Interaction between fish probiotic roseobacters and the natural microbiota in aquaculture settings

Abstract

Aquaculture is the fastest growing protein producing sector in the world and this growth is required to feed the growing world population. Microbial diseases are a major bottle-neck in aquaculture, which must be controlled to avoid great, economic losses. Adult fish can be vaccinated against the most common bacterial diseases. However, the vaccines cannot be used on fish larvae because they have underdeveloped immune systems. Antibiotics are commonly used for acute treatment of infection, however, this increases the risk of antibiotic resistance dissemination. Therefore, more sustainable, preventive measures are sought and probiotics has been proposed as one of the solutions. Probiotics are “live organisms which when administered in adequate amounts confer a health benefit on the host” (FAO and WHO, 2001). Tropodithietic acid (TDA) producing members of the Roseobacter group, such as Ruegeria spp. and Phaeobacter spp., have potential as probiotics in aquaculture. They have repeatedly been isolated from aquaculture environments and they can reduce mortality of fish larvae challenged with pathogens. However, it is uncertain how the probiotic treatment affects the commensal microbiome of the larvae. The purpose of the present PhD project was to determine how probiotic Phaeobacter inhibens affect the natural microbiota in marine eukaryote systems related to aquaculture. Given that roseobacters are commonly found in complex communities of marine eukaryotes in nature and that they are indigenous to the aquaculture environment, the main hypothesis of this work is that P. inhibens can establish itself in microbiomes associated with aquaculturerelated eukaryotes and protect the host with minor impact on the commensal bacteria. In this study, 16S rRNA amplicon taxonomics was used to characterize the microbiota of different trophic levels – Tetraselmis suecica (microalga), Acartia tonsa (copepod), and Scophthalmus maximus (turbot) larvae – and determine the changes in diversity induced by treatment with probiotic P. inhibens. Interestingly, the structure of the microbial community associated with the lower trophic levels were shifted in the presence of P. inhibens, though not for the fish larval community. The effect was specific and targeted taxa closely related to the probiotic bacterium. Despite previous studies suspecting the live-feed to be vectors of infection, these microbiotas had low abundance of Vibrio spp. commonly causing disease in fish larvae. In contrast, the turbot egg microbiome were dominated by vibrios, however, these were suppressed after 24 hours incubation and kept stable - most likely due to inherent roseobacters or the added probiotic. In nature, members of the Roseobacter group are often found in association with marine eukaryotes such as algae and molluscs. Secondary metabolite production is believed to be involved in these interactions, though it is uncertain how they shape the microbiome. In microalgal blooms, roseobacters increase in abundance, which suggests that they play a role in the course of the bloom and they likely impact the microbiome. In this study, two model systems – Emiliania huxleyi (microalga) and Ostrea edulis (European flat oysters) – were used to study how the secondary metabolite producer P. inhibens affects the diversity and composition of the associated microbiomes. Roseobacters were indigenous to both communities and addition of P. inhibens caused substantial changes in the structure of the low-complexity microbiome of E. huxleyi, though not to the more complex oyster microbiomes. The impact was specific to vibrios and pseudoalteromonads, which were decreased in abundance. The role of TDA in host-bacteria, bacteria-bacteria interactions is unknown. A mode of action has been proposed for TDA, but it is based on studies of Escherichia coli rather than marine, non-TDA-producing bacteria which are more likely to encounter TDA in their surroundings. In this study, a transcriptomics approach was used to study how a sub-lethal concentration of TDA affected the fish and human pathogen, Vibrio vulnificus. Exposure to TDA triggered a defense response to reactive oxygen species and iron depletion in V. vulnificus. Furthermore, there were indications of switch to a biofilm phenotype, which could explain why inherent resistance and tolerance is rarely observed. This thesis concludes that TDA-producing P. inhibens causes minor impact on the microbiomes of various marine eukaryotes. The changes are highly specific to the commensal microbiome; in part decreasing related taxa, in part decreasing the abundance of putative pathogens such as vibrios. The molecular mechanism of TDA and role is still uncertain, but these data indicate that TDA induces a phenotypic switch in the target organism to protect the cells. Given the ease of introduction, the targeted effect, and the lack of resistance development, the application of P. inhibens as probiotic in aquaculture is highly promising.