in 15
243
minutes catalyzed by enzymes in 1g tissue protein at 37 °C. AST and ALT activities were
244
determined following the calorimetric method of Reitman and Frankel’s (1957) decided by
245
standard curve acquired by contrasting assay between experimental method and Carmen’s unit (1
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Carmen’s unit = 0.482 IU L-1, 25 °C) at an absorbance peak at 510 nm. MDA was tested by the
247
thiobarbituric acid (TBA) technique in the glacial acetic medium. The decomposition of lipid
248
hydroperoxide products can with TBA condensate producing red compounds at an absorbance
249
peak wavelength of 532 nm.
250
2.8 Histological assay of the midgut
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The midgut being the primary tissue that deals with incoming meal is thought to produce,
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secrete, and absorb most of the digestive enzymes in the body. It serves as a “gateway organ”
253
providing nutrients to the body. It also serves as an important defense site against microbial
254
pathogens and harmful chemicals with ingested food due to its exoskeleton lining making it a
255
site likely to be penetrated for pathogens (Lovett and Felder, 1990; Ruby et al., 1980). Thus, the
256
midgut was chosen as the site for histological assessment. The midgut segments after removal
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from Bouin’s solution were dehydrated with different gradients of alcohol concentration, cleaned
258
in toluene and embedded in paraffin to make solid wax blocks. Sections (5 µm) were made using
259
rotary microtome, stained with hematoxylin and eosin (H&E), and examined under a light
260
microscope (Olympus, model BX51, Serial number: 9K18395, Tokyo, Japan). By using software
261
Image-Pro Plus, 6.3 (Media Cybernetics, Inc., Rockville, USA) following the procedure of
262
Bullerwell et al. (2016), the villus height (VH), villus width (VW) and muscle thickness (MT)
263
answerable to nutrient absorption were measured.
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2.9 Gut Microbiota Community Discovery and Analysis
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2.9.1 Gut DNA extraction and 16S rRNA amplification
266
The total genomic DNA was extracted from stool samples using the E.Z.N.A.™ stool DNA
267
Kit (Omega Bio-Tek, Norcross, GA, U.S.) following the manufacturer’s instructions. The 16S
268
rDNA V3-V4 region of the gene was amplified by PCR. The PCR conditions were 2 min at 95
269
°C followed by 27 cycles of 10 s at 98 °C, 30 s at 62 °C, and 30 s at 68 °C and finally 10 min at
270
68 °C using universal bacterial primers 341F ： CCTACGGGNGGCWGCAG; 806R ：
271
GGACTACHVGGGTATCTAAT, where the barcode is an eight-base sequence unique to
272
individual sample. The PCR reactions were carried out in triplicate 50 µL mixture containing 5
273
µL of 10 × KOD Buffer, 5 µL of 2.5 mM dNTPs, 1.5 µL of each primer (5 µM), 1 µL of KOD
274
Polymerase, and 100 ng of template DNA. High-throughput sequencing was performed using
275
Illumina Hiseq 2500 sequencing.
276
2. 9.2 Illumina Hiseq 2500 sequencing
277
Amplicons were extracted from 2 % agarose gels and purified using the AxyPrep DNA Gel
278
Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s
279
instructions and quantified using QuantiFluor-ST (Promega, U.S.). Purified amplicons were
280
pooled in equimolar and paired-end sequenced (2 × 250) according to standard protocols on an
281
Illumina platform.
282
2. 9.3 Accession number
283
The microbiota sequence data were exported as individual FastQ files and deposited in the
284
Sequence Read Archive (SRA) database of the National Center for Biotechnology Information
285
(NCBI). The obtained accession number was SRP172960.
286
2. 9.4 Sequencing analysis
287
The paired-end clean reads were jointed as raw tags using FLASH (v 1.2.11) (Magoč and
288
Salzberg, 2011) with a minimum 10 bp overlap and mismatch error rates of 2%. Noisy sequences
289
of raw tags were filtered by QIIME (v 1.9.1) (Caporaso et al., 2010) under specific filtering
290
conditions (Bokulich et al., 2013) to obtain high-quality clean tags. Clean tags were searched
291
against the reference database (http://drive5.com/uchime/uchime_download.html) to perform
292
Reference-based
293
(http://www.drive5.com/usearch/manual/uchime_algo.html). All chimeric tags were removed
294
and finally obtained effective tags for further analysis. Subsequently, effective tags were
295
clustered into operational taxonomic units’ (OTUs) of ≥ 97% similarity using UPARSE (Edgar,
296
2013), which led to the tag sequence with the highest abundance being selected as a reprehensive
297
cluster within each cluster. The representative sequences were classified into organisms by a
298
naive Bayesian model using RDP classifier (Wang et al., 2007) (version 2.2) based on SILVA
299
(Pruesse et al., 2007) Database (https://www.arb-silva.de/).
chimera
checking
using
UCHIME
algorithm
300
Taxonomic richness estimators and community diversity metrics were determined for each
301
library in Mothur (version 1.39.1, http://www.mothur.org/). Alpha diversity is applied to analyze
302
the complexity of species diversity for a sample through 6 metrics, including Observed-species,
303
Chao1, Shannon, Simpson, ACE, and Good-coverage. Two alpha diversity metrics such as
304
Chao1 and ACE were selected and used to identify Community richness, whereas another two
305
metrics, Shannon (Shannon, 1948) and Simpson (Mahaffee and Kloepper, 1997) were used in
306
assessing community diversity. LEfSe (Segata et al., 2011) was used to characterize microbial
307
differences of biological relevance between individual experimental groups. The LEfSe was
308
performed using an alpha value of 0.01 for both the factorial Kruskal-Wallis rank-sum test and
309
the pairwise Wilcoxon test. The different taxonomic level results were transformed to relative
310
abundance count to draw the bar plots.
311
2.10 Challenge study
312
2.10.1 Preparation of inoculum bacteria
313
The preparation of the pathogenic bacterium, Vibrio parahaemolyticus (provided by MOE
314
Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Life
315
Sciences, Sun Yat-sen University, Guangzhou, P. R. China) was conducted as previously
316
described (Amoah et al., 2019). Briefly, the bacterium was added to a 100 mL of LB in a 250 mL
317
flat-bottom flask which was then kept in a shaken incubator (180 rpm for 20 h at 37 °C). After
318
obtaining the cell pellets through centrifugation (4,930 × g at 4 °C for 10 m), they were washed
319
twice using PBS, and the concentration adjusted at 600 nm wavelength. Through the serial
320
dilution technique, the supernatant acquired were re-suspended in PBS to obtain graded doses
321
(106, 107, 108 and 109 cfu mL-1).
322
2.10.2 Calculation of fifty percent (50%) endpoint (Lethal Dose50)
323
A prior experiment lasting 14 days was conducted to determine the lethal dose 50 (LD50).
324
Briefly, 40 shrimps of similar sizes to the final experimental shrimp sizes using the graded doses
325
(106, 107, 108 and 109 cfu mL-1) of bacteria were injected with 0.2 ml V. parahaemolyticus (VP)
326
intramuscularly. Mortalities were observed daily throughout the trial to determine the appropriate
327
concentration of VP for the experiment. The LD50 calculated as previously described (Reed and
328
Muench, 1938) was 1.0 × 108 cfu mL-1.
329
2.10.3 Disease resistance test
330
After the eight weeks feeding trial, 10 shrimps were collected from each of the probiotic-
331
supplemented and control tanks and distributed into another 12 fiberglass tanks for a 14-day
332
challenge study. The shrimps were injected intramuscularly at their third abdominal segment
333
with 0.2 ml of the suspended bacteria (108 cfu mL-1) for the disease test. A group of 30 additional
334
shrimps of similar sizes to the experimental shrimps were injected with PBS for the negative
335
control (NC) during the disease test. The challenged shrimp were kept under observation, and the
336
mortality in each group recorded up to the 14th day of the challenge. The physiology changes of
337
the shrimps were checked daily, and the removal of dead shrimps was regularly done. The
338
following formula was used in calculating cumulative mortality (%) and relative percent survival
339
(RPS, %);
340
1. Cumulative mortality, % = 100 ×
341
2. RPS, % =100 × 1 −
342
2.11 Data analysis and statistics
[+
)
# #
+ +
)
)
! )
)
!
)
! "
)
]
;
.
343
All data, presented as mean ± standard error (mean ± S.E.), were subjected to a one-way
344
analysis of variance (ANOVA) using statistical package for social sciences (IBM SPSS v20.0,
345
Inc., 2010, Chicago, USA) followed by the testing of mean differences using Tukey's multiple
346
comparison tests. The value of P < 0.05 was set for statistical significance. The illustration of the
347
number of shared and unique species among groups was done by creating a Venn diagram.
348
Pheatmap was used in generating heatmap which presented the normalized abundance among
349
groups. Bar graphs were generated using GraphPad Prism (version 7, GraphPad Software, La
350
Jolla California USA) for windows.
351
3. Results
352
3.1 Growth performance, feed utilization, Survival, and whole body composition
353
After eight weeks of feeding, the growth performance between shrimps fed probiotic-enriched
354
diet and control diet is illustrated in table 2. Statistically, a significant (P < 0.05) increase in FW,
355
WGR, SGR, PER, and a decrease in FCR of L. vannamei were observed with increasing
356
inclusion of PP in the diet. As a result, the PP3 group obtained the highest compared to the PP0.
357
Correspondingly, a significant (P < 0.05) increase in CF and SR, was observed in the treated
358
group in comparison to the control.
359
The results of proximate whole body composition except moisture were found to be
360
significantly (P < 0.05) higher in the probiotic-treated with better elevation in PP3 group,
361
followed by PP2 and PP1 when compared to PP0. The reverse was recorded in the case of the
362
moisture content (Table 2).
363
3.2 Serum and hepatopancreas biochemical measurements
364
The effects of PP on serum and hepatopancreas immune responses and antioxidant activities
365
are presented in table 3. It was observed that, inclusion of PP in shrimp’s diet resulted in a
366
significant increase (P < 0.05) in T-AOC, LYZ, SOD, ACP in serum; AKP in liver; GSH-Px,
367
GSH in serum and hepatopancreas and a significant decrease (P < 0.05) in TG in serum; AST,
368
ALT and MDA in serum and hepatopancreas in comparison with the control.
369
Correspondingly, it was observed that increasing the inclusion of PP in shrimp’s diet revealed
370
a significant increase in the TP (Fig. 1A), ALB (Fig. 1B), and GLO (Fig. 1C) of the serum.
371
3.3 Evaluation of intestinal enzyme activity
372
Figure 2 illustrates the results of AMS, LPS, and TRP activities in the intestines of individual
373
group shrimps. Compared with the PP0 (control), the levels of the digestive enzyme activities
374
including AMS (Figure 2A), LPS (Figure 2B) and TRP (Figure 2C) were significantly increased
375
(P < 0.05) in the three probiotic treated groups, with PP3 group obtaining the highest.
376
3.4 Histological structure of the midgut
377
The shrimp mid-intestinal cell in the PP3 and PP2 were arranged closely, having a clearer cell
378
gap with their intestinal walls observed to have tall and wide VH and VW, respectively (Fig. 3 -
379
PP3 [Z] and PP2 [Y - a and b]). Though there were some distortions in the intestinal epithelial
380
cells in PP1 like the PP0 from the basement membrane compared to PP2 and PP3, it was
381
observed that the VH and VW were somewhat enhanced like as found in PP2 and PP3 group’s
382
intestine (Fig. 3 – PP1 [X]). Complete separation from the basement membrane and distortions
383
of cells were recorded in the PP0 group alongside its shorter intestinal villi compared to the other
384
groups (Fig. 3 – PP0 [V and W]).
385
Correspondingly, the total VH (Fig. 4A) and MT (Fig. 4C) significantly decreased (P < 0.05)
386
in the untreated group compared to the treated. It was revealed that only the PP3 had a
387
significantly higher (P < 0.05) VW compared to other groups. PP2, PP1, and PP0 had no
388
significant (P > 0.05) difference, although there was an enhancement in the PP2 and PP1
389
compared to the PP0 (Fig. 4B).
390
3.5 Microbiota of the gut analysis
391
3. 5.1 Microbiota of the intestine richness and diversity analysis
392
The results from the LEfSe analysis are presented in a circular cladogram (Figure 5). It was
393
observed that shrimps of a) PPO group had higher relative abundance belonging to the NS10
394
marine group, as well as genera Actibacter, Flavobacterium sp. NBRC 101627, Acinetobacter
395
bayiyi, b) PP1 group had higher relative abundance belonging to class Plantomycetacia, order
396
Plantomycetales, family Plantomycetaceae, Pir4 lineage, c) PP2 group had a higher relative
397
abundance of order Cellvibrionales, family Cellvibrionaceae, and d) PP3 group had a higher
398
relative abundance of phylum Candidatus-Moranbacteria.
399
The OTUs and Alpha diversity metrics of the intestinal microbiota in L. vannamei are
400
presented in table 4. OTUs were observed to be significantly higher in the probiotic-treated
401
group (P < 0.05) than the untreated (PP0). PP0 attained the least OTU (Table 4). The probiotic-
402
treated groups were observed to have a significantly higher (P < 0.05) chao1 and ACE estimator
403
metrics than the untreated. Moreover, the Shannon and Simpson estimator metrics ranging from
404
4.98 to 5.45 and 0.93 to 0.96 respectively saw the probiotic-treated group to be significantly
405
lower (P < 0.05) than the untreated (Table 4). A total of 302 OTUs coincided in all the four
406
treatments with the analysis of a Venn diagram. It was observed that the number of core OTUs in
407
the PP1 (147) was the highest, followed by PP2 (137), then PP3 (122) with the least going for
408
PP0 (77) (Figure 6).
409
3.5.2 Microbial community change analysis and comparison
410
Different bacterial phyla were observed in the intestines of shrimps. The dominant phyla in
411
the intestinal microbiota were Proteobacteria, followed by Bacteriodetes, and Planctomycetes in
412
the clustered four groups. A significant increase (P < 0.05) of the relative abundance of
413
Proteobacteria in PP2 group was observed, which was followed by PP3, PP1 and lastly decreased
414
(P < 0.05) in the PP0 group. The relative abundance of Bacteriodetes was significantly higher (P
415
< 0.05) in the probiotic-treated group but decreased in the untreated group (P < 0.05) which was
416
in contrast to as observed in Firmicutes phylum (Figure 7).
417
At the class level, Gammaproteobacteria, Flavobacteriia, and Alphaproteobacteria were the
418
most relatively abundant bacteria irrespective of the group. PP1 and PP2 group were found to
419
have highly abundant Gammaproteobacteria, followed by PP3, with PP0 experiencing the least
420
abundance. However, Flavobacteriia was observed to be highly significant (P < 0.05) in L.
421
vannamei’s intestine of PP0 group, which was followed by the PP2, PP3 groups and then lastly
422
the PP1 group (Figure 8).
423
The heatmap analysis of L. vannamei’s intestinal bacterial abundance at the genus level
424
indicated that Spongiimonas, Shewanella, Muricauda, Halobacteriovorax, Shimia, and Kangiella
425
bacteria were more abundant in the untreated group than in the three probiotic-treated groups
426
(Figure 9). It was realized at the genus level that, relative to all the groups, Vibrio and Ruegeria
427
were the most abundant (Figure 10). Among these genera, the abundances of Vibrio,
428
Photobacterium, Tenacibaculum, Kangiella, Spongiimonas, Muricauda, Halobacteriovorax, and
429
Shewanella significantly decreased in the probiotic-enriched group relative to the control (P <
430
0.05). In dissimilarity to this, the abundance of Ruegeria, and Pseudoalteromonas significantly
431
increased in shrimp groups exposed to probiotic treatment (P < 0.05) (Figure 10).
432
3.6 Challenge test
433
After challenge with V. parahaemolyticus for 14 days, the cumulative mortality rates of the
434
untreated group (PP0) was significantly higher (P < 0.05) than those fed with probiotic-treated
435
diets (Fig. 11). At the end of the challenge test, the cumulative mortality was significantly (P <
436
0.05) lower in the probiotic-treated groups than in the control group and in the order 77 %, 43 %,
437
30 %, and 17 % for shrimp fed with PP0, PP1, PP2, PP3 diets respectively (Fig. 11). The relative
438
percent survival (RPS %) was highest in the PP3 (78.3 %), followed by PP2 (60.9 %), and PP1
439
(43.5%) group.
440
4 Discussion
441
Probiotics’ supplementations in diets have recently been increased in meeting the demands as
442
the best substitute for antibiotics. Undeniably, field research, aside unveiling the improvement
443
probionts have towards growth, modulation of immune response, antioxidant activities, and
444
disease resistance to host aquatic organisms (Buruiană et al., 2014; Farzanfar, 2006) have
445
established that the gut microbial composition is strongly affected by dietary supplementation of
446
these same probionts (Lazado et al., 2015). Paenibacillus polymyxa (PP) has been reported to
447
improve growth performance, immune response, and disease resistance in aquatic animals
448
(Gupta et al., 2016a, 2014). Nonetheless, there is a paucity of information on PP’s ability in the
449
aquaculture industry. It is thought that PP’s supplementation owing to the beneficial properties
450
they possess can improve the intestinal microbial composition and morphology. Thus, in this
451
study, L. vannamei was fed different doses of P. polymyxa to evaluate its effect on the growth,
452
immune and antioxidant enzyme activities, resistance against V. parahaemolyticus infection,
453
intestinal morphology and microbiota.
454
Following eight weeks supplementation with PP in L. vannamei’s diets, it became known that
455
increasing inclusion of PP caused a significant increase in the SR, FBW, WGR, SGR, CF, PER,
456
whole body proximate analysis (crude protein, crude lipid, ash) and a significant decrease in the
457
FCR and moisture content of the whole body composition analysis. The whole body proximate
458
composition analysis serves as useful pointers of physiological wellness of animals and the meat
459
quality as well (Vijayavel and Balasubramanian, 2006). Corresponding works showing increase
460
in growth and whole body composition have been previously documented on Fenneropenaeus
461
indicus (Ziaei-Nejad et al., 2006), Penaeus monodon (Boonthai et al., 2011), Litopenaeus
462
vannamei (Balcázar et al., 2007; Shen et al., 2010; Wang, 2007; Zokaeifar et al., 2014),
463
Macrobrachium resenbergii (Saad et al., 2009), Cyprinus carpio (Gupta et al., 2016a), Labeo
464
rohita (Kumar et al., 2006) and Oreochromis niloticus (Sutthi et al., 2018). Further studies on the
465
gut morphology and microbiota, digestive enzyme activities, immune response, and antioxidant
466
activities were elucidated to ascertain what led to such changes stated above.
467
In the present study, the analysis of serum and hepatopancreas immune and antioxidant
468
indices such as TP, ALB, GLO, TG, LYZ, SOD, T-AOC, GSH, GSH-Px, AST, ALT, ACP,
469
AKP, and MDA which is known to play crucial roles in infectious agents’ defense were
470
conducted. Serum TP, ALB and GLO were observed to be significantly higher in the probiotic-
471
treated group than the untreated which was in agreement with other studies on L. vannamei using
472
B. coagulans Sc8168 (Zhou et al., 2009) and Lactobacillus sp. (Mg and Ammani, 2015). Serum
473
TG levels are intensely associated with components of lipid in diets. Thus, decreasing the lipid
474
composition of human food is vital in reducing the cardiovascular disorder rates. In this study,
475
serum TG in the probiotic-treated group significantly decreased with increasing inclusion levels
476
which was in agreement with other findings (Wu et al., 2015). Little information on serum TG is
477
known; hence, we suggest much more research using probiotics be done.
478
ACP and AKP play vital roles in regulating the immune system. ACP is noted for intracellular
479
breaking down of phagocytized antigens whiles AKP also hydrolyzes bounds of phosphoesters in
480
diverse organic compounds such as lipids, proteins, and carbohydrates (Mohapatra et al., 2013;
481
Roubaty and Portmann, 1988). Yi et al. (2018) reported a significant increase in the ACP and
482
AKP after dietary supplementation of Bacillus velezensis JW than the control. Similarly, PP
483
supplementation increased the serum ACP and hepatopancreas AKP activity in L. vannamei in a
484
dose-dependent manner.
485
The damage of tissues in aquatic animals is a developing problem due to their underlying liver
486
injury. AST and ALT activities are regarded as reliable biomarkers of hepatotoxicity in the
487
serum and tissues, including liver (Kamada et al., 2016). Higher AST and ALT levels in serum
488
or liver tissues depict tissue dysfunctioning and damage (Cheng et al., 2018). This study showed
489
a decreasing trend of serum and hepatopancreas AST and ALT activities with increasing
490
inclusion of probiotics in diet indicating the ability of PP to reduce the toxicity in shrimps,
491
similar to as previously described (Sutthi et al., 2018).
492
LYZ being a humoral component, has bactericidal activity against infectious diseases because
493
they can break down the polysaccharide bacteria wall (Li et al., 2015). Shrimps, like other
494
aquatic animals, are susceptible to reactive oxygen attacks. Antioxidant indices such as T-AOC,
495
SOD, GSH, and GSH-Px aid in repairing the damages caused by free radicals (Martínez-Álvarez
496
et al., 2005). T-AOC is used in detecting oxidative stress in the serum and tissues including liver
497
(Dawood et al., 2016) whereas SOD generates cellular H2O2 to defend organisms against
498
microbial infection (Shen et al., 2010). GSH and GSH-Px mainly exploit the detoxification of
499
toxic hydrogen peroxides and other peroxides such as lipid hydroperoxides (Couto et al., 2013;
500
Wang et al., 2017) whereas MDA content is considered to be an indicator of the extent of lipid
501
peroxidation portraying the toxic processes caused as a result of free radicals (Yang et al., 2017).
502
In the present 8-weeks study, all probiotic-treated groups exhibited a significant increase of LYZ
503
and SOD in the serum; GSH, GSH-Px in both serum and hepatopancreas, and a significant
504
decrease in the MDA in both serum and hepatopancreas. This signifies the efficiency of PP
505
supplementation in enhancing immune and antioxidant activities such as observed in previous
506
studies (Gupta et al., 2016a; Liu et al., 2017; Shen et al., 2010; Zokaeifar et al., 2014).
507
In spite of the importance of the intestine in digestion, absorption, and defense, scanty of
508
information on probiotics’ effect on the morphological changes of the intestinal structure has
509
been recognized (Merrifield et al., 2010). A significant increase in the midgut MT, VH, and VW
510
of the probiotic-treated group in animals have been established (Asaduzzaman et al., 2018;
511
Ranadheera et al., 2014) similar to the observation in this study. Since taller and wider intestinal
512
microvilli have been reported as indications for higher absorption of nutrients (Kristiansen et al.,
513
2011), the increase in .midgut MT, VH, and VW of the probiotic-treated group can be the reason
514
why there were improved growth performance, feed utilization, and whole body composition in
515
the treated group.
516
The gut microbiota owing to the immense contributions towards shaping the intestinal
517
structure by digestion of food, absorption of nutrients, competing and conquering of other
518
unwanted microbes to improve the survival and health status of organisms has gained much
519
attention recently (Claus et al., 2016; Sugita et al., 1997). A lot of research has established
520
probiotics’ ability to change the microbial composition of the gut concerning the abundance of
521
opportunistic pathogens and or beneficial microbes (O’shea et al., 2012). In this study, barcoded
522
16S rRNA gene Illumina high-throughput sequencing was used to study the intestinal microbial
523
composition and relative abundance. Our results showed that dietary PP shaped the diversity of
524
the gut microbiota.
525
Following the eight weeks supplementation with PP, it was observed that the most relatively
526
abundant bacterial species in the intestine of shrimps at the phylum level were Proteobacteria and
527
Bacteriodetes, which also are portrayed to be the ordinarily dominant bacteria in most growth
528
stages of shrimps (Hou et al., 2017; Huang et al., 2016; Suo et al., 2017). Additionally, in
529
dissimilarity to the abundance of Bacteriodetes, the bacterial abundance in Firmicutes and
530
Cyanobacteria were observed to decrease significantly in the probiotic-treated group, unlike the
531
untreated. Higher Firmicutes abundance was witnessed in obese mice, which were concurrent
532
with a relatively low abundance of Bacteriodetes (Backhed et al., 2004; Turnbaugh et al., 2008).
533
Moreso, within the Bacteriodetes phylum, Bacteroides thetaiotaomicron has also been observed
534
to improve the host’s nutrient uptake and processing (Hooper, 2001). Cyanobacteria, known as
535
“blue-green bacteria” are photosynthetic microorganisms occurring naturally on the surface of
536
water (Zhao et al., 2006). Reports suggest that low abundance of Cyanobacteria is advantageous
537
to the growth of host organism (Jia et al., 2016) whereas higher abundance is detrimental to
538
aquatic animals’ health as a result of the production of hepatotoxic microcystins (Kang et al.,
539
2012). Research also suggest them to produce lipopeptides that are cytotoxic, which in turn
540
causes cell necrosis (Howard et al., 2012). Therefore the increase in Bacteriodetes and the
541
decrease in Firmicutes and Cyanobacteria may explicate that PP supplementation can interrupt
542
and shape the gut microbial composition in L. vannamei positively.
543
At the class level, regardless of the experimental treatment, Gammaproteobacteria accounted
544
for the majority of bacteria, which was followed by Flavobacteriia and Alphaproteobacteria
545
abundance. This was in accordance to previous research conducted on shrimps (Buchan et al.,
546
2005; Duan et al., 2018; Gao et al., 2014; Xiong et al., 2015; Zheng et al., 2016) signifying the
547
abundance of these bacteria in the gut of shrimps.
548
It was observed at the genus level in our study that irrespective of the treatment fed to L.
549
vannamei, Vibrio, Ruegeria, Photobacterium, Pseudoalteromonas, Tenacibaculum, and
550
Maribacter bacteria were observed to be relatively abundant, similarly to as previously reported
551
(Amoah et al., 2019; Moss et al., 2000; Suo et al., 2017; Zheng et al., 2016). However, some of
552
the genera found in other studies including Meridianimaribacter (Zheng et al., 2016), and
553
Acinetobacter, Candidatus, Aeromonas, Pseudomonas, Gemmobacter, Bacilloplasma, Shinella,
554
Rhodobacter (Zhang et al., 2014) were absent in the present study. Vibrio, Photobacterium,
555
Tenacibaculum, and Shewanella genera which were found to be highly significant in the
556
untreated group than the treated, have been noted in previous studies as opportunistic pathogens
557
causing stress and disease infection in animals (Alderkamp et al., 2007; Miao et al., 2018; Zheng
558
et al., 2016). For example, Tenacibaculum maritimum found in diseased fish has been associated
559
with causing tenacibaculosis (Avendaño-Herrera et al., 2006). Moreso, Shewanella genus found
560
in the Shewanellaceae family are observed to possess foul-smelling compounds such as H2S and
561
are also known to cause spoilage of seafood (Vogel et al., 2005). Wright et al. (2016) also noted
562
Shewanella spp. as having the ability to reduce the amount of manganese, a microelement
563
necessary for bone formation and regeneration of red blood cells. Thus, the increase in its
564
abundance in the untreated group’s gut might be the reason for lower growth performance and
565
feed utilization.
566
Also, Ruegeria and Pseudoalteromonas genera regarded as beneficial microbes were
567
significantly higher in the probiotic-treated group, unlike the untreated in the present study.
568
Ruegeria shows triestherase activity (Yamaguchi et al., 2016) and antagonistic activity on Vibrio
569
anguillarium (Porsby et al., 2008) signifying how beneficial they are to host organisms. Also,
570
reports show Pseudoalteromonas species being used as probiotics (ten Doeschate and Coyne,
571
2008) because they produce chemical compounds such as amylase, protease, phospholipases and
572
extracellular materials which aid in inhibiting the settlement and metamorphosis Shewanella
573
(Cadman and Eichberg, 1983; Gavrilovic et al., 1982; Venkateswaran and Dohmoto, 2000).
574
Consequently, we suggest that Ruegeria and Pseudoalteromonas abundance in the probiotic-
575
treated diet might be due to the probiotic’s ability to compete and conquer the growth of
576
pathogenic bacteria present, which led to the beneficial bacteria’s abundance (O’shea et al.,
577
2012).
578
Digestive enzymes including AMS, LPS, and TRP, play a vital role in breaking down
579
polymeric macromolecules into smaller building blocks to facilitate nutrient absorption (Duan et
580
al., 2017; Gobi et al., 2018; Svendsen, 2000). In our study, dietary PP supplementation positively
581
increased the digestive enzymes AMS, LPS, and TRP, which is supported by other findings
582
(Gobi et al., 2018; Ziaei-Nejad et al., 2006; Zokaeifar et al., 2014). This increase can be
583
attributed to the presence of beneficial bacteria such as Ruegeria which shows triestherase
584
activity (Yamaguchi et al., 2016) and Pseudoalteromonas which produces amylase, protease,
585
phospholipases (Cadman and Eichberg, 1983; Gavrilovic et al., 1982; Venkateswaran and
586
Dohmoto, 2000). Accordingly, the higher increase in the digestive enzymes of the probiotic-
587
treated group can be the reason for the improvement of their growth (Duan et al., 2017).
588
Numerous studies reveal probiotics’ ability to grant shrimps natural resistance against
589
pathogens providing higher survivability (Das et al., 2013; Vaseeharan and Ramasamy, 2003).
590
Vibrio species noted as opportunistic pathogens which have a dominant flora in the various
591
stages of L. vannamei’s developments are reported to cause lots of disease to aquatic organisms
592
and humans (Moss et al., 2000; Suo et al., 2017). The early mortality syndrome (EMS) or acute
593
hepatopancreatic necrosis syndrome (AHPNS) reported to be an emerging serious disease of
594
cultured shrimp including L. vannamei and P. monodon, caused by the Vibrio parahaemolyticus
595
strain, has affected Southeastern Asian shrimp farms since its occurrence (FAO/MARD, 2015;
596
NACA-FAO, 2012; Tran et al., 2013). A fourteen-day challenge using V. parahaemolyticus after
597
the 8 weeks feeding trial led to a significant increase in the cumulative mortality of the untreated
598
group compared to the probiotic-treated which was as well in line other research conducted
599
(Balcázar et al., 2007; Ramalingam and Shyamala, 2006; Zokaeifar et al., 2012). The reason can
600
be attributed to the higher abundance of opportunistic pathogens in the untreated group after the
601
microbial intestinal composition analysis.
602
Conclusion
603
This study demonstrated that the supplementation of PP modulated the growth performance,
604
whole body proximate composition, the immune and antioxidant activities, the intestinal
605
morphology, the composition of the intestine microbiome in L. vannamei with some species of
606
beneficial bacteria becoming more abundant whiles opportunistic pathogens decreased in
607
abundance. Also, PP increased the resistance activity against V. parahaemolyticus infection.
608
These results suggest that PP may benefit intestine health regulation in L. vannamei, and is a
609
candidate for use as a feed additive in shrimp culture. The optimal dietary PP supplementation
610
dosage 108 cfu g-1 feed should be used.
611 612
613
Acknowledgment
614
We are grateful to the support given by Professor Sun Cheng-Bo (Key Laboratory of
615
Crustacean Genetic Breeding and Aquaculture Research Center of Guangdong Ocean
616
University) for providing us with the shrimps used in this current study. We are also appreciative
617
to the support given by Dr. E. D. Abarike (Key Laboratory of Control for Diseases of Aquatic
618
Animals of Guangdong Higher Education Institutes) who helped in the bacterial preparation for
619
feed and disease test. Also, the authors thank the laboratory members for experimental material
620
preparation and all authors to whom we cited in this research work. We are also grateful to
621
Editor and Reviewers for their reasonable criticisms and suggestions, making this study a
622
genuine one.
623
Funding
624
This study was financially supported by the Science and Technology Department of
625
Guangdong Province (2015A020209170); Marine & Fisheries Department of Guangdong
626
Province (A201608C06); the Science and Technology Bureau of Zhanjiang (2016A3022); and
627
the China Agriculture Research System (CARS-47).
628
Additional Information
629
Competing Interests:
630 631
The authors affirm that the experiment was conducted in the absence of any commercial or financial relationships that might be construed as a potential conflict of interest.
632
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Tables Table 1: Dietary formulation and nutritional composition of the basal diet Ingredients
Composition (%)
Brown fish meal
a
26.0
a
Soybean meal
13.0
Peanut meal
8.0
Wheat flour
a
Corn gluten meal
26.0 a
Shrimp shell meal Soybean oil Fish oil
9.0 a
a
a
6.0 1.0 1.0
a
Soy lecithin
1.0
Vitamin premixb
1.0
Mineral premixc
1.0
Choline chloridec
0.5
Vitamin Ca
0.1
Ca (H2PO4)2d
1.5
Carboxymethyl cellulosee
1.0
Microcrystalline Cellulosee
3.9
Total
100
Nutrient Index (Dry matter) Crude protein
Proximate composition (%) 40.65
Crude lipid
7.7
Crude ash
10.9
Moisture
10.31
a
Ingredients acquired from Zhanjiang HaiBao Feed Factory, Zhanjiang, Guangdong, China.
b
Vitamin premix supplied the following per kg of the diet: vitamin A, 22,500 IU; vitamin D3, 6,000 IU; vitamin E,
200 mg; vitamin K3, 40 mg; vitamin B1, 30 mg; vitamin B2, 45 mg; vitamin B6, 35 mg; vitamin B12, 0.25mg; calcium pantothenate, 150 mg; niacin, 225 mg; folic acid, 12.5 mg; biotin, 0.5 mg; inositol, 500 mg (Acquired from Zhanjiang Yuehai Feed Co. Ltd., Guangdong, China)
c
Mineral premix provided the following per kilogram of diet: Fe, 60 mg; Zn, 24 mg; Mn, 16 mg; Cu, 1.4 mg; Co,
0.2 mg; Se, 0.1 mg; I, 0.2 mg (Acquired from Zhanjiang Yuehai Feed Co. Ltd., Guangdong, China) d e
Acquired from Shanghai Macklin Biochemical Co. Ltd., 1288 Canggong Rd., Shanghai, China.
Acquired from Shantou Xilong Chemical Factory, Guangdong, China.
Table 2: Effects of different supplementation levels of P. polymyxa on the growth performance of L. vannamei. Indices
PP0 (Control)
PP1
PP2
PP3
Growth index IBW (g)
0.58 ± 0.001
0.58 ± 0.001
0.58 ± 0.000
0.58 ± 0.001
FBW (g)
6.45 ± 0.02a
6.69 ± 0.11ab
6.99 ± 0.14b
7.46 ± 0.04c
1018.75 ± 2.64a
1059.70 ± 19.02ab
1111.07 ± 24.13b
1193.27 ± 6.15c
SGR (%/day)
4.31 ± 0.00a
4.38 ± 0.03ab
4.45 ± 0.04b
4.57 ± 0.01c
FCR
2.71 ± 0.03c
2.29 ± 0.06b
2.25 ± 0.02b
1.97 ± 0.02a
SR (%)
81.67 ± 2.20a
95.83 ± 2.20b
95.00 ± 1.44b
98.33 ± 1.67b
PER
0.98 ± 0.01a
1.16 ± 0.03b
1.19 ± 0.01b
1.37 ± 0.01c
CF (%)
0.76 ± 0.03a
1.25 ± 0.02bc
0.96 ± 0.11ab
1.45 ± 0.12c
WGR (%)
Body composition (% DM) Moisture
77.04 ± 0.26b
73.05 ± 0.37a
74.09 ± 0.46a
72.90 ± 0.38a
Crude protein
71.78 ± 1.00a
77.60 ± 1.10bc
75.02 ± 0.24ab
81.21 ± 0.85c
Crude lipid
6.88 ± 0.06a
7.46 ± 0.06b
7.67 ± 0.05b
8.00 ± 0.10c
Ash
12.98 ± 0.04a
14.35 ± 0.05b
14.35 ± 0.06b
14.92 ± 0.41b
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Where; IBW, initial body weight; FBW, final body weight; WGR, weight gain rate; SGR, specific growth rate; FCR, feed conversion ratio; CF, condition factor; PER, protein efficiency ratio; SR, survival rate. PPO = basal diet void of P. polymycoxa supplementation; PP1 = basal diet with 106 cfu P. polymycoxa g-1 feed; PP2 = basal diet with 107 cfu P. polymycoxa g-1 feed; PP3 = basal diet with 108 cfu P. polymycoxa g-1 feed.
Table 3: Effects of different supplementation levels of P. polymyxa on immune and antioxidant factors in serum and hepatopancreas of L. vannamei. Indices
PP0 (Control)
PP1
PP2
PP3
TG (mmol L-1)
0.56 ± 0.02c
0.47 ± 0.00b
0.44 ± 0.01ab
0.40 ± 0.02a
T-AOC (mmol L-1)
0.59 ± 0.01a
0.65 ± 0.01b
0.62 ± 0.01ab
0.68 ± 0.01c
GSH-Px (U mL-1)
71.36 ± 0.87a
79.82 ± 2.87a
124.01 ± 6.74b
113.02 ± 3.23b
GSH (µmol L-1)
19.08 ± 0.46a
26.79 ± 1.23b
25.50 ± 0.83b
25.75 ± 1.55b
LYZ (U mL-1)
10.67 ± 0.67a
13.67 ± 0.88b
13.00 ± 0.58ab
15.67 ± 0.33b
SOD (U mL-1)
782.46 ± 1.41a
1215.04 ± 19.72c
959.40 ± 21.05b
1377.44 ± 23.49d
ACP (U 100mL-1)
14.33 ± 0.51a
22.77 ± 0.08b
20.79 ± 1.48b
28.87 ± 0.15c
AST (U L-1)
13.27 ± 0.23b
10.42 ± 1.09a
10.00± 0.63a
11.71 ± 0.47ab
ALT (U L-1)
23.19 ± 1.12b
15.85 ± 0.62a
13.31 ± 1.11a
14.32 ± 0.92a
MDA (nmol mL-1)
5.00 ± 0.12c
1.16 ± 0.12a
1.69 ± 0.16ab
1.99 ± 0.07b
GSH-Px (U mgprot-1)
325.78 ± 4.30a
368.28 ± 19.75ab
341.02 ± 3.73a
413.99 ± 15.78b
GSH (µmol gprot-1)
15.87 ± 0.83a
19.79 ± 1.57ab
17.74 ± 1.32ab
22.17 ± 1.05b
AKP (KU gprot-1)
11.76 ± 0.65a
16.83 ± 0.63b
15.93 ± 1.08b
16.93± 0.72b
AST (U gprot-1)
1.79 ± 0.09b
1.05 ± 0.09a
0.89 ± 0.02a
0.74 ± 0.5a
ALT (U gprot-1)
1.29 ± 0.11b
0.67 ± 0.09a
0.70 ± 0.08a
0.61 ± 0.02a
MDA (nmol mgprot1 )
4.96 ± 0.08a
1.27 ± 0.02a
1.38 ± 0.07a
1.15 ± 0.02a
Serum
Hepatopancreas
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Where; TG, triglyceride; TAOC, Total antioxidant capacity; GSH-Px, glutathione peroxidase; GSH, glutathione; LYZ, Lysozyme; SOD, superoxide dismutase; ACP, Acid phosphatase; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; AKP, Alkaline phosphatase; MDA, malondialdehyde. Abbreviations are as defined in table 2.
Table 4: OTUs, diversity metrics estimated OUT richness for the intestinal bacterial analysis of L. vannamei fed different levels of P. polymyxa. Reads Groups
Richness estimators Tags
Raw
Valid
PPO (Control)
70880
69131
65883
PP1
120615
117394
PP2
123795
PP3
112840
Diversity estimators
OTUs Chao1
ACE
Shannon
Simpson
309.67 6.57a
432.12 ± 33.91a
440.92 ± 37.76a
5.45 ± 0.15c
0.96 ± 0.002c
112064
525.33 ±3.18c
667.71 ± 30.01b
621.99 ± 38.12b
4.19 ± 0.03a
0.88 ± 0.002a
120245
109649
510.00 ± 5.51c
659.34 ± 6.14b
640.98 ± 2.38b
4.79 ± 0.06b
0.93 ± 0.006b
109523
103148
470.67 ± 13.02b
623.79 ± 7.47b
593.50 ± 35.74b
4.98 ± 0.01ab
0.93 ± 0.009ab
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Abbreviations are as defined in table
Figure Legends: Fig. 1. Effects of different supplementation levels of P. polymyxa on serum total protein (A), albumin (B), and globulin (C) contents of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. PPO = basal diet void of P. polymyxa supplementation; PP1 = basal diet with 106 cfu P. polymyxa g-1 feed; PP2 = basal diet with 107 cfu P. polymyxa g-1 feed; PP3 = basal diet with 108 cfu P. polymyxa g-1 feed. Fig. 2. Effects of different supplementation levels of P. polymyxa on the amylase (A), lipase (B), and trypsin (C) intestinal enzyme activities of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 3. Photomicrographs of the midgut tract cross-cutting of L. vannamei fed at different PP concentration with hematoxylin and eosin staining to show changes in the intestinal epithelial cells and microvillus. Arrows show the pathological changes. Magnification was 200×, and the scale represents 200 µm. PP0: arrow V shows the intestinal epithelial cells completely detached from the basement membrane and arrow W also shows some distortions in the epithelial cells; PP1: X shows some detachment of intestinal epithelial cells from the basement, however, there are some tall villus height and width observed; PP2: Y shows the close integration of the intestinal epithelial cells and the basement membrane, a shows the villus height, b shows the villus width; PP3: Z shows the close integration of the intestinal epithelial cells and the basement membrane. Abbreviations are as defined in figure 1. Fig. 4. Effects of different supplementation levels of P. polymyxa on the villi height (A), villi width (B), and muscle thickness (C) measurements of the midgut tract cross-cutting of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 5. Circular cladogram of the intestinal microbiota reporting results of the uppermost relative abundance from the LEfSe analysis. The identified OTU are distributed according to phylogenetic characteristics around the circle. The dots close to the center represent the OTU on phylum level while the outer circle of dots characterizes the OTU on the genus level. Node size is proportional to the average abundance; color indicates the relative concentration of the clusters. Abbreviations are as defined in figure 1. Fig. 6. Venn diagram displaying compartmental OTU distribution of the core intestinal microbiota shared by L. vannamei fed four diets supplemented with P. polymyxa probiotic bacteria. Three hundred and two OTUs were identified as core microbiota for all diets in the intestines of L. vannamei. Abbreviations are as defined in figure 1.
Fig. 7. Relative abundance of the major intestinal bacteria of L. vannamei at the Phylum level. Vertical bars represent the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P < 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 8. Relative abundance of the major intestinal bacteria of L. vannamei at the Class level. Vertical bars represent the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P < 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 9. Heatmap of the abundance of L. vannamei intestinal bacteria at the genus level at different P. polymyxa concentration. Phylogenetic positions are projected by the OTUs, and the taxa of OTUs are listed on the right. Color intensity indicates the relative abundance of OTUs. Abbreviations are as defined in figure 1. Fig. 10. Relative abundance of major intestinal bacteria of L. vannamei at the genus level. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 11. Cumulative mortality (%) of L. vannamei after 14 days post challenge with V. parahaemolyticus. NC, Negative control injected with PBS. Each line graph represents mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Abbreviations are as defined in figure 1.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
c
75
c b
50 a 25 7 6 5 4 3 2 1
c a
b b ab aba b
PP0 PP2
PP1 PP3
c b
a
c
a c b a abb
0.04 0.02 0.00
aba b
b ab a
bb a a
e a a t a a eri teriia cteri etaci obia hagia oidia robii plas ther sified t c r O las a a p c ro ic er ic ac c eob vob oteob tomy com Cyto Bact idim Chlo t o Un c r c rru l a pr p n F A a a Pla Ve lph mm A a G
Figure 8
Figure 9
Figure 10
NC
90
PP0
PP1
PP2
PP3
d
Cumnulative mortality (%)
80 70 60
c
50 40
bc
30
b
20 10
a
0 1
2
3
4
5
6
7
8
9
Days after challenge infection
Figure 11
10
11
12
13
14
Highlights The effects of potential probiotic Paenibacillus polymyxa ATCC 842 (PP) on shrimp, Litopeneaus vannamei were assessed. Dietary supplementation of PP enhanced fish growth, serum and hepatopancreas immune and antioxidant activities. Probiotic PP improved the digestive enzyme activities, intestinal morphology and shaped the gut microbiota composition. Probiotic PP increased the survival of shrimps challenged with V. parahaemolyticus infection. 108 cfu g-1 diet of PP was the optimal dietary supplementation level.
S0044-8486(19)30903-2
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734563
Reference:
AQUA 734563
To appear in:
Aquaculture
Received Date: 16 April 2019 Revised Date:
1 September 2019
Accepted Date: 1 October 2019
Please cite this article as: Amoah, K., Huang, Q.-c., Dong, X.-h., Tan, B.-p., Zhang, S., Chi, S.-y., Yang, Q.-h., Liu, H.-y., Yang, Y.-z., Paenibacillus polymyxa, improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus., Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734563. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1 2 3
Paenibacillus polymyxa, improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus.
4 5
Kwaku Amoahab, Qin-cheng Huangab, Xiao-hui Dongabc*, Bei-ping Tanabc*, Shuang Zhangabc,
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Shu-yan Chiabc, Qi-hui Yangabc, Hong-yu Liuabc, Yuan-zhi Yanga
7
a
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Zhanjiang, Guangdong 524088, PR China
9
b
Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University,
Aquatic Animals Precision Nutrition and High-Efficiency Feed Engineering Research Centre of Guangdong
10
Province, Zhanjiang, Guangdong 524088, PR China
11
c
12
Agriculture, Zhanjiang, Guangdong 524000, China
13
* Correspondence: X-H Dong, Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries,
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Guangdong Ocean University, Zhanjiang, Guangdong 524088, China.
15
E-mail address: [email protected]
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* Correspondence: B-P Tan, Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries,
17
Guangdong Ocean University, Zhanjiang, Guangdong 524088, China.
18
E-mail address: [email protected]
19 20 21 22 23
Key Laboratory of Aquatic, Livestock and Poultry Feed Science and Technology in South China, Ministry of
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ABSTRACT
25
This present study was performed to examine the effects of different levels of dietary
26
Paenibacillus polymyxa ATCC 842 (PP) on the growth, immune response, antioxidant activities,
27
intestinal morphology and microbiota, and defense against Vibrio parahaemolyticus (VP)
28
infection in white leg shrimp, Litopeneaus vannamei. Shrimps (initial weight of 0.58 ± 0.001g)
29
were fed diet containing 0 (control, PP0), 106 (PP1), 107 (PP2) and 108 (PP3) cfu g-1 P. polymyxa.
30
After 8 weeks feeding trial, shrimps fed the PP treated diet displayed a synergistic effect which
31
significantly enhanced (P < 0.05) the final body weight, survival rate, weight gain rate, specific
32
growth rate, protein efficiency ratio, condition factor; total protein, albumin, globulin,
33
triglyceride, lysozyme, total antioxidant capacity, superoxide dismutase, acid phosphatase
34
activity in the serum; alkaline phosphatase activity in the hepatopancreas; glutathione,
35
glutathione peroxidase activity in both the serum and hepatopancreas, with decreased feed
36
conversion rate; aspartate aminotransferase, alanine aminotransferase, and malondialdehyde
37
levels in both serum and hepatopancreas compared to the control group (PP0). Correspondingly,
38
the probiotic-treated group experienced significantly improved (P < 0.05) mid-intestinal
39
morphological structures such as villus height, villus width, muscle thickness and digestive
40
enzyme activities including amylase, trypsin, and lipase than the untreated group with the PP3
41
group obtaining the highest. Dietary PP supplementation in diets again was observed to change
42
the shrimps’ intestinal microbial composition. Explicitly, the most dominant bacterial phyla,
43
Proteobacteria, Bacteriodetes, and Planctomycetes observed in this study were significantly
44
higher (P < 0.05) in the probiotic-enriched group compared to the control. At the genus level, the
45
relative
46
Tenacibaculum, and Shewanella) were significantly decreased (P < 0.05), whereas beneficial
47
bacteria (Ruegeria and Pseudoalteromonas) were significantly enhanced (P < 0.05) in the
48
probiotic-treated group than the untreated. Additionally, dietary supplementation of PP in L.
49
vannamei’s diet significantly improved (P < 0.05) the protection against VP infections with PP3
50
treatment group obtaining the highest relative percentage survival of 78.3 %. These results
51
collectively suggest that dietary PP had a positive effect on the intestinal health of L. vannamei
52
via the modulation of the microbial composition; thus, promoting the digestion and absorption of
53
nutrients in boosting shrimps’ immunity. The optimal supplementation dosage in diets was found
54
to be 108 cfu g-1 diet.
abundance
of
opportunistic
bacterial
pathogens
(Vibrio,
Photobacterium,
55
KEYWORDS: Paenibacillus polymyxa ATCC 842, Litopenaeus vannamei, midgut, relative
56
percentage survival, microbiota.
57
1. Introduction
58
Pacific white shrimps, Litopenaeus vannamei, owing to their higher benefits have been
59
reported as the most cultured shrimp species (Chiu et al., 2007; FAO, 2012). Aquaculturists upon
60
realizing the benefits these shrimp possess and also having the desire of meeting the increasing
61
demand for shrimp food have shifted from the extensive system of culturing to the semi-
62
intensive and intensive system causing challenges such as bacterial and viral disease infections
63
as a result of the higher stocking densities (Bondad-Reantaso et al., 2005). Shrimps’
64
susceptibility to bacterial, viral, fungal and parasitic infections are very wide. Bachère et al.
65
(2004) in their work reported that shrimps lack adaptive immune features in removing pathogens
66
hence are easily infected, causing poor growth and development, as well as high mortality. This
67
has led researchers into finding lasting solutions to the disease menace to prevent the re-
68
occurrence of the previously described disease outbreaks (Flegel, 2006; Lightner et al., 2012).
69
Antibiotic usage in shrimp aquaculture, though proven to help combat these diseases by
70
improving growth, feed utilization and disease resistance, have been critiqued because of their
71
potential adverse effects on humans, the environment such as the development and spread of
72
drug-resistant pathogens (Das et al., 2013), destroying of microbial populations in the
73
aquaculture environment and the suppression of aquatic animal immune systems (Li et al., 2007;
74
Zhou et al., 2009). Probiotics which have proven otherwise is being investigated due to the
75
positive results it has concerning the immune response, antioxidant defense system, disease
76
resistance and the gut microbiota (Spinler et al., 2008; Verschuere et al., 2000). Among the
77
commercial probiotics being tested currently, Bacillus spp. have been noted as the fore of recent
78
research in shrimp culture due to the colossal sum of beneficial properties they possess as
79
compared to others (Hong et al., 2005; Verschuere et al., 2000). Paenibacillus polymyxa
80
formerly recognized as Bacillus polymyxa is a Gram-positive, rod-shaped bacterium that
81
distinctly swells its sporangium (Lal and Tabacchioni, 2009) with their spores able to resist harsh
82
environmental conditions, including extreme pH, temperature, high pressure, UV irradiation,
83
aridity and chemical infiltration (Huo et al., 2012). It is worth mentioning that their spores are
84
known to produce a variety of beneficial bioactive substances including fusaricidins,
85
antimicrobial polypeptides and polymyxin antibiotic compounds as a result of the presence of
86
non-ribosomal peptide synthetase systems in them (Shaheen et al., 2011; Grady et al., 2016).
87
Studies show P. polymyxa to be the source of dispase enzyme which is reported to be used in the
88
isolation of tissues of animals (Ono et al., 1977; Stenn et al., 1989). The principal role of this
89
wholesome strain P. polymyxa is described as the phytopathogen disease control in numerous
90
crops and aquatic animals (Zhou et al., 2016; Gupta et al., 2016a). For example, P. polymyxa
91
NSY50 was reported to effectively reduce Fusarium wilt (56.4%) by altering the soil
92
physicochemical properties and all enzyme activities of the soil (Shi et al., 2017). It is
93
noteworthy this strain is comparatively used as the last remedy for infections caused by multiple-
94
drug-resistant, Pseudomonas aeruginosa (Velkov et al., 2013). Isolated surfactant from P.
95
polymyxa has been reported to disrupt biofilms of Bacillus subtilis, Pseudomonas aeruginosa,
96
Micrococcus luteus, Streptococcus bovis, and Staphylococcus aureus effectively (Quinn et al.,
97
2012). P. polymyxa ATCC 842 bacterium has been noted to improve on the digestibility of
98
nutrients and gut microflora in broiler chicken when added to fermented palm kernel cake
99
(FPKC) (Alshelmani et al., 2016). Furthermore, reports of P. polymyxa has been noted as being
100
able to improve the growth, enhance the immune and antioxidant activities of aquatic animals
101
(Gupta et al., 2016a, 2014). However, to the best of our knowledge, no research has been
102
conducted to investigate the effects of P. polymyxa on the gut microbiota and morphology
103
changes, as well as the disease resistance using Vibrio parahaemolyticus on L. vannamei.
104
Moreover, the efficacy during the application of probiotic research depends on many factors
105
including species composition and viability, probiotic inclusion level, mode, and frequency of
106
probiotic supplementation and environmental conditions (Gomez-Gil et al., 2000; Gupta et al.,
107
2014; Lara-flores, 2011). Hence, here in this study, we report the impact of dietary Paenibacillus
108
polymyxa ATCC 842 (PP) on the growth, immune response, digestive enzyme activity, intestinal
109
morphology and microbiota, and defense against V. parahaemolyticus infection of Pacific white
110
shrimp, L. vannamei.
111
2. Materials and Methods
112
2.1 Bacterial strain
113
The potential probiotic strain, P. polymyxa ATCC 842 (PP) bacterium was acquired from the
114
Guangdong Microbial Culture Center (GDMCC), Guangdong, China. Under sterile conditions,
115
deionized water was added to the probiotic PP, streaked on Luria-Bertani (LB media, Sangon
116
Biotech) agar plate and incubated at 37 °C for 48 h. In confirming the purity of the strain,
117
colonies were identified by their morphological and biochemical characterization. Cell density
118
was calculated from optical density (OD) at 600 nm and correlated with a colony-forming unit
119
(cfu) counts using serial dilution and spread plating technique on LB agar. The quantified
120
bacteria were maintained at 4 °C in a suspended form with phosphate-buffered saline (PBS, pH
121
7.5). Cells were re-suspended in the same buffer before use.
122
2.2 Experimental set-up and shrimp management
123
Pacific white shrimp, L. vannamei, not displaying signs of disease (gross examination of the
124
gills of individual samples, carapace, thoracic and abdominal segments, pods (uro, pere and
125
pleo), without previous history of parasitic infections were collected from the shrimp farm of the
126
College of Fisheries, Guangdong Ocean University (Zhanjiang, Guangdong province, China) and
127
maintained in aerated cement pools (4.5 m (l) × 3.45 m (w) × 1.8 m (h)) for two weeks where
128
they were hand-fed four times daily (07:00, 11:00, 17:00 and 21:00) with commercial diets
129
(purchased from Zhanjiang Aohua Feed Co. Ltd., Guangdong, China). After adaptation, shrimps
130
were starved for 24 h and a total of 480 shrimps of similar size (0.58 ± 0.001g) were randomly
131
distributed into (40 shrimps per tank) 12 fiberglass (four treatments in triplicates) tanks (0.3 m3)
132
in an indoor facility of the Marine Biological Research Base of Guangdong Ocean University for
133
the experiment. The treatments consisted of three P. polymyxa concentrations at 106 (PP1), 107
134
(PP2) and 108 (PP3) cfu g-1 feed and one control (PP0, without any probiotic). The selected strain
135
was grown in LB media in a shaking incubator (190 rpm) at 37 °C for 24 - 28 h and cells
136
harvested by centrifugation (4,300 × g at 4 °C for 10 min) to obtain the microbial pellet. The
137
pellet was washed twice with PBS (pH 7.2) and re-suspended in the same buffer. The optical
138
density (OD) measured at 600 nm (Gupta et al., 2014) were found to be 1.021. Serial dilutions
139
were prepared, and the concentration of microbial pellet adjusted to suit the required dose for the
140
various feed preparation. The shrimps were fed the basal pelleted diets at 10 % of their body
141
weight for an acclimatization period of one week, which shifted to their experimental diet
142
feeding. The experiment lasted 56 days (under 12 h light per 12 h dark photoperiod regime) and
143
siphoning done daily to remove uneaten food which was weighed and recorded. Single-air stones
144
were used for aeration. Water quality was maintained daily by a renewal of 50 % of the water
145
with a maintained temperature range of 28 – 30 °C, pH range of 7.8 – 8.1, dissolved oxygen
146
concentration of ≥ 6 mg L-1, and a salinity range of 28– 32 ‰ (YSI 556 MPS, YSI, Inc., USA).
147
2.3 Experimental diets
148
Table 1 shows the nutritional composition and proximate analysis of the basal diet in the
149
current study, which was also in conformity to our previous experiment formulation (Amoah et
150
al., 2019). Fish meal, soybean meal, and corn gluten served as the main source of protein while
151
fish oil, soy lecithin oil, and soybean oil also served as the primary lipid sources. The proximate
152
composition analysis of the basal diet was assessed following the AOAC (2002, 1995) method.
153
Crude protein (40.65) was measured by the Kjeldahl system (8400-Autoanalyzer, FOSS), crude
154
lipid (7.7) was extracted with ether by the Soxhlet method, crude ash content (10.9) was
155
determined by combustion of muffle oven which involves oven incineration at 550 °C for 5 hrs,
156
and moisture (10.31) content also identified through oven drying at 105 °C. With continuous
157
mixing of ingredients sieved with 80 meshes/Inch (0.2 mm side length/square hole) in the
158
Hobart-type mixer, the bacterial suspensions made previously (see section 2.2) were slowly
159
added to the dough to achieve final concentrations in the diet. Each diet was then made to pass
160
through a mincer with its resulting pellets air-dried until moisture levels were around 10%.
161
Pellets after drying were broken up, sieved into pellets of similar sizes and stored at -20 °C in
162
sealed Ziploc bags until used. All the processes were done under sterile conditions. After
163
assessing the survival of the bacteria in diets for 8 weeks following the methods of Irianto and
164
Austin (2002) and Gupta et al. (2016b), fresh diets were prepared every week to ensure high
165
probiotic level in the experimental diets.
166
2.4 Sample collection
167
2.4.1
Assessment of growth performance
168
After the 56-day feeding trial, the shrimps were being fasted for 24 h before harvesting. Total
169
remaining numbers of shrimps were counted, and their mean body weight measured. Based on
170
the recording of their weight, growth parameters such as survival rate (SR), weight gain rate
171
(WGR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER),
172
and condition factor (CF) were calculated as described below;
173
1. SR, % = 100 ×
174
2. WGR, % = 100 ×
175
3. SGR, % = 100 ×
176
4. FCR =
177
5. PER =
178
6. CF, % = 100 ×
179
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2.4.2 Serum, hepatopancreas and intestinal collection
180
After being fasted for 24 h, shrimps per tank were collectively weighed. Hemolymph and
181
hepatopancreas samples from five randomly sampled shrimps in each tank were collected. The
182
hemolymph was collected at their ventral sinus with 1-mL sterile syringes into 1.5-mL
183
Eppendorf tubes and stored at 4 °C overnight. The supernatant of stored hemolymph samples at
184
–80 °C after centrifugation (1,252 × g for 10 min at 4 °C), were used for subsequent serum
185
biochemical analysis. In analyzing the intestinal microbiota, the same shrimp samples used for
186
the collection of hemolymph and hepatopancreas were subsequently dissected under sterile
187
conditions to obtain the intestines with their stool samples. All samples from shrimps fed the
188
same diet were gathered into one sterile Eppendorf tube and immediately stored in liquid
189
nitrogen until usage. For histological evaluation, five shrimps from each tank were randomly
190
selected to remove the intestinal samples. The shrimps’ midguts were then cut and kept in
191
Bouin’s fluid for future analysis. Five shrimps from each tank were again selected randomly and
192
kept wholly in sealed Ziploc bags and stored at -20 °C for whole body composition analysis.
193
2.5 Whole body composition analysis
194
The proximate analysis of the whole shrimp body was determined using methods proposed by
195
the AOAC (2002, 1995). Thus, crude protein, crude lipid, ash contents, and the moisture were
196
determined as earlier described (see section 2.3).
197
2.6 Digestive enzyme assays
198
The digestive enzyme activity of the intestines was measured as previously described (Amoah
199
et al., 2019). Briefly, the shrimps’ intestinal samples after weighing were homogenized by
200
adding sterile 0.9% saline solution in preparing 10 % (w:v) homogenates of which the
201
homogenate was centrifuged at 4 °C for 10 min (489 × g) and the supernatant collected in a 1.5-
202
mL Eppendorf tubes for the digestive enzyme activity analysis. The protein concentration used to
203
calculate the standard of the digestive enzyme activities was determined following the
204
manufacturer’s instructions using the Coomassie brilliant blue kit (provided by the Nanjing
205
Jiancheng Biological Engineering Research Institute, P. R. China) by reading in a
206
spectrophotometer (OD595nm). Trypsin (TRP), amylase (AMS), and lipase (LPS) activities were
207
analyzed by using the colorimetric method with procured commercial kits (Nanjing Jiancheng
208
Biological Engineering Research Institute, P. R. China) which was later read in a
209
spectrophotometer at 253, 660 and 540 nm wavelengths respectively following the company’s
210
instructions.
211
2.7 Activity of immune-related enzymes
212
Serum and hepatic immunological assays were measured separately for probiotic treated and
213
control shrimp. By using bead homogenizer, hepatic samples after weighing were homogenized
214
in sterile 0.9 % saline (1:9 (w:v) ratio) and centrifuged (959 × g for 10 min at 4 °C). The
215
supernatant was collected and stored for later hepatopancreas analysis. Serum total protein (TP)
216
(OD540nm) and albumin (ALB) (OD628nm) were determined by applying the biuret method (Lowry
217
et al., 1951) and bromocresol green calorimetric technique (Webster, 1974), respectively.
218
Deducting of ALB from the TP led to the determination of globulin protein (GLO) content.
219
Lysozyme (LYZ) activity was measured using the turbidimetric assay following Ellis (1990)
220
methodology with partial modification. Briefly, 100 µL of serum was added to 1 ml lyophilized
221
Micrococcus lysodeikticus (cultured in LB media at 37 °C, centrifuged at 3,622 × g (4 °C for 5
222
min), washed two times, and re-suspended in PBS) which was later adjusted to 0.243 mg mL-1
223
(c:v) concentration in PBS (pH 6.4). The OD was recorded at 530 nm at 1, and 20 min at 22 °C.
224
LYZ activity was expressed as units/mL, where a unit was defined as the amount of serum that
225
caused a reduction in absorbance of 0.001 units min-1. Serum triglyceride (TG) content was
226
carried out using a commercial kit (Nanjing Jiancheng Biological Engineering Institute, China)
227
measured at an OD of 510 nm following the company’s protocol.
228
Glutathione (GSH), glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), acid
229
phosphatase (ACP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and
230
malondialdehyde (MDA) were determined using commercial kits (Nanjing Jiancheng Biological
231
Engineering Institute, China) following the company’s protocol. Based on the reaction ability of
232
ditho-dinitrobenzoic acid with sulfhydryl compounds at 405 nm absorption peak in producing a
233
relatively stable yellow color, GSH activity was measured. GSH-Px is preferably represented by
234
catalyzed GSH reaction rate by measuring absorbance at 412 nm. The SOD activity was
235
determined according to Oyanagui (1984) with the reaction based on its inhibitory effect on the
236
scale of superoxide anion generating by xanthine and xanthine oxidase reaction system. A SOD
237
unit was measured as the amount of enzyme that led to a 50 % inhibition of the nitroblue
238
tetrazolium reduction rate measured at 550 nm using ELISA microplate reader. ACP activity
239
measurement was spectrophotometrically measured by the use of disodium phenyl phosphate
240
being the substrate with an acid phosphate detection kit at an absorbance of 530 nm. AKP
241
catalyzes the hydrolysis of disodium phenyl phosphate and was measured at 520 nm absorbance
242
peak with its unit being KU g-1. One king unit is defined as one-milligram phenol generated in 15
243
minutes catalyzed by enzymes in 1g tissue protein at 37 °C. AST and ALT activities were
244
determined following the calorimetric method of Reitman and Frankel’s (1957) decided by
245
standard curve acquired by contrasting assay between experimental method and Carmen’s unit (1
246
Carmen’s unit = 0.482 IU L-1, 25 °C) at an absorbance peak at 510 nm. MDA was tested by the
247
thiobarbituric acid (TBA) technique in the glacial acetic medium. The decomposition of lipid
248
hydroperoxide products can with TBA condensate producing red compounds at an absorbance
249
peak wavelength of 532 nm.
250
2.8 Histological assay of the midgut
251
The midgut being the primary tissue that deals with incoming meal is thought to produce,
252
secrete, and absorb most of the digestive enzymes in the body. It serves as a “gateway organ”
253
providing nutrients to the body. It also serves as an important defense site against microbial
254
pathogens and harmful chemicals with ingested food due to its exoskeleton lining making it a
255
site likely to be penetrated for pathogens (Lovett and Felder, 1990; Ruby et al., 1980). Thus, the
256
midgut was chosen as the site for histological assessment. The midgut segments after removal
257
from Bouin’s solution were dehydrated with different gradients of alcohol concentration, cleaned
258
in toluene and embedded in paraffin to make solid wax blocks. Sections (5 µm) were made using
259
rotary microtome, stained with hematoxylin and eosin (H&E), and examined under a light
260
microscope (Olympus, model BX51, Serial number: 9K18395, Tokyo, Japan). By using software
261
Image-Pro Plus, 6.3 (Media Cybernetics, Inc., Rockville, USA) following the procedure of
262
Bullerwell et al. (2016), the villus height (VH), villus width (VW) and muscle thickness (MT)
263
answerable to nutrient absorption were measured.
264
2.9 Gut Microbiota Community Discovery and Analysis
265
2.9.1 Gut DNA extraction and 16S rRNA amplification
266
The total genomic DNA was extracted from stool samples using the E.Z.N.A.™ stool DNA
267
Kit (Omega Bio-Tek, Norcross, GA, U.S.) following the manufacturer’s instructions. The 16S
268
rDNA V3-V4 region of the gene was amplified by PCR. The PCR conditions were 2 min at 95
269
°C followed by 27 cycles of 10 s at 98 °C, 30 s at 62 °C, and 30 s at 68 °C and finally 10 min at
270
68 °C using universal bacterial primers 341F ： CCTACGGGNGGCWGCAG; 806R ：
271
GGACTACHVGGGTATCTAAT, where the barcode is an eight-base sequence unique to
272
individual sample. The PCR reactions were carried out in triplicate 50 µL mixture containing 5
273
µL of 10 × KOD Buffer, 5 µL of 2.5 mM dNTPs, 1.5 µL of each primer (5 µM), 1 µL of KOD
274
Polymerase, and 100 ng of template DNA. High-throughput sequencing was performed using
275
Illumina Hiseq 2500 sequencing.
276
2. 9.2 Illumina Hiseq 2500 sequencing
277
Amplicons were extracted from 2 % agarose gels and purified using the AxyPrep DNA Gel
278
Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s
279
instructions and quantified using QuantiFluor-ST (Promega, U.S.). Purified amplicons were
280
pooled in equimolar and paired-end sequenced (2 × 250) according to standard protocols on an
281
Illumina platform.
282
2. 9.3 Accession number
283
The microbiota sequence data were exported as individual FastQ files and deposited in the
284
Sequence Read Archive (SRA) database of the National Center for Biotechnology Information
285
(NCBI). The obtained accession number was SRP172960.
286
2. 9.4 Sequencing analysis
287
The paired-end clean reads were jointed as raw tags using FLASH (v 1.2.11) (Magoč and
288
Salzberg, 2011) with a minimum 10 bp overlap and mismatch error rates of 2%. Noisy sequences
289
of raw tags were filtered by QIIME (v 1.9.1) (Caporaso et al., 2010) under specific filtering
290
conditions (Bokulich et al., 2013) to obtain high-quality clean tags. Clean tags were searched
291
against the reference database (http://drive5.com/uchime/uchime_download.html) to perform
292
Reference-based
293
(http://www.drive5.com/usearch/manual/uchime_algo.html). All chimeric tags were removed
294
and finally obtained effective tags for further analysis. Subsequently, effective tags were
295
clustered into operational taxonomic units’ (OTUs) of ≥ 97% similarity using UPARSE (Edgar,
296
2013), which led to the tag sequence with the highest abundance being selected as a reprehensive
297
cluster within each cluster. The representative sequences were classified into organisms by a
298
naive Bayesian model using RDP classifier (Wang et al., 2007) (version 2.2) based on SILVA
299
(Pruesse et al., 2007) Database (https://www.arb-silva.de/).
chimera
checking
using
UCHIME
algorithm
300
Taxonomic richness estimators and community diversity metrics were determined for each
301
library in Mothur (version 1.39.1, http://www.mothur.org/). Alpha diversity is applied to analyze
302
the complexity of species diversity for a sample through 6 metrics, including Observed-species,
303
Chao1, Shannon, Simpson, ACE, and Good-coverage. Two alpha diversity metrics such as
304
Chao1 and ACE were selected and used to identify Community richness, whereas another two
305
metrics, Shannon (Shannon, 1948) and Simpson (Mahaffee and Kloepper, 1997) were used in
306
assessing community diversity. LEfSe (Segata et al., 2011) was used to characterize microbial
307
differences of biological relevance between individual experimental groups. The LEfSe was
308
performed using an alpha value of 0.01 for both the factorial Kruskal-Wallis rank-sum test and
309
the pairwise Wilcoxon test. The different taxonomic level results were transformed to relative
310
abundance count to draw the bar plots.
311
2.10 Challenge study
312
2.10.1 Preparation of inoculum bacteria
313
The preparation of the pathogenic bacterium, Vibrio parahaemolyticus (provided by MOE
314
Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Life
315
Sciences, Sun Yat-sen University, Guangzhou, P. R. China) was conducted as previously
316
described (Amoah et al., 2019). Briefly, the bacterium was added to a 100 mL of LB in a 250 mL
317
flat-bottom flask which was then kept in a shaken incubator (180 rpm for 20 h at 37 °C). After
318
obtaining the cell pellets through centrifugation (4,930 × g at 4 °C for 10 m), they were washed
319
twice using PBS, and the concentration adjusted at 600 nm wavelength. Through the serial
320
dilution technique, the supernatant acquired were re-suspended in PBS to obtain graded doses
321
(106, 107, 108 and 109 cfu mL-1).
322
2.10.2 Calculation of fifty percent (50%) endpoint (Lethal Dose50)
323
A prior experiment lasting 14 days was conducted to determine the lethal dose 50 (LD50).
324
Briefly, 40 shrimps of similar sizes to the final experimental shrimp sizes using the graded doses
325
(106, 107, 108 and 109 cfu mL-1) of bacteria were injected with 0.2 ml V. parahaemolyticus (VP)
326
intramuscularly. Mortalities were observed daily throughout the trial to determine the appropriate
327
concentration of VP for the experiment. The LD50 calculated as previously described (Reed and
328
Muench, 1938) was 1.0 × 108 cfu mL-1.
329
2.10.3 Disease resistance test
330
After the eight weeks feeding trial, 10 shrimps were collected from each of the probiotic-
331
supplemented and control tanks and distributed into another 12 fiberglass tanks for a 14-day
332
challenge study. The shrimps were injected intramuscularly at their third abdominal segment
333
with 0.2 ml of the suspended bacteria (108 cfu mL-1) for the disease test. A group of 30 additional
334
shrimps of similar sizes to the experimental shrimps were injected with PBS for the negative
335
control (NC) during the disease test. The challenged shrimp were kept under observation, and the
336
mortality in each group recorded up to the 14th day of the challenge. The physiology changes of
337
the shrimps were checked daily, and the removal of dead shrimps was regularly done. The
338
following formula was used in calculating cumulative mortality (%) and relative percent survival
339
(RPS, %);
340
1. Cumulative mortality, % = 100 ×
341
2. RPS, % =100 × 1 −
342
2.11 Data analysis and statistics
[+
)
# #
+ +
)
)
! )
)
!
)
! "
)
]
;
.
343
All data, presented as mean ± standard error (mean ± S.E.), were subjected to a one-way
344
analysis of variance (ANOVA) using statistical package for social sciences (IBM SPSS v20.0,
345
Inc., 2010, Chicago, USA) followed by the testing of mean differences using Tukey's multiple
346
comparison tests. The value of P < 0.05 was set for statistical significance. The illustration of the
347
number of shared and unique species among groups was done by creating a Venn diagram.
348
Pheatmap was used in generating heatmap which presented the normalized abundance among
349
groups. Bar graphs were generated using GraphPad Prism (version 7, GraphPad Software, La
350
Jolla California USA) for windows.
351
3. Results
352
3.1 Growth performance, feed utilization, Survival, and whole body composition
353
After eight weeks of feeding, the growth performance between shrimps fed probiotic-enriched
354
diet and control diet is illustrated in table 2. Statistically, a significant (P < 0.05) increase in FW,
355
WGR, SGR, PER, and a decrease in FCR of L. vannamei were observed with increasing
356
inclusion of PP in the diet. As a result, the PP3 group obtained the highest compared to the PP0.
357
Correspondingly, a significant (P < 0.05) increase in CF and SR, was observed in the treated
358
group in comparison to the control.
359
The results of proximate whole body composition except moisture were found to be
360
significantly (P < 0.05) higher in the probiotic-treated with better elevation in PP3 group,
361
followed by PP2 and PP1 when compared to PP0. The reverse was recorded in the case of the
362
moisture content (Table 2).
363
3.2 Serum and hepatopancreas biochemical measurements
364
The effects of PP on serum and hepatopancreas immune responses and antioxidant activities
365
are presented in table 3. It was observed that, inclusion of PP in shrimp’s diet resulted in a
366
significant increase (P < 0.05) in T-AOC, LYZ, SOD, ACP in serum; AKP in liver; GSH-Px,
367
GSH in serum and hepatopancreas and a significant decrease (P < 0.05) in TG in serum; AST,
368
ALT and MDA in serum and hepatopancreas in comparison with the control.
369
Correspondingly, it was observed that increasing the inclusion of PP in shrimp’s diet revealed
370
a significant increase in the TP (Fig. 1A), ALB (Fig. 1B), and GLO (Fig. 1C) of the serum.
371
3.3 Evaluation of intestinal enzyme activity
372
Figure 2 illustrates the results of AMS, LPS, and TRP activities in the intestines of individual
373
group shrimps. Compared with the PP0 (control), the levels of the digestive enzyme activities
374
including AMS (Figure 2A), LPS (Figure 2B) and TRP (Figure 2C) were significantly increased
375
(P < 0.05) in the three probiotic treated groups, with PP3 group obtaining the highest.
376
3.4 Histological structure of the midgut
377
The shrimp mid-intestinal cell in the PP3 and PP2 were arranged closely, having a clearer cell
378
gap with their intestinal walls observed to have tall and wide VH and VW, respectively (Fig. 3 -
379
PP3 [Z] and PP2 [Y - a and b]). Though there were some distortions in the intestinal epithelial
380
cells in PP1 like the PP0 from the basement membrane compared to PP2 and PP3, it was
381
observed that the VH and VW were somewhat enhanced like as found in PP2 and PP3 group’s
382
intestine (Fig. 3 – PP1 [X]). Complete separation from the basement membrane and distortions
383
of cells were recorded in the PP0 group alongside its shorter intestinal villi compared to the other
384
groups (Fig. 3 – PP0 [V and W]).
385
Correspondingly, the total VH (Fig. 4A) and MT (Fig. 4C) significantly decreased (P < 0.05)
386
in the untreated group compared to the treated. It was revealed that only the PP3 had a
387
significantly higher (P < 0.05) VW compared to other groups. PP2, PP1, and PP0 had no
388
significant (P > 0.05) difference, although there was an enhancement in the PP2 and PP1
389
compared to the PP0 (Fig. 4B).
390
3.5 Microbiota of the gut analysis
391
3. 5.1 Microbiota of the intestine richness and diversity analysis
392
The results from the LEfSe analysis are presented in a circular cladogram (Figure 5). It was
393
observed that shrimps of a) PPO group had higher relative abundance belonging to the NS10
394
marine group, as well as genera Actibacter, Flavobacterium sp. NBRC 101627, Acinetobacter
395
bayiyi, b) PP1 group had higher relative abundance belonging to class Plantomycetacia, order
396
Plantomycetales, family Plantomycetaceae, Pir4 lineage, c) PP2 group had a higher relative
397
abundance of order Cellvibrionales, family Cellvibrionaceae, and d) PP3 group had a higher
398
relative abundance of phylum Candidatus-Moranbacteria.
399
The OTUs and Alpha diversity metrics of the intestinal microbiota in L. vannamei are
400
presented in table 4. OTUs were observed to be significantly higher in the probiotic-treated
401
group (P < 0.05) than the untreated (PP0). PP0 attained the least OTU (Table 4). The probiotic-
402
treated groups were observed to have a significantly higher (P < 0.05) chao1 and ACE estimator
403
metrics than the untreated. Moreover, the Shannon and Simpson estimator metrics ranging from
404
4.98 to 5.45 and 0.93 to 0.96 respectively saw the probiotic-treated group to be significantly
405
lower (P < 0.05) than the untreated (Table 4). A total of 302 OTUs coincided in all the four
406
treatments with the analysis of a Venn diagram. It was observed that the number of core OTUs in
407
the PP1 (147) was the highest, followed by PP2 (137), then PP3 (122) with the least going for
408
PP0 (77) (Figure 6).
409
3.5.2 Microbial community change analysis and comparison
410
Different bacterial phyla were observed in the intestines of shrimps. The dominant phyla in
411
the intestinal microbiota were Proteobacteria, followed by Bacteriodetes, and Planctomycetes in
412
the clustered four groups. A significant increase (P < 0.05) of the relative abundance of
413
Proteobacteria in PP2 group was observed, which was followed by PP3, PP1 and lastly decreased
414
(P < 0.05) in the PP0 group. The relative abundance of Bacteriodetes was significantly higher (P
415
< 0.05) in the probiotic-treated group but decreased in the untreated group (P < 0.05) which was
416
in contrast to as observed in Firmicutes phylum (Figure 7).
417
At the class level, Gammaproteobacteria, Flavobacteriia, and Alphaproteobacteria were the
418
most relatively abundant bacteria irrespective of the group. PP1 and PP2 group were found to
419
have highly abundant Gammaproteobacteria, followed by PP3, with PP0 experiencing the least
420
abundance. However, Flavobacteriia was observed to be highly significant (P < 0.05) in L.
421
vannamei’s intestine of PP0 group, which was followed by the PP2, PP3 groups and then lastly
422
the PP1 group (Figure 8).
423
The heatmap analysis of L. vannamei’s intestinal bacterial abundance at the genus level
424
indicated that Spongiimonas, Shewanella, Muricauda, Halobacteriovorax, Shimia, and Kangiella
425
bacteria were more abundant in the untreated group than in the three probiotic-treated groups
426
(Figure 9). It was realized at the genus level that, relative to all the groups, Vibrio and Ruegeria
427
were the most abundant (Figure 10). Among these genera, the abundances of Vibrio,
428
Photobacterium, Tenacibaculum, Kangiella, Spongiimonas, Muricauda, Halobacteriovorax, and
429
Shewanella significantly decreased in the probiotic-enriched group relative to the control (P <
430
0.05). In dissimilarity to this, the abundance of Ruegeria, and Pseudoalteromonas significantly
431
increased in shrimp groups exposed to probiotic treatment (P < 0.05) (Figure 10).
432
3.6 Challenge test
433
After challenge with V. parahaemolyticus for 14 days, the cumulative mortality rates of the
434
untreated group (PP0) was significantly higher (P < 0.05) than those fed with probiotic-treated
435
diets (Fig. 11). At the end of the challenge test, the cumulative mortality was significantly (P <
436
0.05) lower in the probiotic-treated groups than in the control group and in the order 77 %, 43 %,
437
30 %, and 17 % for shrimp fed with PP0, PP1, PP2, PP3 diets respectively (Fig. 11). The relative
438
percent survival (RPS %) was highest in the PP3 (78.3 %), followed by PP2 (60.9 %), and PP1
439
(43.5%) group.
440
4 Discussion
441
Probiotics’ supplementations in diets have recently been increased in meeting the demands as
442
the best substitute for antibiotics. Undeniably, field research, aside unveiling the improvement
443
probionts have towards growth, modulation of immune response, antioxidant activities, and
444
disease resistance to host aquatic organisms (Buruiană et al., 2014; Farzanfar, 2006) have
445
established that the gut microbial composition is strongly affected by dietary supplementation of
446
these same probionts (Lazado et al., 2015). Paenibacillus polymyxa (PP) has been reported to
447
improve growth performance, immune response, and disease resistance in aquatic animals
448
(Gupta et al., 2016a, 2014). Nonetheless, there is a paucity of information on PP’s ability in the
449
aquaculture industry. It is thought that PP’s supplementation owing to the beneficial properties
450
they possess can improve the intestinal microbial composition and morphology. Thus, in this
451
study, L. vannamei was fed different doses of P. polymyxa to evaluate its effect on the growth,
452
immune and antioxidant enzyme activities, resistance against V. parahaemolyticus infection,
453
intestinal morphology and microbiota.
454
Following eight weeks supplementation with PP in L. vannamei’s diets, it became known that
455
increasing inclusion of PP caused a significant increase in the SR, FBW, WGR, SGR, CF, PER,
456
whole body proximate analysis (crude protein, crude lipid, ash) and a significant decrease in the
457
FCR and moisture content of the whole body composition analysis. The whole body proximate
458
composition analysis serves as useful pointers of physiological wellness of animals and the meat
459
quality as well (Vijayavel and Balasubramanian, 2006). Corresponding works showing increase
460
in growth and whole body composition have been previously documented on Fenneropenaeus
461
indicus (Ziaei-Nejad et al., 2006), Penaeus monodon (Boonthai et al., 2011), Litopenaeus
462
vannamei (Balcázar et al., 2007; Shen et al., 2010; Wang, 2007; Zokaeifar et al., 2014),
463
Macrobrachium resenbergii (Saad et al., 2009), Cyprinus carpio (Gupta et al., 2016a), Labeo
464
rohita (Kumar et al., 2006) and Oreochromis niloticus (Sutthi et al., 2018). Further studies on the
465
gut morphology and microbiota, digestive enzyme activities, immune response, and antioxidant
466
activities were elucidated to ascertain what led to such changes stated above.
467
In the present study, the analysis of serum and hepatopancreas immune and antioxidant
468
indices such as TP, ALB, GLO, TG, LYZ, SOD, T-AOC, GSH, GSH-Px, AST, ALT, ACP,
469
AKP, and MDA which is known to play crucial roles in infectious agents’ defense were
470
conducted. Serum TP, ALB and GLO were observed to be significantly higher in the probiotic-
471
treated group than the untreated which was in agreement with other studies on L. vannamei using
472
B. coagulans Sc8168 (Zhou et al., 2009) and Lactobacillus sp. (Mg and Ammani, 2015). Serum
473
TG levels are intensely associated with components of lipid in diets. Thus, decreasing the lipid
474
composition of human food is vital in reducing the cardiovascular disorder rates. In this study,
475
serum TG in the probiotic-treated group significantly decreased with increasing inclusion levels
476
which was in agreement with other findings (Wu et al., 2015). Little information on serum TG is
477
known; hence, we suggest much more research using probiotics be done.
478
ACP and AKP play vital roles in regulating the immune system. ACP is noted for intracellular
479
breaking down of phagocytized antigens whiles AKP also hydrolyzes bounds of phosphoesters in
480
diverse organic compounds such as lipids, proteins, and carbohydrates (Mohapatra et al., 2013;
481
Roubaty and Portmann, 1988). Yi et al. (2018) reported a significant increase in the ACP and
482
AKP after dietary supplementation of Bacillus velezensis JW than the control. Similarly, PP
483
supplementation increased the serum ACP and hepatopancreas AKP activity in L. vannamei in a
484
dose-dependent manner.
485
The damage of tissues in aquatic animals is a developing problem due to their underlying liver
486
injury. AST and ALT activities are regarded as reliable biomarkers of hepatotoxicity in the
487
serum and tissues, including liver (Kamada et al., 2016). Higher AST and ALT levels in serum
488
or liver tissues depict tissue dysfunctioning and damage (Cheng et al., 2018). This study showed
489
a decreasing trend of serum and hepatopancreas AST and ALT activities with increasing
490
inclusion of probiotics in diet indicating the ability of PP to reduce the toxicity in shrimps,
491
similar to as previously described (Sutthi et al., 2018).
492
LYZ being a humoral component, has bactericidal activity against infectious diseases because
493
they can break down the polysaccharide bacteria wall (Li et al., 2015). Shrimps, like other
494
aquatic animals, are susceptible to reactive oxygen attacks. Antioxidant indices such as T-AOC,
495
SOD, GSH, and GSH-Px aid in repairing the damages caused by free radicals (Martínez-Álvarez
496
et al., 2005). T-AOC is used in detecting oxidative stress in the serum and tissues including liver
497
(Dawood et al., 2016) whereas SOD generates cellular H2O2 to defend organisms against
498
microbial infection (Shen et al., 2010). GSH and GSH-Px mainly exploit the detoxification of
499
toxic hydrogen peroxides and other peroxides such as lipid hydroperoxides (Couto et al., 2013;
500
Wang et al., 2017) whereas MDA content is considered to be an indicator of the extent of lipid
501
peroxidation portraying the toxic processes caused as a result of free radicals (Yang et al., 2017).
502
In the present 8-weeks study, all probiotic-treated groups exhibited a significant increase of LYZ
503
and SOD in the serum; GSH, GSH-Px in both serum and hepatopancreas, and a significant
504
decrease in the MDA in both serum and hepatopancreas. This signifies the efficiency of PP
505
supplementation in enhancing immune and antioxidant activities such as observed in previous
506
studies (Gupta et al., 2016a; Liu et al., 2017; Shen et al., 2010; Zokaeifar et al., 2014).
507
In spite of the importance of the intestine in digestion, absorption, and defense, scanty of
508
information on probiotics’ effect on the morphological changes of the intestinal structure has
509
been recognized (Merrifield et al., 2010). A significant increase in the midgut MT, VH, and VW
510
of the probiotic-treated group in animals have been established (Asaduzzaman et al., 2018;
511
Ranadheera et al., 2014) similar to the observation in this study. Since taller and wider intestinal
512
microvilli have been reported as indications for higher absorption of nutrients (Kristiansen et al.,
513
2011), the increase in .midgut MT, VH, and VW of the probiotic-treated group can be the reason
514
why there were improved growth performance, feed utilization, and whole body composition in
515
the treated group.
516
The gut microbiota owing to the immense contributions towards shaping the intestinal
517
structure by digestion of food, absorption of nutrients, competing and conquering of other
518
unwanted microbes to improve the survival and health status of organisms has gained much
519
attention recently (Claus et al., 2016; Sugita et al., 1997). A lot of research has established
520
probiotics’ ability to change the microbial composition of the gut concerning the abundance of
521
opportunistic pathogens and or beneficial microbes (O’shea et al., 2012). In this study, barcoded
522
16S rRNA gene Illumina high-throughput sequencing was used to study the intestinal microbial
523
composition and relative abundance. Our results showed that dietary PP shaped the diversity of
524
the gut microbiota.
525
Following the eight weeks supplementation with PP, it was observed that the most relatively
526
abundant bacterial species in the intestine of shrimps at the phylum level were Proteobacteria and
527
Bacteriodetes, which also are portrayed to be the ordinarily dominant bacteria in most growth
528
stages of shrimps (Hou et al., 2017; Huang et al., 2016; Suo et al., 2017). Additionally, in
529
dissimilarity to the abundance of Bacteriodetes, the bacterial abundance in Firmicutes and
530
Cyanobacteria were observed to decrease significantly in the probiotic-treated group, unlike the
531
untreated. Higher Firmicutes abundance was witnessed in obese mice, which were concurrent
532
with a relatively low abundance of Bacteriodetes (Backhed et al., 2004; Turnbaugh et al., 2008).
533
Moreso, within the Bacteriodetes phylum, Bacteroides thetaiotaomicron has also been observed
534
to improve the host’s nutrient uptake and processing (Hooper, 2001). Cyanobacteria, known as
535
“blue-green bacteria” are photosynthetic microorganisms occurring naturally on the surface of
536
water (Zhao et al., 2006). Reports suggest that low abundance of Cyanobacteria is advantageous
537
to the growth of host organism (Jia et al., 2016) whereas higher abundance is detrimental to
538
aquatic animals’ health as a result of the production of hepatotoxic microcystins (Kang et al.,
539
2012). Research also suggest them to produce lipopeptides that are cytotoxic, which in turn
540
causes cell necrosis (Howard et al., 2012). Therefore the increase in Bacteriodetes and the
541
decrease in Firmicutes and Cyanobacteria may explicate that PP supplementation can interrupt
542
and shape the gut microbial composition in L. vannamei positively.
543
At the class level, regardless of the experimental treatment, Gammaproteobacteria accounted
544
for the majority of bacteria, which was followed by Flavobacteriia and Alphaproteobacteria
545
abundance. This was in accordance to previous research conducted on shrimps (Buchan et al.,
546
2005; Duan et al., 2018; Gao et al., 2014; Xiong et al., 2015; Zheng et al., 2016) signifying the
547
abundance of these bacteria in the gut of shrimps.
548
It was observed at the genus level in our study that irrespective of the treatment fed to L.
549
vannamei, Vibrio, Ruegeria, Photobacterium, Pseudoalteromonas, Tenacibaculum, and
550
Maribacter bacteria were observed to be relatively abundant, similarly to as previously reported
551
(Amoah et al., 2019; Moss et al., 2000; Suo et al., 2017; Zheng et al., 2016). However, some of
552
the genera found in other studies including Meridianimaribacter (Zheng et al., 2016), and
553
Acinetobacter, Candidatus, Aeromonas, Pseudomonas, Gemmobacter, Bacilloplasma, Shinella,
554
Rhodobacter (Zhang et al., 2014) were absent in the present study. Vibrio, Photobacterium,
555
Tenacibaculum, and Shewanella genera which were found to be highly significant in the
556
untreated group than the treated, have been noted in previous studies as opportunistic pathogens
557
causing stress and disease infection in animals (Alderkamp et al., 2007; Miao et al., 2018; Zheng
558
et al., 2016). For example, Tenacibaculum maritimum found in diseased fish has been associated
559
with causing tenacibaculosis (Avendaño-Herrera et al., 2006). Moreso, Shewanella genus found
560
in the Shewanellaceae family are observed to possess foul-smelling compounds such as H2S and
561
are also known to cause spoilage of seafood (Vogel et al., 2005). Wright et al. (2016) also noted
562
Shewanella spp. as having the ability to reduce the amount of manganese, a microelement
563
necessary for bone formation and regeneration of red blood cells. Thus, the increase in its
564
abundance in the untreated group’s gut might be the reason for lower growth performance and
565
feed utilization.
566
Also, Ruegeria and Pseudoalteromonas genera regarded as beneficial microbes were
567
significantly higher in the probiotic-treated group, unlike the untreated in the present study.
568
Ruegeria shows triestherase activity (Yamaguchi et al., 2016) and antagonistic activity on Vibrio
569
anguillarium (Porsby et al., 2008) signifying how beneficial they are to host organisms. Also,
570
reports show Pseudoalteromonas species being used as probiotics (ten Doeschate and Coyne,
571
2008) because they produce chemical compounds such as amylase, protease, phospholipases and
572
extracellular materials which aid in inhibiting the settlement and metamorphosis Shewanella
573
(Cadman and Eichberg, 1983; Gavrilovic et al., 1982; Venkateswaran and Dohmoto, 2000).
574
Consequently, we suggest that Ruegeria and Pseudoalteromonas abundance in the probiotic-
575
treated diet might be due to the probiotic’s ability to compete and conquer the growth of
576
pathogenic bacteria present, which led to the beneficial bacteria’s abundance (O’shea et al.,
577
2012).
578
Digestive enzymes including AMS, LPS, and TRP, play a vital role in breaking down
579
polymeric macromolecules into smaller building blocks to facilitate nutrient absorption (Duan et
580
al., 2017; Gobi et al., 2018; Svendsen, 2000). In our study, dietary PP supplementation positively
581
increased the digestive enzymes AMS, LPS, and TRP, which is supported by other findings
582
(Gobi et al., 2018; Ziaei-Nejad et al., 2006; Zokaeifar et al., 2014). This increase can be
583
attributed to the presence of beneficial bacteria such as Ruegeria which shows triestherase
584
activity (Yamaguchi et al., 2016) and Pseudoalteromonas which produces amylase, protease,
585
phospholipases (Cadman and Eichberg, 1983; Gavrilovic et al., 1982; Venkateswaran and
586
Dohmoto, 2000). Accordingly, the higher increase in the digestive enzymes of the probiotic-
587
treated group can be the reason for the improvement of their growth (Duan et al., 2017).
588
Numerous studies reveal probiotics’ ability to grant shrimps natural resistance against
589
pathogens providing higher survivability (Das et al., 2013; Vaseeharan and Ramasamy, 2003).
590
Vibrio species noted as opportunistic pathogens which have a dominant flora in the various
591
stages of L. vannamei’s developments are reported to cause lots of disease to aquatic organisms
592
and humans (Moss et al., 2000; Suo et al., 2017). The early mortality syndrome (EMS) or acute
593
hepatopancreatic necrosis syndrome (AHPNS) reported to be an emerging serious disease of
594
cultured shrimp including L. vannamei and P. monodon, caused by the Vibrio parahaemolyticus
595
strain, has affected Southeastern Asian shrimp farms since its occurrence (FAO/MARD, 2015;
596
NACA-FAO, 2012; Tran et al., 2013). A fourteen-day challenge using V. parahaemolyticus after
597
the 8 weeks feeding trial led to a significant increase in the cumulative mortality of the untreated
598
group compared to the probiotic-treated which was as well in line other research conducted
599
(Balcázar et al., 2007; Ramalingam and Shyamala, 2006; Zokaeifar et al., 2012). The reason can
600
be attributed to the higher abundance of opportunistic pathogens in the untreated group after the
601
microbial intestinal composition analysis.
602
Conclusion
603
This study demonstrated that the supplementation of PP modulated the growth performance,
604
whole body proximate composition, the immune and antioxidant activities, the intestinal
605
morphology, the composition of the intestine microbiome in L. vannamei with some species of
606
beneficial bacteria becoming more abundant whiles opportunistic pathogens decreased in
607
abundance. Also, PP increased the resistance activity against V. parahaemolyticus infection.
608
These results suggest that PP may benefit intestine health regulation in L. vannamei, and is a
609
candidate for use as a feed additive in shrimp culture. The optimal dietary PP supplementation
610
dosage 108 cfu g-1 feed should be used.
611 612
613
Acknowledgment
614
We are grateful to the support given by Professor Sun Cheng-Bo (Key Laboratory of
615
Crustacean Genetic Breeding and Aquaculture Research Center of Guangdong Ocean
616
University) for providing us with the shrimps used in this current study. We are also appreciative
617
to the support given by Dr. E. D. Abarike (Key Laboratory of Control for Diseases of Aquatic
618
Animals of Guangdong Higher Education Institutes) who helped in the bacterial preparation for
619
feed and disease test. Also, the authors thank the laboratory members for experimental material
620
preparation and all authors to whom we cited in this research work. We are also grateful to
621
Editor and Reviewers for their reasonable criticisms and suggestions, making this study a
622
genuine one.
623
Funding
624
This study was financially supported by the Science and Technology Department of
625
Guangdong Province (2015A020209170); Marine & Fisheries Department of Guangdong
626
Province (A201608C06); the Science and Technology Bureau of Zhanjiang (2016A3022); and
627
the China Agriculture Research System (CARS-47).
628
Additional Information
629
Competing Interests:
630 631
The authors affirm that the experiment was conducted in the absence of any commercial or financial relationships that might be construed as a potential conflict of interest.
632
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633
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Tables Table 1: Dietary formulation and nutritional composition of the basal diet Ingredients
Composition (%)
Brown fish meal
a
26.0
a
Soybean meal
13.0
Peanut meal
8.0
Wheat flour
a
Corn gluten meal
26.0 a
Shrimp shell meal Soybean oil Fish oil
9.0 a
a
a
6.0 1.0 1.0
a
Soy lecithin
1.0
Vitamin premixb
1.0
Mineral premixc
1.0
Choline chloridec
0.5
Vitamin Ca
0.1
Ca (H2PO4)2d
1.5
Carboxymethyl cellulosee
1.0
Microcrystalline Cellulosee
3.9
Total
100
Nutrient Index (Dry matter) Crude protein
Proximate composition (%) 40.65
Crude lipid
7.7
Crude ash
10.9
Moisture
10.31
a
Ingredients acquired from Zhanjiang HaiBao Feed Factory, Zhanjiang, Guangdong, China.
b
Vitamin premix supplied the following per kg of the diet: vitamin A, 22,500 IU; vitamin D3, 6,000 IU; vitamin E,
200 mg; vitamin K3, 40 mg; vitamin B1, 30 mg; vitamin B2, 45 mg; vitamin B6, 35 mg; vitamin B12, 0.25mg; calcium pantothenate, 150 mg; niacin, 225 mg; folic acid, 12.5 mg; biotin, 0.5 mg; inositol, 500 mg (Acquired from Zhanjiang Yuehai Feed Co. Ltd., Guangdong, China)
c
Mineral premix provided the following per kilogram of diet: Fe, 60 mg; Zn, 24 mg; Mn, 16 mg; Cu, 1.4 mg; Co,
0.2 mg; Se, 0.1 mg; I, 0.2 mg (Acquired from Zhanjiang Yuehai Feed Co. Ltd., Guangdong, China) d e
Acquired from Shanghai Macklin Biochemical Co. Ltd., 1288 Canggong Rd., Shanghai, China.
Acquired from Shantou Xilong Chemical Factory, Guangdong, China.
Table 2: Effects of different supplementation levels of P. polymyxa on the growth performance of L. vannamei. Indices
PP0 (Control)
PP1
PP2
PP3
Growth index IBW (g)
0.58 ± 0.001
0.58 ± 0.001
0.58 ± 0.000
0.58 ± 0.001
FBW (g)
6.45 ± 0.02a
6.69 ± 0.11ab
6.99 ± 0.14b
7.46 ± 0.04c
1018.75 ± 2.64a
1059.70 ± 19.02ab
1111.07 ± 24.13b
1193.27 ± 6.15c
SGR (%/day)
4.31 ± 0.00a
4.38 ± 0.03ab
4.45 ± 0.04b
4.57 ± 0.01c
FCR
2.71 ± 0.03c
2.29 ± 0.06b
2.25 ± 0.02b
1.97 ± 0.02a
SR (%)
81.67 ± 2.20a
95.83 ± 2.20b
95.00 ± 1.44b
98.33 ± 1.67b
PER
0.98 ± 0.01a
1.16 ± 0.03b
1.19 ± 0.01b
1.37 ± 0.01c
CF (%)
0.76 ± 0.03a
1.25 ± 0.02bc
0.96 ± 0.11ab
1.45 ± 0.12c
WGR (%)
Body composition (% DM) Moisture
77.04 ± 0.26b
73.05 ± 0.37a
74.09 ± 0.46a
72.90 ± 0.38a
Crude protein
71.78 ± 1.00a
77.60 ± 1.10bc
75.02 ± 0.24ab
81.21 ± 0.85c
Crude lipid
6.88 ± 0.06a
7.46 ± 0.06b
7.67 ± 0.05b
8.00 ± 0.10c
Ash
12.98 ± 0.04a
14.35 ± 0.05b
14.35 ± 0.06b
14.92 ± 0.41b
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Where; IBW, initial body weight; FBW, final body weight; WGR, weight gain rate; SGR, specific growth rate; FCR, feed conversion ratio; CF, condition factor; PER, protein efficiency ratio; SR, survival rate. PPO = basal diet void of P. polymycoxa supplementation; PP1 = basal diet with 106 cfu P. polymycoxa g-1 feed; PP2 = basal diet with 107 cfu P. polymycoxa g-1 feed; PP3 = basal diet with 108 cfu P. polymycoxa g-1 feed.
Table 3: Effects of different supplementation levels of P. polymyxa on immune and antioxidant factors in serum and hepatopancreas of L. vannamei. Indices
PP0 (Control)
PP1
PP2
PP3
TG (mmol L-1)
0.56 ± 0.02c
0.47 ± 0.00b
0.44 ± 0.01ab
0.40 ± 0.02a
T-AOC (mmol L-1)
0.59 ± 0.01a
0.65 ± 0.01b
0.62 ± 0.01ab
0.68 ± 0.01c
GSH-Px (U mL-1)
71.36 ± 0.87a
79.82 ± 2.87a
124.01 ± 6.74b
113.02 ± 3.23b
GSH (µmol L-1)
19.08 ± 0.46a
26.79 ± 1.23b
25.50 ± 0.83b
25.75 ± 1.55b
LYZ (U mL-1)
10.67 ± 0.67a
13.67 ± 0.88b
13.00 ± 0.58ab
15.67 ± 0.33b
SOD (U mL-1)
782.46 ± 1.41a
1215.04 ± 19.72c
959.40 ± 21.05b
1377.44 ± 23.49d
ACP (U 100mL-1)
14.33 ± 0.51a
22.77 ± 0.08b
20.79 ± 1.48b
28.87 ± 0.15c
AST (U L-1)
13.27 ± 0.23b
10.42 ± 1.09a
10.00± 0.63a
11.71 ± 0.47ab
ALT (U L-1)
23.19 ± 1.12b
15.85 ± 0.62a
13.31 ± 1.11a
14.32 ± 0.92a
MDA (nmol mL-1)
5.00 ± 0.12c
1.16 ± 0.12a
1.69 ± 0.16ab
1.99 ± 0.07b
GSH-Px (U mgprot-1)
325.78 ± 4.30a
368.28 ± 19.75ab
341.02 ± 3.73a
413.99 ± 15.78b
GSH (µmol gprot-1)
15.87 ± 0.83a
19.79 ± 1.57ab
17.74 ± 1.32ab
22.17 ± 1.05b
AKP (KU gprot-1)
11.76 ± 0.65a
16.83 ± 0.63b
15.93 ± 1.08b
16.93± 0.72b
AST (U gprot-1)
1.79 ± 0.09b
1.05 ± 0.09a
0.89 ± 0.02a
0.74 ± 0.5a
ALT (U gprot-1)
1.29 ± 0.11b
0.67 ± 0.09a
0.70 ± 0.08a
0.61 ± 0.02a
MDA (nmol mgprot1 )
4.96 ± 0.08a
1.27 ± 0.02a
1.38 ± 0.07a
1.15 ± 0.02a
Serum
Hepatopancreas
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Where; TG, triglyceride; TAOC, Total antioxidant capacity; GSH-Px, glutathione peroxidase; GSH, glutathione; LYZ, Lysozyme; SOD, superoxide dismutase; ACP, Acid phosphatase; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; AKP, Alkaline phosphatase; MDA, malondialdehyde. Abbreviations are as defined in table 2.
Table 4: OTUs, diversity metrics estimated OUT richness for the intestinal bacterial analysis of L. vannamei fed different levels of P. polymyxa. Reads Groups
Richness estimators Tags
Raw
Valid
PPO (Control)
70880
69131
65883
PP1
120615
117394
PP2
123795
PP3
112840
Diversity estimators
OTUs Chao1
ACE
Shannon
Simpson
309.67 6.57a
432.12 ± 33.91a
440.92 ± 37.76a
5.45 ± 0.15c
0.96 ± 0.002c
112064
525.33 ±3.18c
667.71 ± 30.01b
621.99 ± 38.12b
4.19 ± 0.03a
0.88 ± 0.002a
120245
109649
510.00 ± 5.51c
659.34 ± 6.14b
640.98 ± 2.38b
4.79 ± 0.06b
0.93 ± 0.006b
109523
103148
470.67 ± 13.02b
623.79 ± 7.47b
593.50 ± 35.74b
4.98 ± 0.01ab
0.93 ± 0.009ab
Note: Data are mean values of three replicates ± S.E. Means in the same row with no superscripts do not differ significantly (P > 0.05) by Tukey’s HSD test. Abbreviations are as defined in table
Figure Legends: Fig. 1. Effects of different supplementation levels of P. polymyxa on serum total protein (A), albumin (B), and globulin (C) contents of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. PPO = basal diet void of P. polymyxa supplementation; PP1 = basal diet with 106 cfu P. polymyxa g-1 feed; PP2 = basal diet with 107 cfu P. polymyxa g-1 feed; PP3 = basal diet with 108 cfu P. polymyxa g-1 feed. Fig. 2. Effects of different supplementation levels of P. polymyxa on the amylase (A), lipase (B), and trypsin (C) intestinal enzyme activities of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 3. Photomicrographs of the midgut tract cross-cutting of L. vannamei fed at different PP concentration with hematoxylin and eosin staining to show changes in the intestinal epithelial cells and microvillus. Arrows show the pathological changes. Magnification was 200×, and the scale represents 200 µm. PP0: arrow V shows the intestinal epithelial cells completely detached from the basement membrane and arrow W also shows some distortions in the epithelial cells; PP1: X shows some detachment of intestinal epithelial cells from the basement, however, there are some tall villus height and width observed; PP2: Y shows the close integration of the intestinal epithelial cells and the basement membrane, a shows the villus height, b shows the villus width; PP3: Z shows the close integration of the intestinal epithelial cells and the basement membrane. Abbreviations are as defined in figure 1. Fig. 4. Effects of different supplementation levels of P. polymyxa on the villi height (A), villi width (B), and muscle thickness (C) measurements of the midgut tract cross-cutting of L. vannamei. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 5. Circular cladogram of the intestinal microbiota reporting results of the uppermost relative abundance from the LEfSe analysis. The identified OTU are distributed according to phylogenetic characteristics around the circle. The dots close to the center represent the OTU on phylum level while the outer circle of dots characterizes the OTU on the genus level. Node size is proportional to the average abundance; color indicates the relative concentration of the clusters. Abbreviations are as defined in figure 1. Fig. 6. Venn diagram displaying compartmental OTU distribution of the core intestinal microbiota shared by L. vannamei fed four diets supplemented with P. polymyxa probiotic bacteria. Three hundred and two OTUs were identified as core microbiota for all diets in the intestines of L. vannamei. Abbreviations are as defined in figure 1.
Fig. 7. Relative abundance of the major intestinal bacteria of L. vannamei at the Phylum level. Vertical bars represent the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P < 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 8. Relative abundance of the major intestinal bacteria of L. vannamei at the Class level. Vertical bars represent the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P < 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 9. Heatmap of the abundance of L. vannamei intestinal bacteria at the genus level at different P. polymyxa concentration. Phylogenetic positions are projected by the OTUs, and the taxa of OTUs are listed on the right. Color intensity indicates the relative abundance of OTUs. Abbreviations are as defined in figure 1. Fig. 10. Relative abundance of major intestinal bacteria of L. vannamei at the genus level. Vertical bars represented the mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Data marked with no letters do not differ significantly (P > 0.05) among groups. Abbreviations are as defined in figure 1. Fig. 11. Cumulative mortality (%) of L. vannamei after 14 days post challenge with V. parahaemolyticus. NC, Negative control injected with PBS. Each line graph represents mean ± S.E. (Tukey’s HSD, P < 0.05; n = 3). Abbreviations are as defined in figure 1.
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Highlights The effects of potential probiotic Paenibacillus polymyxa ATCC 842 (PP) on shrimp, Litopeneaus vannamei were assessed. Dietary supplementation of PP enhanced fish growth, serum and hepatopancreas immune and antioxidant activities. Probiotic PP improved the digestive enzyme activities, intestinal morphology and shaped the gut microbiota composition. Probiotic PP increased the survival of shrimps challenged with V. parahaemolyticus infection. 108 cfu g-1 diet of PP was the optimal dietary supplementation level.