Document Type

Thesis - Open Access

Award Date

2019

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

William Gibbons

Keywords

Aquaculture, Litopenaeus vannamei, microbiome, Pacific Whiteleg shrimp

Abstract

Knowledge of the functional role of the gut microbiome in animal health and nutrition may provide solutions to shrimp aquaculture challenges, such as improving disease resistance and optimizing growth particularly with low cost feeds. Successful manipulation of bacteria found in the gut requires a deeper understanding of shrimp microbial communities and how their compositional structure is influenced by environmental conditions, and inherent host factors such as genetics. The initial research investigated the intestinal bacterial communities of the Pacific whiteleg shrimp (Litopenaeus vannamei) reared in pond systems compared to indoor aquaculture facilities as an exploration of the effects of aquaculture practices on the acquired gut microbiome. Ponds averaged a depth of 1.5 meters, with stocking densities maintained within a range typical of intensive production systems (30–60 shrimp/m3). Water chemistry testing was conducted weekly to monitor levels of TAN, nitrite, nitrate and alkalinity. These parameters were used to determine the rates of water exchange to maintain water quality, which ranged from 0% to 20%. Feed was offered at scheduled times during the day (5 a.m., 12 p.m. and 5 p.m.). Indoor production shrimp were maintained at 28 ± 1 °C in temperature-controlled tanks. Water management was carried out using separate recirculating aquaculture systems, one for each tank, utilizing fresh water processed by reverse osmosis, and then mixed with 28 g Marinemix (Marine Enterprises International, LLC., Baltimore, MD, USA) per liter of production water. Total ammonia nitrogen (TAN) levels were maintained at less than 3.0 mg/mL (NH3 ≤ 0.2), nitrite levels below 4.5 mg/mL, and nitrate levels never exceeded 100 mg/mL. Evaporated water was replaced with fresh water as needed to maintain salinity at 28 parts per thousand (ppt). Intestinal bacterial community profiles were different between each production system. Bacteria affiliated with Rhodobacteraceae (Proteobacteria) and Actinobacteria were significantly more abundant in indoor cultured shrimp (84.4% vs. 5.1%; 3.0% vs. 0.06%, respectively), while Vibrionaceae (Proteobacteria) (0.03% vs. 44.8%), Firmicutes (0.7% vs. 36.0%), Fusobacteria (0.0% vs. 7.9%), and Cyanobacteria (0.001% vs. 1.6%) were predominant in pond raised shrimp. The results indicate that aquaculture practices greatly influence the intestinal bacterial profile of whiteleg shrimp, and further suggest the bacterial communities of this economically important crustacean could be effectively manipulated using diet composition or environmental factors such as water chemistries. A subsequent research consisted of two experiments focused on two genetic families of Pacific whiteleg shrimp. One family was selected for specific pathogen resistance (Shrimp Improvement Systems, SIS), while the other genetic line was selected for growth (Oceanic Institute, OI). Both stocks of postlarvae juvenile shrimp were reared in a biosecure / indoor aquaculture facility under the same practices described in the prior study. Two genetic lines of Litopenaeus vannamei were used: Shrimp Improvement Systems (SIS, Islamorada, Florida, USA), selected for disease resistance, and Oceanic Institute (OI, Oahu, Hawaii, USA), selected for growth. During each trial, three replicate tanks were supplemented with a commercial probiotic, while the remaining three tanks did not receive any supplementation (controls). Stocking densities were maintained in the standard range of intensive production systems at 30–60 shrimp per cubic meter, with feed offered continuously. Production tanks (2,921 L) were maintained for 72 days with six tanks for each genetic family, three replicate tanks receiving probiotic infusions and three without. A commercial product, BioWish 3P, was obtained from BioWish Technologies (Cincinnati, Ohio). BioWish 3P contains Pediococcus acidilactici ≥ 1 x 108 cfu/g, Pediococcus pentosaceus ≥ 1 x 108 cfu/g, Lactobacillus plantarum ≥ 1 x 108 cfu/g, Bacillus subtilis ≥ 1 x 107 cfu/g. The freeze-dried bacterial cultures are delivered in an inert carrier, cereal food fines. BioWish 3P was added a rate of 0.73 ± 0.05 g per trial tank daily and was dosed with the feed offered over 24h. Microbiome sampling occurred on day 43 (prior to probiotic additions), day 57, and day 71 (28 days following initial probiotic introduction). Water management was maintained at 28 ± 1 °C and 28 ppt salinity utilizing artificial seawater. Total ammonia nitrogen, nitrite, and nitrate levels were maintained at less than 3.0 mg/mL (NH3 ≤ 0.2), below 4.5 mg/mL, below 100 mg/mL respectively. Considering that the gut microbiome of shrimp begins to develop immediately after hatching and is dependent on the initial diets and environmental conditions, it was not surprising to discover the initial phylum abundance between groups on day 43 differed, with a range of 72.3- 82.5% being Proteobacteria in the SIS shrimp compared to 61.1-65.1% in OI shrimp. The two genetic lines of shrimp revealed significant differences in gut microbiome by family and sampling time points following the inclusion of probiotics into the diet. After 28 days of treatment on tenure day 71, significant variation became evident in the second most abundant phylum in the SIS shrimp with Bacteroidetes increasing from 4.0% on day 43 to 8.6% on day 71 with probiotics, and from 5.6% on day 43 to 30.6% on day 71 without probiotics. By comparison, the second most abundant phylum in the OI shrimp was Firmicutes, increasing from 0.5% on day 43 to 22.1% on day 71 with probiotics, and from 2.8% to 36.7% without probiotics. As Pediococcus, Lactobacillus, and Bacillus-affiliated OTUs were found in only very low abundance or were undetectable in the gut of probiotic supplemented shrimp, bacterial species from the commercial probiotic formulation did not appear to efficiently colonize the shrimp gut in this study. However, their presence or absence did impact the development of gut bacterial communities through abundances. While future investigations will be necessary to uncover the mechanisms involved, it could be hypothesized that, even if probiotic bacterial species do not become established in high density in the gut of exposed shrimp, they produce metabolites that favor the establishment of certain bacterial species or OTUs over others. These results suggest that bacterial communities of this economically important crustacean can be effectively manipulated utilizing environmental conditions. They further indicate that development of direct fed microbial strategies to effectively manipulate the microbiome of this important seafood will likely need to take into serious consideration the genetic background of the shrimp genetic lines used in aquaculture production.

Library of Congress Subject Headings

Whiteleg shrimp.
Intestines -- Microbiology.
Microbial ecology.
Aquaculture industry.

Format

application/pdf

Number of Pages

122

Publisher

South Dakota State University

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Rights Statement

In Copyright