Deploying an In Vitro Gut Model to Assay the Impact of the Mannan-Oligosaccharide Prebiotic Bio-Mos on the Atlantic Salmon (Salmo salar) Gut Microbiome.

Alpha mannose-oligosaccharide (MOS) prebiotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and their associated metabolism. Bio-Mos is a commercially available prebiotic currently used in the agri-feed industry, but studies show contrasting results of its effect on fish performance and feed efficiency. Thus, detailed studies are needed to investigate the effect of MOS supplements on the fish microbiome to enhance our understanding of the link between MOS and gut health. To assess Bio-Mos for potential use as a prebiotic growth promoter in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. The microbial communities obtained from ceca compartments from four adult farmed salmon were inoculated in biological triplicate reactors in SalmoSim. Prebiotic treatment was supplemented for 20 days, followed by a 6-day washout period. Inclusion of Bio-Mos in the media resulted in a significant increase in formate (P = 0.001), propionate (P = 0.037) and 3-methyl butanoic acid (P = 0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to Bio-Mos supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to complementin vivo trials of microbiome modulators. IMPORTANCE In this paper we report the results of the impact of a prebiotic (alpha-MOS supplementation) on microbial communities, using an in vitro simulator of the gut microbial environment of the Atlantic salmon. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production by the microbiota from salmon pyloric ceca and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

SalmoSim: the development of a three-compartment in vitro simulator of the Atlantic salmon GI tract and associated microbial communities.

BACKGROUND: The aquaculture sector now accounts for almost 50% of all fish for human consumption and is anticipated to provide 62% by 2030. Innovative strategies are being sought to improve fish feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the aquaculture industry. There is still a lack of knowledge surrounding the importance and functionality of the teleost gut microbiome in fish nutrition. In vitro gut model systems might prove a valuable tool to study the effect of feed, and additives, on the host's microbial communities. Several in vitro gut models targeted at monogastric vertebrates are now in operation. Here, we report the development of an Atlantic salmon gut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum, and midgut) and associated microbial communities. RESULTS: The gut model was established in a series of linked bioreactors seeded with biological material derived from farmed adult marine-phase salmon. We first aimed to achieve a stable microbiome composition representative of founding microbial communities derived from Atlantic salmon. Then, in biological triplicate, the response of the in vitro system to two distinct dietary formulations (fishmeal and fishmeal free) was compared to a parallel in vivo trial over 40 days. Metabarcoding based on 16S rDNA sequencing qPCR, ammoniacal nitrogen, and volatile fatty acid measurements were undertaken to survey the microbial community dynamics and function. SalmoSim microbiomes were indistinguishable (p = 0.230) from their founding inocula at 20 days and the most abundant genera (e.g., Psycrobacter, Staphylococcus, Pseudomonas) proliferated within SalmoSim (OTUs accounting for 98% of all reads shared with founding communities). Real salmon and SalmoSim responded similarly to the introduction of novel feed, with majority of the taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa (e.g., a small fraction of Psychrobacter) was differentially affected across both systems. Consistent with a low impact of the novel feed on microbial fermentative activity, volatile fatty acid profiles were not significantly different in SalmoSim pre- and post-feed switch. CONCLUSION: By establishing stable and representative salmon gut communities, this study represents an important step in the development of an in vitro gut system as a tool for the improvement of fish nutrition and welfare. The steps of the system development described in this paper can be used as guidelines to develop various other systems representing other fish species. These systems, including SalmoSim, aim to be utilised as a prescreening tool for new feed ingredients and additives, as well as being used to study antimicrobial resistance and transfer and fundamental ecological processes that underpin microbiome dynamics and assembly. Video abstract.

Drosophila Sexual Attractiveness in Older Males Is Mediated by Their Microbiota.

Age is well known to be a basis for female preference of males. However, the mechanisms underlying age-based choices are not well understood, with several competing theories and little consensus. The idea that the microbiota can affect host mate choice is gaining traction, and in this study we examine whether the male microbiota influences female preference for older individuals in the fruit fly Drosophila pseudoobscura. We find that an intact microbiota is a key component of attractiveness in older males. However, we found no evidence that this decrease in older male attractiveness was simply due to impaired microbiota generally reducing male quality. Instead, we suggest that the microbiota underlies an honest signal used by females to assess male age, and that impaired microbiota disrupt this signal. This suggests that age-based preferences may break down in environments where the microbiota is impaired, for example when individuals are exposed to naturally occurring antibiotics, extreme temperatures, or in animals reared in laboratories on antibiotic supplemented diet.

The effect of gut microbiota elimination in Drosophila melanogaster: A how-to guide for host-microbiota studies.

In recent years, there has been a surge in interest in the effects of the microbiota on the host. Increasingly, we are coming to understand the importance of the gut microbiota in modulating host physiology, ecology, behavior, and evolution. One method utilized to evaluate the effect of the microbiota is to suppress or eliminate it, and compare the effect on the host with that of untreated individuals. In this study, we evaluate some of these commonly used methods in the model organism, Drosophila melanogaster. We test the efficacy of a low-dose streptomycin diet, egg dechorionation, and an axenic or sterile diet, in the removal of gut bacteria within this species in a fully factorial design. We further determine potential side effects of these methods on host physiology by performing a series of standard physiological assays. Our results showed that individuals from all treatments took significantly longer to develop, and weighed less, compared to normal flies. Males and females that had undergone egg dechorionation weighed significantly less than streptomycin reared individuals. Similarly, axenic female flies, but not males, were much less active when analyzed in a locomotion assay. All methods decreased the egg to adult survival, with egg dechorionation inducing significantly higher mortality. We conclude that low-dose streptomycin added to the dietary media is more effective at removing the gut bacteria than egg dechorionation and has somewhat less detrimental effects to host physiology. More importantly, this method is the most practical and reliable for use in behavioral research. Our study raises the important issue that the efficacy of and impacts on the host of these methods require investigation in a case-by-case manner, rather than assuming homogeneity across species and laboratories.

Fifty important research questions in microbial ecology.

Microbial ecology provides insights into the ecological and evolutionary dynamics of microbial communities underpinning every ecosystem on Earth. Microbial communities can now be investigated in unprecedented detail, although there is still a wealth of open questions to be tackled. Here we identify 50 research questions of fundamental importance to the science or application of microbial ecology, with the intention of summarising the field and bringing focus to new research avenues. Questions are categorised into seven themes: host-microbiome interactions; health and infectious diseases; human health and food security; microbial ecology in a changing world; environmental processes; functional diversity; and evolutionary processes. Many questions recognise that microbes provide an extraordinary array of functional diversity that can be harnessed to solve real-world problems. Our limited knowledge of spatial and temporal variation in microbial diversity and function is also reflected, as is the need to integrate micro- and macro-ecological concepts, and knowledge derived from studies with humans and other diverse organisms. Although not exhaustive, the questions presented are intended to stimulate discussion and provide focus for researchers, funders and policy makers, informing the future research agenda in microbial ecology.

Dyeing insects for behavioral assays: the mating behavior of anesthetized Drosophila.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

You are what you eat.

Gut bacteria is now considered as an additional host organ, and it has been shown that they have important influences on host developmental and physiological processes. More recently gut bacteria have additionally been implicated in behavioral processes. We showed that in two species of Drosophila, gut bacteria variation affects behavior, altering partner investment in copulation in relation to whether individuals were familiar (i.e., having developed in the same environment), or were related. This suggests that gut bacteria play a role in kin recognition in these species. We suggest that this phenomenon is associated with gut bacteria influencing the scent profiles used by Drosophila in mate choice.

Extreme cost of rivalry in a monandrous species: male-male interactions result in failure to acquire mates and reduced longevity.

Mating system variation is profound in animals. In insects, female willingness to remate varies from mating with hundreds of males (extreme polyandry) to never remating (monandry). This variation in female behaviour is predicted to affect the pattern of selection on males, with intense pre-copulatory sexual selection under monandry compared to a mix of pre- and post-copulatory forces affecting fitness under polyandry. We tested the hypothesis that differences in female mating biology would be reflected in different costs of pre-copulatory competition between males. We observed that exposure to rival males early in life was highly costly for males of a monandrous species, but had lower costs in the polyandrous species. Males from the monandrous species housed with competitors showed reduced ability to obtain a mate and decreased longevity. These effects were specific to exposure to rivals compared with other types of social interactions (heterospecific male and mated female) and were either absent or weaker in males of the polyandrous species. We conclude that males in monandrous species suffer severe physiological costs from interactions with rivals and note the significance of male-male interactions as a source of stress in laboratory culture.