Rearing Water Treatment Induces Microbial Selection Influencing the Microbiota and Pathogen Associated Transcripts of Cod (Gadus morhua) Larvae.
ABSTRACT: We have previously shown that K-selection and microbial stability in the rearing water increases survival and growth of Atlantic cod (Gadus morhua) larvae, and that recirculating aquaculture systems (RAS) are compatible with this. Here, we have assessed how water treatment influenced the larval microbiota and host responses at the gene expression level. Cod larvae were reared with two different rearing water systems: a RAS and a flow-through system (FTS). The water microbiota was examined using a 16S rDNA PCR/DGGE strategy. RNA extracted from larvae at 8, 13, and 17 days post hatching was used for microbiota and microarray gene expression analysis. Bacterial cDNA was synthesized and used for 16S rRNA amplicon 454 pyrosequencing of larval microbiota. Both water and larval microbiota differed significantly between the systems, and the larval microbiota appeared to become more dissimilar between systems with time. In total 4 phyla were identified for all larvae: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The most profound difference in larval microbiota was a high abundance of Arcobacter (Epsilonproteobacteria) in FTS larvae (34 ± 9% of total reads). Arcobacter includes several species that are known pathogens for humans and animals. Cod larval transcriptome responses were investigated using an oligonucleotide gene expression microarray covering approximately 24,000 genes. Interestingly, FTS larvae transcriptional profiles revealed an overrepresentation of upregulated transcripts associated with responses to pathogens and infections, such as c1ql3-like, pglyrp-2-like and zg16, compared to RAS larvae. In conclusion, distinct water treatment systems induced differences in the larval microbiota. FTS larvae showed up-regulation of transcripts associated with responses to microbial stress. These results are consistent with the hypothesis that RAS promotes K-selection and microbial stability by maintaining a microbial load close to the carrying capacity of the system, and ensuring long retention times for both bacteria and water in the system.
Project description:The roles of host-associated bacteria have gained attention lately, and we now recognise that the microbiota is essential in processes such as digestion, development of the immune system and gut function. In this study, Atlantic cod larvae were reared under germ-free, gnotobiotic and conventional conditions. Water and fish microbiota were characterised by 16S rRNA gene analyses. The cod larvae's transcriptional responses to the different microbial conditions were analysed by a custom Agilent 44 k oligo microarray. Gut development was assessed by transmission electron microscopy (TEM). Water and fish microbiota differed significantly in the conventional treatment and were dominated by different fast-growing bacteria. Our study indicates that components of the innate immune system of cod larvae are downregulated by the presence of non-pathogenic bacteria, and thus may be turned on by default in the early larval stages. We see indications of decreased nutrient uptake in the absence of bacteria. The bacteria also influence the gut morphology, reflected in shorter microvilli with higher density in the conventional larvae than in the germ-free larvae. The fact that the microbiota alters innate immune responses and gut morphology demonstrates its important role in marine larval development.
Project description:Many studies demonstrate the importance of the commensal microbiomes to animal health and development. However, the initial community assembly process is poorly understood. It is unclear to what extent the hosts select for their commensal microbiota, whether stochastic processes contribute, and how environmental conditions affect the community assembly. We investigated community assembly in Atlantic cod larvae exposed to distinct microbial metacommunities. We aimed to quantify ecological processes influencing community assembly in cod larvae and to elucidate the complex relationship between the bacteria of the environment and the fish. Selection within the fish was the major determinant for community assembly, but drift resulted in inter-individual variation. The environmental bacterial communities were highly dissimilar from those associated with the fish. Still, differences in the environmental bacterial communities strongly influenced the fish communities. The most striking difference was an excessive dominance of a single OTU (Arcobacter) for larvae reared in two of the three systems. These larvae were exposed to environments with higher fractions of opportunistic bacteria, and we hypothesise that detrimental host-microbe interactions might have made the fish susceptible to Arcobacter colonisation. Despite strong selection within the host, this points to a possibility to steer the metacommunity towards mutualistic host-microbe interactions and improved fish health and survival.
Project description:<h4>Background</h4>Fish gut microbial colonisation starts during larval stage and plays an important role in host's growth and health. To what extent first colonisation could influence the gut microbiome succession and growth in later life remains unknown. In this study, Nile tilapia embryos were incubated in two different environments, a flow-through system (FTS) and a biofloc system (BFS); hatched larvae were subsequently cultured in the systems for 14 days of feeding (dof). Fish were then transferred to one common recirculating aquaculture system (RAS1, common garden, 15-62 dof), followed by a growth trial in another RAS (RAS2, growth trial, 63-105 dof). In RAS2, fish were fed with two types of diet, differing in non-starch polysaccharide content. Our aim was to test the effect of rearing environment on the gut microbiome development, nutrient digestibility and growth performance of Nile tilapia during post-larvae stages.<h4>Results</h4>Larvae cultured in the BFS showed better growth and different gut microbiome, compared to FTS. After the common garden, the gut microbiome still showed differences in species composition, while body weight was similar. Long-term effects of early life rearing history on fish gut microbiome composition, nutrient digestibility, nitrogen and energy balances were not observed. Still, BFS-reared fish had more gut microbial interactions than FTS-reared fish. A temporal effect was observed in gut microbiome succession during fish development, although a distinct number of core microbiome remained present throughout the experimental period.<h4>Conclusion</h4>Our results indicated that the legacy effect of first microbial colonisation of the fish gut gradually disappeared during host development, with no differences in gut microbiome composition and growth performance observed in later life after culture in a common environment. However, early life exposure of larvae to biofloc consistently increased the microbial interactions in the gut of juvenile Nile tilapia and might possibly benefit gut health.
Project description:Little information is available on the link between host development (growth rate and ontogeny) and the composition of the microbiota in fish larvae. This study was carried out to examine potential correlations of microbiota composition with age and growth rate of Atlantic cod larvae. Small and large cod larvae of the same age, representing slow and fast growing individuals, were sampled 10 times during a period of 42 days post hatching (dph), and the composition of the larval microbiota was investigated using a PCR/DGGE (Denaturing Gradient Gel Electrophoresis) strategy. We found significant differences in the intestinal microbiota of small and large larvae of the same age for 4 of the 10 age stages studied. We further found that the variation in the composition of the larval microbiota was more strongly correlated to age than to growth rate for larvae up to 28 dph, whereas for the older larvae growth rate and age was equally correlated to the composition of the microbiota. These results indicate that larval development may structure the microbiota through a change in selection pressure due to host-microbe and microbe-microbe interactions, and that the composition of the microbiota may influence larval development through improved energy gain.
Project description:The gut microbiota of fish larvae evolves fast towards a complex community. Both host and environment affect the development of the gut microbiota; however, the relative importance of both is poorly understood. Determining specific changes in gut microbial populations in response to a change in an environmental factor is very complicated. Interactions between factors are difficult to separate and any response could be masked due to high inter-individual variation even for individuals that share a common environment. In this study we characterized and quantified the spatio-temporal variation in the gut microbiota of tilapia larvae, reared in recirculating aquaculture systems (RAS) or active suspension tanks (AS). Our results showed that variation in gut microbiota between replicate tanks was not significantly higher than within tank variation, suggesting that there is no tank effect on water and gut microbiota. However, when individuals were reared in replicate RAS, gut microbiota differed significantly. The highest variation was observed between individuals reared in different types of system (RAS vs. AS). Our data suggest that under experimental conditions in which the roles of deterministic and stochastic factors have not been precisely determined, compositional replication of the microbial communities of an ecosystem is not predictable.
Project description:The mosquito body hosts highly diverse microbes, which influence different physiological traits of both larvae and adults. The composition of adult mosquito microbiota is tightly linked to that of larvae, which are aquatic and feed on organic detritus, algae and prokaryotic microorganisms present in their breeding sites. Unraveling the ecological features of larval habitats that shape the structure of bacterial communities and their interactions with the mosquito host is still a poorly investigated topic in the Asian tiger mosquito <i>Aedes albopictus</i>, a highly invasive species that is vector of numerous arboviruses, including Dengue, Chikungunya, and Zika viruses. In this study, we investigated the composition of the bacterial community present in the water from a natural larval breeding site in which we separately reared wild-collected larvae and hatched eggs of the Foshan reference laboratory strain. Using sequence analysis of bacterial 16S rRNA gene amplicons, we comparatively analyzed the microbiota of the larvae and that of adult mosquitoes, deriving information about the relative impact of the breeding site water on shaping mosquito microbiota. We observed a higher bacterial diversity in breeding site water than in larvae or adults, irrespective of the origin of the sample. Moreover, larvae displayed a significantly different and most diversified microbial community than newly emerged adults, which appeared to be dominated by Proteobacteria. The microbiota of breeding site water significantly increased its diversity over time, suggesting the presence of a dynamic interaction among bacterial communities, breeding sites and mosquito hosts. The analysis of <i>Wolbachia</i> prevalence in adults from Foshan and five additional strains with different geographic origins confirmed the described pattern of dual <i>w</i>AlbA and <i>w</i>AlbB strain infection. However, differences in <i>Wolbachia</i> prevalence were detected, with one strain from La Reunion Island showing up to 18% uninfected individuals. These findings contribute in further understanding the dynamic interactions between the ecology of larval habitats and the structure of host microbiota, as well as providing additional information relative to the patterns of <i>Wolbachia</i> infection.
Project description:Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) .
Project description:Ciliate protists and rotifers are ubiquitous in aquatic habitats and can comprise a significant portion of the microbial food resources available to larval mosquitoes, often showing substantial declines in abundance in the presence of mosquito larvae. This top-down regulation of protists is reported to be strong for mosquitoes inhabiting small aquatic containers such as pitcher plants or tree holes, but the nature of these interactions with larval mosquitoes developing in other aquatic habitats is poorly understood. We examined the effects of these two microbial groups on lower trophic level microbial food resources, such as bacteria, small flagellates, and organic particles, in the water column, and on <i>Culex</i> larval development and adult production. In three independent laboratory experiments using two microeukaryote species (one ciliate protist and one rotifer) acquired from field larval mosquito habitats and cultured in the laboratory, we determined the effects of <i>Culex nigripalpus</i> larval grazing on water column microbial dynamics, while simultaneously monitoring larval growth and development. The results revealed previously unknown interactions that were different from the top-down regulation of microbial groups by mosquito larvae in other systems. Both ciliates and rotifers, singly or in combination, altered other microbial populations and inhibited mosquito growth. It is likely that these microeukaryotes, instead of serving as food resources, competed with early instar mosquito larvae for microbes such as small flagellates and bacteria in a density-dependent manner. These findings help our understanding of the basic larval biology of <i>Culex</i> mosquitoes, variation in mosquito production among various larval habitats, and may have implications for existing vector control strategies and for developing novel microbial-based control methods.
Project description:Arthropod-borne viruses comprise a significant global disease burden. Surveillance and mitigation of arboviruses like Zika virus (ZIKV) require accurate estimates of transmissibility by vector mosquitoes. Although <i>Aedes</i> species mosquitoes are established as competent ZIKV vectors, differences in experimental protocols across studies prevent direct comparisons of relative transmissibility. An understudied factor complicating these comparisons is differential environmental microbiota exposures, where most vector competence studies use mosquitoes reared in laboratory tap water, which does not represent the microbial complexity of environmental water where wild larvae develop. We simulated natural larval development by rearing Californian Aedes aegypti larvae with microbes obtained from cemetery headstone water compared to conventional tap water. A. aegypti larvae reared in environmental cemetery water pupated 3 days faster and at higher rates. Mosquitoes reared in environmental water were less competent vectors of ZIKV than laboratory water-reared A. aegypti, as evidenced by significantly reduced infection and transmission rates. Microbiome comparisons of laboratory water- and environment water-reared mosquitoes and their rearing water showed significantly higher bacterial diversity in environment water. Despite this pattern, corresponding differences in bacterial diversity were not consistently observed between the respective adult mosquitoes. We also observed that the microbial compositions of adult mosquitoes differed more by whether they ingested a bloodmeal than by larval water type. Together, these results highlight the role of transient microbes in the larval environment in modulating A. aegypti vector competence for ZIKV. Laboratory vector competence likely overestimates the true transmissibility of arboviruses like ZIKV when conventional laboratory water is used for rearing. <b>IMPORTANCE</b> We observed that A. aegypti mosquitoes reared in water from cemetery headstones instead of the laboratory tap exhibited a reduced capacity to become infected with and transmit Zika virus. Water from the environment contained more bacterial species than tap water, but these bacteria were not consistently detected in adult mosquitoes. Our results suggest that rearing mosquito larvae in water collected from local environments as opposed to laboratory tap water, as is conventional, could provide a more realistic assessment of ZIKV vector competence since it better recapitulates the natural environment in which larvae develop. Given that laboratory vector competence is used to define the species to target for control, the use of environmental water to rear larvae could better approximate the microbial exposures of wild mosquitoes, lessening the potential for overestimating ZIKV transmission risk. These studies raise the question of whether rearing larvae in natural water sources also reduces vector competence for other mosquito-borne viruses.
Project description:Chironomids are aquatic insects that undergo a complete metamorphosis of four life stages. Here we studied, for the first time, the microbiota composition of Chironomus circumdatus, a tropical midge species, both from the Mula and Mutha Rivers in Pune, India and as a laboratory-reared culture. We generated a comparative microbial profile of the eggs, larvae and pupae, the three aquatic life stages of C. circumdatus. Non-metric multidimensional scaling analysis (NMDS) demonstrated that the developmental stage had a more prominent effect on the microbiota composition compared to the sampling location. Notably, the microbiota composition of the egg masses from the different sampling points clustered together and differed from laboratory culture larvae. Proteobacteria was the dominant phylum in all the environmental and laboratory-reared egg masses and pupal samples, and in the laboratory-reared larvae, while Fusobacteria was the dominant phylum in the larvae collected from the field environment. The most abundant genera were Cetobacterium, Aeromonas, Dysgonomonas, Vibrio, and Flavobacterium. The ten amplicon sequence variants (ASVs) that most significantly contributed to differences in microbiota composition between the three sampled locations were: Burkholderiaceae (ASVs 04 and 37), C39 (Rhodocyclaceae, ASV 14), Vibrio (ASV 07), Arcobacter (ASV 21), Sphaerotilus (ASV 22), Bacteroidia (ASVs 12 and 28), Flavobacterium (ASV 29), and Gottschalkia (ASV 10). No significant differences were found in the microbial richness (Chao1) or diversity (Shannon H') of the three sampled locations. In contrast, significant differences were found between the microbial richness of the three life stages. Studying the microbiota of this Chironomus species may contribute to a better understanding of the association of C. circumdatus and its microbial inhabitants.