Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell and Molecular Biology (CMB)

Department

Fisheries, Animal and Veterinary Science

First Advisor

Marta Gomez-Chiarri

Abstract

Oysters are described as keystone species serving an important ecological role. As filter-feeders they help in maintaining water quality. Oyster reefs provide refuge and support to different organisms. The eastern oyster, Crassostrea virginica, native to the East Coast of United States and Gulf of Mexico is a part of the rapidly growing aquaculture industry. Aquaculture production depends on a healthy and constant supply of oyster larvae that are provided by hatcheries. Several hatcheries on the east coast that provide C. virginica seed to oyster farms face significant losses owing to Vibrio infections causing massive larval mortalities. Use of antibiotics is avoided due to possibility of development of antibiotic resistance. The probiotic bacteria, Phaeobacter inhibens S4 and Bacillus pumilus RI06-95 have been shown to successfully protect C. virginica larvae from V. coralliilyticus RE22 infection. Use of these probiotics in hatcheries can reduce mortalities due to disease thereby avoiding significant economic losses. In order to design best practices for probiotic use it is crucial to understand their mechanisms of action. There has been great progress in understanding the components of oyster immune system, its functioning in response to various stimuli and its uniqueness as compared to other organisms. This is in part due to availability of sophisticated tools like high throughput sequencing and various –omics analyses such as proteomics, genomics and transcriptomics and partly due to interest in controlling diseases affecting aquaculture. As such most of our knowledge is based on studies that focus on oyster-pathogen or oyster-environmental stimuli interaction. Little is known about the effect of bacteria other than pathogens on the oysters. Moreover, very little about larval immunity of eastern oyster, C. virginica. This is the first study to investigate the effect of both pathogen and probiotic bacteria on C. virginica larval immunity using transcriptomes. The aim of this study is to test the safety and efficacy of formulated probiotic Bacillus pumilus RI06-95 in a hatchery, understand the mechanisms of action of both probiotics and to characterize the effect of V. coralliilyticus RE22 infection on the larval immune system of eastern oysters.

Chapter 1 reviews the current knowledge of oyster immune system and the mechanisms of action of probiotics especially mechanisms related to immunomodulation of innate immunity. Previous studies have demonstrated successful protection of C. virginica larvae from V. coralliilyticus RE22 infection in a laboratory based setting as well as in a hatchery using laboratory grown cultures of probiotics. The ultimate use of the probiotics is in a hatchery setting, which would require easy to use and stable formulation of the probiotics instead of time consuming laboratory-grown probiotic cultures that are viable for only a short duration of time.

Chapter 2 discusses methods of formulation of probiotic Bacillus pumilus RI06- 95, testing the formulation in a hatchery and its effect on larval survival at the hatchery and post V. coralliilyticus RE22 experimental challenge in the laboratory. A spray dried formulation of Bacillus pumilus RI06-95 was both shelf-stable and effective in protecting C. virginica larvae from V. coralliilyticus RE22 challenge. The formulation did not show any adverse effects on the larvae during the course of the trial.

Chapter 3 investigates the host–pathogen interaction between C. virginica larvae and V. coralliilyticus RE22 using transcriptomes produced after experimental challenge. Exposure of larvae to the pathogen for 6 hours provided information of the changes in the larval oysterimmune system brought about by the pathogen in the early stages of disease. Overall, despite upregulation of several pattern recognition receptors, immune signaling pathways leading to the production of antimicrobial effectors, such as protease inhibitors and the pore forming protein perforin-2, were suppressed by V. coralliilyticus RE22. The transcriptomic evidence suggests that lack of an adequate immune response to thwart the infection of RE22, combined with a high metabolic load and decreased feeding, leads to large-scale mortalities of C. virginica larvae. This research allows for a better understanding of the disease process caused by V. coralliilyticus RE22 in larval eastern oysters.

Chapter 4 investigates the effect of exposure to non-pathogenic probiotic bacteria P. inhibens S4 and B. pumilus RI06-95 on the immune system of the host, C. virginica larvae. It presents evidence of immunomodulation of C. virginica larval immunity by both probiotic organisms. High upregulation of immune effectors such as serine protease inhibitors is seen in larval oysters after short exposures to the probiotic (6 and 24h) in the laboratory as well as after exposure for several days during a hatchery trial. Other important modulations that help larvae protect themselves from V. coralliilyticus RE22 infection include activation of pathogen receptors and signaling pathways, modulation of mucin genes, and upregulation of pore-forming protein perforin-2.

Chapter 5 summarizes and advocates the of use of probiotics in the larviculture of C. virginica and suggests their potential role in limiting vibriosis.

Available for download on Thursday, May 30, 2019

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