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Elucidating the role of microbial communities in cod larval death and survival

Final Report Summary - COD LARVAL SURVIVAL (Elucidating the role of microbial communities in cod larval death and survival)

The contribution of aquaculture to world food production has increased significantly over the last few decades and this sector now supplies nearly half of the total fish and shellfish used for human consumption. Atlantic cod (Gadus morhua) has been a candidate for aquaculture for some decades in countries around the North Atlantic Ocean. Norway, the leading aquaculture producer in continental Europe, has taken a great interest in developing a cod farming industry, but has experienced a variety of bottlenecks at all life stages, resulting in large economic losses. For most marine aquaculture species, a major biological challenge is to overcome bottlenecks with regards to producing high quality juveniles suitable for the intensive cultivation environment. Unfavorable host–microbe interactions are a common phenomenon in aquaculture fish production. Especially the youngest life stages of cultivated fish are prone to negative interactions with opportunistic bacteria, resulting in infections and often high mortalities. To establish a more sustainable juvenile production a detailed understanding of the characteristics and the role of the indigenous microflora of fish larvae is essential to improve conditions for the intensive mass rearing of healthy fish.

Due to its complexity, this demands an interdisciplinary approach, combining concepts and methods from microbial ecology, marine larviculture and a variety of state-of-the-art imaging methods. We studied cod larvae reared under three conditions: germ-free, containing a known and defined microbial flora (gnotobiotic) thought to be beneficial for cod larvae survival, and cod larvae grown conventionally with a less controlled microbial community. We utilized correlative light and electron microscopy to characterize in detail the bacterial colonization and also the associated changes in their community structure resulting from stimulation with probiotic bacteria and infection by a common fish pathogenic bacterium, Vibrio anguillarum. We hypothesized that the microbial composition in the intestine of cod can increase the host’s resistance against pathogenic infection.

We re-designed the experimental rearing system for gnotobiotic grown cod larvae, which now allows easier removal of excess water, feed and dead larvae, and improved the aeration system by making it better controllable.

During the experiments sterility-checks were performed using flow cytometry. Rearing water was incubated with SYBR Green I, a DNA stain, to detect any bacteria within the rearing water. No analyzed samples show any contamination in germ-free larvae bottles nor feed cultures. Further, no growth was visible on agar plates used to check for culturable bacteria.

We ran our experiments up to 21 days. In general, within our experiments, there was lower survival of cod larvae grown conventionally compared to germ-free and gnotobiotic reared larvae. Survival was highest for cod larvae grown under gnotobiotic conditions. For example, on day 16 post hatch the dry weight of germ-free grown larvae was significantly lower than for gnotobiotic reared fish larvae (p=0.017 and 0.059 respectively at 16 dph; see also Fig.1) indicating an important role of bacteria in nutrient absorptions.

We developed, adopted and optimized fixation and embedding protocols for cod larvae and microbes for transmission electron microscopy. Fluorescence in situ hybridization (FISH) probes to target the 16S rRNA sequences of beneficial and pathogenic bacteria were designed. For morphological structural studies, paraffin sections of biological tissues were analyzed by using special staining techniques combined with light microscopy. We also performed FISH on paraffin sections and could therefore correlate morphological/structural information of the host with distribution and taxonomic information of the bacteria.

In order to determine the 3D community organization, modes of interactions between microbes, as well as microbe-host interactions, focused ion beam scanning electron microscopy (FIB-SEM) was originally planned to apply. Due to logistical reasons, we used a different volume electron microscopy (volume EM) technique, namely Teneo volume scope (serial block face imaging, SBF-SEM). SBF-SEM and FIB-SEM have their advantages and share some commonalities in aspects of sample preparation, imaging parameters and data analysis. But the major difference is in the Z resolution (slice thickness/depth) that each of the system can provide. With FIB-SEM small isotropic voxels are achievable but only in relatively small volumes; SBF-SEM can handle much larger volumes but is limited in Z resolution. The strength of volume EM is to study spatial relationships of macromolecular complexes while retaining the context of the surrounding cellular and tissue architecture within large 3D volumes (tens to hundreds of microns in each dimension). Back-scatter electron detection, the mode of imaging for volume EM, requires a higher amount of heavy metal staining than typical for conventional TEM in biological samples. Therefore, protocols for staining procedures of fish larvae were developed.

By correlating light and electron microscopy we obtained integrated information on the scale of one hundred micrometers up to a few nm and thus defined the identity and distribution of organisms, their morphologies, and the spatial organization between organisms. We characterized bacteria-host interactions, changes in membership ratios and relationships.

So far, successful aquaculture of cod and many other species is still hampered by low survival at the larval stage caused by microbial problems. To our knowledge, this was the first study designed to investigate the complex microbe-host and microbe-microbe interactions in cod larvae by correlating different microscopic techniques at different resolutions and thus providing the foundations for analysis of more complex communities. The initial colonization process in cod larvae is still poorly understood. There is an urgent need to understand the characteristics and the role of the indigenous microflora of cod larvae to improve conditions for the intensive mass rearing of healthy fish.
Further, strategies for microbial control in aquaculture that can effectively replace the use of antibiotics have high environmental impact by avoiding the spread of antibiotics resistance. Antibiotics are still extensively used in aquaculture outside of the European Union. Furthermore, increased survival and more predictable outcomes in marine larval rearing will lead to a better use of resources (feed and larvae).
The depletion of certain wild stocks of cod gives rise to supply difficulties due to high demand in Europe. An in-depth mechanistic understanding of such microbial communities and their interaction with the host will be invaluable to maximize the survival of the fish larvae, therefore significantly increasing the yield in Atlantic cod aquaculture, and thus be of high value to the Norwegian Fishing industry as well as the European Community at large.

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