Elsevier

Fish & Shellfish Immunology

Volume 86, March 2019, Pages 497-506
Fish & Shellfish Immunology

Full length article
Analysis of the gut and gill microbiome of resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss)

https://doi.org/10.1016/j.fsi.2018.11.079Get rights and content

Highlights

  • Selective breeding results in enhanced resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to F. psychrophiluminfection.

  • The gut microbiome of the ARS-Fp-R line is less diverse than that of the ARS-Fp-S. line.

  • Mycoplasma sp.constituted a majority of the gut microbiome in both lines, although it was more abundant in ARS-Fp-R.

  • An increased abundance of potential opportunistic pathogens was observed in the ARS-Fp-S line.

  • Stocking density had an impact on the trout microbial communities.

Abstract

Commensal microorganisms present at mucosal surfaces play a vital role in protecting the host organism from bacterial infection. There are multiple factors that contribute to selecting for the microbiome, including host genetics. Flavobacterium psychrophilum, the causative agent of Bacterial Cold Water Disease in salmonids, accounts for acute losses in wild and farmed rainbow trout (Oncorhynchus mykiss). The U.S. National Center for Cool and Cold Water Aquaculture has used family-based selective breeding to generate a line of rainbow trout with enhanced resistance to F. psychrophilum. The goal of this study is to determine whether selective breeding impacts the gut and gill microbiome of the F. psychrophilum-resistant as compared to a background matched susceptible trout line. Mid-gut and gill samples were collected from juvenile fish maintained at high or low stocking densities and microbial diversity assessed by 16S rDNA amplicon sequencing. Results indicate that alpha diversity was significantly higher in the mid-gut of the susceptible line compared to the resistant line, while no significant differences in alpha diversity were observed in the gills. Mycoplasma sp. was the dominant taxon in the mid-gut of both groups, although it was present at a decreased abundance in the susceptible line. We also observed an increased abundance of the potential opportunistic pathogen Brevinema andersonii in the susceptible line. Within the gills, both lines exhibited similar microbial profiles, with Candidatus Branchiomonas being the dominant taxon. Together, these results suggest that selectively bred F. psychrophilum-resistant trout may harness a more resilient gut microbiome, attributing to the disease resistant phenotype. Importantly, interactions between host genetics and environmental factors such as stocking density have a significant impact in shaping trout microbial communities.

Introduction

The microbiome has well established roles in pathogen exclusion and host immunity, including systemic and mucosal innate and adaptive immune responses and development of the immune system [[1], [2], [3]]. Across species, the intestinal microbiome is established at nascent developmental stages upon exposure to external environments. Under homeostatic conditions, primordial commensal microbes colonizing host mucosal surfaces must outcompete any other microorganisms present in the environment. Resident microbes possess an advantage in resource acquisition driving their evolution to better adapt in a specific host microenvironment, which in some cases provides benefits to the host organism. In response to these phenomena, species can adapt to select for those microorganisms that are most beneficial, resulting in microbial assemblies that are to a large degree unique to each species. Studies in teleost fish have supported this by identifying a 'core' gut microbiota in zebrafish (Danio rerio) [4], Atlantic salmon (Salmo salar) [5], and rainbow trout (O. mykiss) cultured in water recirculation systems [6].

Host genetics have been proposed to play a supporting role in the selection of the gut microbiome in humans and other mammals [[7], [8], [9], [10], [11]], while environmental factors have also been shown to largely contribute to host microbiome assembly [[12], [13], [14], [15]]. There have been some efforts to investigate factors that intrinsically influence microbiome assembly in fish, providing support for both host-associated and environmental factors. Host genetic factors that contribute to microbiome assembly have been well characterized in stickleback, as differences in MHC genotype have been shown to affect microbiome composition [16]. Additionally, a longitudinal microbiome analysis conducted on channel catfish (Ictalurus punctatus) characterized how the intestinal microbiota shifts during ontogeny and how diet and environmental microbes influence microbiota in this species [17]. Further work by this group showed that host genetic factors had a minimal impact on microbial composition [18]. Additionally, a study conducted on hatchery-reared and wild caught Atlantic salmon across various regions highlights diet and genetic factors as major contributors to microbiome assembly, particularly in the gut [19]. To further disentangle the contribution of host genetics and environmental factors shaping the fish microbiome, here we utilize a rainbow trout model in which two genetic lines of rainbow trout have been established by selective breeding that differ in susceptibility to a common environmental gram-negative pathogen, Flavobacterium psychrophilum.

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCWD), which is a major concern in the United States aquaculture industry affecting a range of cold-water fish species, including the commercially relevant rainbow trout (O. mykiss). Outbreaks of this pathogen have been reported across all areas of the world that contribute to salmonid aquaculture, posing a substantial threat to the future of this industry [20]. F. psychrophilum is a mucosal pathogen that typically infects the skin and gills of fish but also has the ability to adhere to and damage the intestinal epithelium [21,22]. Additionally, supplementing rainbow trout with probiotic bacteria that are able to colonize the gastrointestinal tract has been shown to decrease F. psychrophilum induced mortality [23]. Symptoms of BCWD in developed fish include necrosis of the caudal region, skin lesions, eroded fin tips, and loss of appetite. F. psychrophilum has a more pronounced effect on young fry, a condition referred to as rainbow trout fry syndrome. Rainbow trout fry syndrome is responsible for acute losses in trout farms worldwide, as the associated mortality rate is reported to be greater than 50% [20]. BCWD is becoming an increasingly difficult disease to treat, as F. psychrophilum strains have developed resistance to several commonly used antibiotics [[24], [25], [26]], and there is currently no commercially available licensed vaccine.

The National Center for Cool and Cold Water Aquaculture (NCCCWA) utilized family-based selective breeding to develop two distinctive genetic lines of rainbow trout that confer enhanced resistance (ARS-Fp-R), or susceptibility (ARS-Fp-S) to the pathogen F. psychrophilum [27]. Enhanced resistance to F. psychrophilum-induced mortality in the ARS-Fp-R line has been described, both in the laboratory setting and on trout farms [28,29]. Previous studies have begun to investigate possible host mechanisms that attribute to enhanced resistance. For instance, a strong correlation between resistance to F. psychrophilum and increased spleen size has been described, although this relation does not appear to translate to other common fish pathogens, such as Yersinia ruckeri [30]. Additionally, whole-body transcriptome analysis has identified numerous acute phase proteins and inflammatory cytokines that are differentially expressed in each line following challenge with F. psychrophilum [31]. Further work is needed to better characterize the mechanism(s) by which enhanced resistance is achieved in the ARS-Fp-R line.

In this paper, we begin to investigate whether ARS-Fp-R and ARS-Fp-S trout lines have different microbial assemblages associated with the gut and gills. Using 16S rDNA amplicon sequencing, we evaluate the effect of host genetics (F. psychrophilum resistance or susceptibility) as well as the effect of different tank conditions on trout microbiome assembly.

Section snippets

Animals and sampling

The Institutional Animal Care and Use Committee (Leetown, WV) reviewed and approved all animal husbandry practices and disease challenge protocols per standards set forth in the USDA, ARS Policies and Procedures 130.4.v.3 titled ‘Institutional Animal Care and Use 84 Committee’. Fish used in these experiments were from the 2017 Year Class and maintained as specific pathogen free as determined by biannual testing as previously described [28]. A total of 33 single sire-dam matings contributed to

Phenotype confirmation of disease resistance/susceptibility

The relative phenotype of the two genetic lines was evaluated at time-points either before or after microbiome sampling. At both time points, the survival of the ARS-Fp-R genetic line was significantly higher (P < 0.001) than the ARS-Fp-S line and consistent with estimated mid-point breeding values. In the first evaluation, a total of 3/120 (3%) ARS-Fp-R line fish died compared to 82/119 (69%) ARS-Fp-S line fish. In the second evaluation, 2/70 (3%) ARS-Fp-R fish died, while 58/70 (83%) ARS-Fp-S

Discussion

Commensal microbes have co-evolved with their eukaryotic counterparts, forming an intricate relationship that benefits both parties involved. Several studies have revealed that host genetics influences gut microbiota composition in a variety of species, including humans and rodents [7,8], chickens [43], and Drosophila [44]. However, other factors such as host diet and environmental conditions are also deeply intertwined and are crucial in determining host microbial communities [[12], [13], [14]

Conclusions

In conclusion, the present study reveals the impact of host genetics and environmental factors on the microbial community composition of rainbow trout, Host genetics shaped the microbial composition of the gut but not the gills of two rainbow trout lines with differential susceptibility to F. psychrophilum infection. Disease susceptibility was associated with a more diverse gut microbiome and the presence of potentially pathogenic taxa although important stocking density effects were also

Data availability

All datasets obtained have been deposited at NCBI BioProject and are publicly available under BioProject ID number PRJNA488363.

Acknowledgements

We thank Kurt Schwalm and Dr. Darrell Dinwiddie for handling the Illumina MiSeq runs. We thank Dr. Timothy Leeds for breeding the ARS-Fp-R and ARS-Fp-S genetic lines and Travis Moreland for fish rearing and Jeremy Everson for assistance with fish sampling. This work was supported in part by the U.S Agricultural Research Service Project 1930-32000-006. Ryan Brown received support from the University of New Mexico PREP program. Mention of trade names or commercial products in this publication is

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