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Here we published a correlative study of bacterial assemblages on naturally-spawned brown trout (Salmo trutta) eggs. During my first year as a PhD student I collected brown trout eggs within a big river system in the canton of Berne. I joined Joachim Guthruf, a passionate brown trout specialist, who monitored natural spawning in the main river Aare and its tributaries. He studied the fish for days in order to see them spawning and mark the burial sites of the eggs. About three weeks later he would go back and dig out the eggs, count them and analyze the spawning success at the different spawning sites. I had the opportunity to join and help him during most his trips. While I helped him with his data acquisition, I also collected eggs at the late-eyed developmental stage for my own studies. I was interested in investigating the spatial pattern of host-associated bacterial communities in brown trout eggs across a rivers system. Recent experiments with salmonid embryos established important principles of microbial colonization, maternal transmission of bacteria, bacterial virulence factors, and host genetic responses to bacterial infections. This progress stands in sharp contrast to what is known about the diversity of host-associated bacteria of fish in their natural environment.
For this study we characterized bacteria at nine different locations. Eight locations were within the river Aare system and one location was in Sils/Maria, within a non-connected river system of a different river, the river Inn. Bacterial communities on brown trout eggs differed markedly from the composition of bacteria found in their water environment. However, they were strikingly similar across different habitats and rivers regardless of (i) geographic distance among spawning places (isolation-by-distance) and (ii) host genetic and morphological differentiation. These findings strongly suggest that brown trout have egg-specific microbiomes. In the paper we describe the trout egg-associated microbiomes in detail and report evidence that bacterial diversity increases with water temperature.
I worked a very long time on this manuscript and I learned some very important conceptual points about working with environmental sampling of bacterial communities. First, I think it is extremely important to collect water samples or any kind of environmental samples when characterizing host-associated bacteria in their natural environment. This allows the discrimination of bacteria that are specifically associated with a host and bacteria that seem to be omnipresent in their environment. Second, I find it important to sample blank samples and sequence them. It is an illusion to think that we can work under sterile conditions in the field. So why not capturing all kinds of contaminations that we collect in the course of the study and subtract this from the real samples? We already followed this approach in our Aquatic Sciences paper earlier this year (blog post about it). Recently, Noah Fierer also tweeted about the importance of this approach and I must admit that I was very proud I already have published some articles where we took care of this issue. Third, we were dealing with some pretty low sample sizes for some locations. As we had to find a way to optimize the rather expensive sequencing technique (454 Pyrosequencing, Roche), we ended up pooling all the different sites where brown trout bury their eggs for each spawning location so the comparisons between locations could not be based anymore on variance estimates. Consequently, our data only allowed for (i) correlations of water temperature with bacterial community diversity, (ii) correlations of geographic and host genetic distance among spawning places with phylogenetic distance of bacterial communities, (iii) characterizations of core bacterial communities on all brown trout eggs, and (iv) comparisons of egg samples and water samples with regard to bacterial composition and its putative function. With regard to the comparison between the bacterial communities of the main river Aare with its tributaries we added a bootstrapping approach that is similar to a power analysis. Here we increased the number of samples for the main river Aare and its tributaries sequentially, using simulations, and investigated how many samples would be needed to find a significant difference in bacterial composition given the observed distribution of bacteria in our dataset. With regard to relationships between phylogenetic distance of bacterial communities among spawning places with geographic distance and host genetic distance respectively, we also analyzed the effect of reducing the number of pairwise comparisons on slope estimates and discussed this in the light of low sample sizes.
At this point I would like to thank Joachim for his time in the field with me, his patience and his generosity, sharing all this insider information about the local brown trout populations. I am also grateful to Claus and Luca for letting me follow my own curiosity and helping me a great deal with the writing for this manuscript. Aude, my first Master student at the University of Lausanne helped me a lot with the wet lab work for this manuscript, which was very time consuming and also under time pressure as I was expecting my daughter Linnea at that time. Finally, Frédéric supported me with the statistical analysis after the first revision by two very competent Scientific Reports reviewers. He backed up my statistical approaches and pointed out some weaknesses. I am very happy that this article is out now!
Wunderschöner, gemütlicher Tag im Regen. Der alte Rhein reisst und tobt. Wir haben in der Stube gezeltet und Spiele gespielt. Jetzt schlafen Donny und Linnea. Jacoby und ich schreiben an einem Manuskript. Jacoby zappelt und streckt sich. So soll es sein.
Hello? Can you hear me?
Please don’t go. Surround me with your love. Understand me. I need you now. Surround me with your words. Understand me. I need your love. I need your love. I need your love. After a week without sleep. The submission of three major fellowship proposals. The data analysis for another manuscript. And hours and hours of teaching lab practices to a beginner. My body full of stress hormones. I need to hide in your arms. My energy is limited. Look at our beautiful daughter. Surround me. I need you now. I do not want anything more than sleeping in the living room while you are talking and she is playing. Paradise.
I guess I should keep this going and write a little story for everybody about each of my manuscripts that some nice editors decide to accept.
This one here is about gene expression in whitefish embryos. Regarding the blog I wrote about the Aquatic Science paper, this could be interpreted as a continuation, a different perspective of our study system. Here we collected sperm and unfertilized eggs from whitefish that can be found in lake Geneva (Coregonus palaea). We fertilized the eggs in vitro and in a full-factorial breeding design. That means that we took the eggs of 4 females and the sperm of 4 males and then crossed them in all possible combinations. This gave us 16 little Tupperware buckets with freshly fertilized eggs. These we brought to the University where we have climate chambers at a constant temperature of 6.5° Celsius. All eggs were filled into individual wells with 2ml of standardized water. In their little microcosmos they grew and developed into embryos. At the late-eyed developmental stage when the blood circulation system is fully functional, we infected 13 replicates of each family with a nasty bacterial pathogen that can be found across many Swiss lakes and rivers Pseudomonas fluorescens. We also added some nutrients that these bacteria like. As a control we added only the nutrients without the pathogen to another 13 replicates of the same families. We already knew from previous studies that at this stage of development it matters who the father of the embryos was with regard to mortality and performance of the embryos under stress. Since the father only provided sperm and no paternal care, father effects can be interpreted as genetic effects. That means, he contributes genes to the embryos only. Once the embryo is old enough it starts expressing these genes and they have an impact on how it will perform under stress. However, what we did not know yet was which genes might be involved. So my goal for this study was to give these significant genetic effects a name.
Accordingly two days after treatment, we collected 3 embryos from each family and treatment and extracted all the gene expression products in their whole body, the so-called RNA. These are the gene products in all animals and plants that will be translated into proteins, which make up what we are. With the use of next-generation sequencing, we digitalized all this information. That means, that only messenger RNAs are filtered out, tagged according to which individual they belong to, pooled and then translated into letters. For this process we collaborated with a Swiss company in Geneva Fasteris. Months later, I received a huuuuuge text file that I could then use to satisfy my hunger for learning bioinformatics tools. With the help of a very friendly guy at Fasteris I found most overlapping sequences of text and aligned everything to a collection of longer text segments (contigs). These were then compared to an online reference of gene expression reads. The amount of data we produced was extremely big. Since I was only interested in genes that are differentially expressed between embryos in our treatment and control group, I decided to quantify the reads first and only compare the ones to a reference, that are also different between the two groups. Many of the reads did not result in a match, however, 1096 did and those could be characterized further. They told us which functional pathways are already active at this early stage of fish development and they gave us some insight into what defense mechanisms these embryos already have.
There is some background information to this paper. First, I must admit that I convinced my PhD supervisor to conduct this experiment because I wanted to get the chance to learn more about bioinformatics. I was the one responsible for the experimental treatment, the laboratory analysis of extracting RNA, and the one who did all the bioinformatics and data analyses. It was also the first manuscript that I wrote mostly on my own. It is not a very ground-breaking story. I would call it a lesson.
This project was always a side project during my PhD. We started with the fieldwork in 2010 when I just began my doctoral studies in Lausanne. The lab work was done in the end of 2011. I had serious troubles getting RNA of good quality from these embryos. We did not extract RNA directly but froze the embryos first for a couple of months at -80° Celsius. I would not recommend that to anybody. I was very busy working on different projects, some of them I considered my main projects at that time, and therefore this side project had to wait several times.
From the 16 families I managed to get a rather high amount of good quality RNA from 4 families. I made sure that I would have gene products from the same mother, crossed with 4 different fathers. I would say that this is the heart of this study. We could reduce variation in gene expression due to maternal effects. Different genotypes from the fathers were investigated against a constant background of the same mother. The whitefish external breeding system allowed us to control for host genotypes and contrast environment-induced changes in gene expression. I am discussing this strength of the study in the paper and encourage other scientists to use the same host system of fish with an external breeding system to investigate gene expression due to different treatments. It does not only present a way to reduce variation in expression due to maternal and environmental effects, it also provides the possibility to study gene expression in natural populations and their ecologically-relevant context.
I also would like to add that it was very pleasant to work with Laurent Farinelli at Fasteris. He is one of the founders of the Illumina sequencing methodology and he had the balls to start his own company. Now he is collaborating on many very exciting projects and he delivers high quality data and an exceptional service. I enjoyed my few meetings in Geneva at his company.
To end the story of this paper I have to mention that I finally sent the extracted RNA for sequencing in summer 2012. I received the data during my maternity leave and already started playing with it. In 2013 I managed to assemble all reads. I had to digest quite a bit of theory about partly assembled gene expression reads (transcriptomes) and I learned how to use a high-performance computing system (clusters at the SIB). In 2014 I did the differential gene expression analysis and compared reads of interest to different online reference databanks. Here I could rely on the theory about differential gene expression in lung cancer datasets that I was exposed to during my internship at Novartis. This project would not have been possible without the help of several co-workers at UNIL, such as Oksana Riba, Kate Ridout, Paris Veltsos and my co-author Emily Clark. At this point I would like to thank them again for their help and advice.
At the moment I am supervising a Master student who is applying the same experimental set-up in grayling (Thymallus thymallus). He is looking at sex-specific gene expression of grayling embryos under estrogen stress. In this project we also did all the steps from fieldwork until bioinformatics ourselves. However, he can rely on several collaborators who are specialized in the different aspects of the project and as a Master student he concentrates on only one project at a time. I am very excited to see him advancing so fast.
This post goes out for Martin Kapun.
0.5 dl milk
salt and nutmeg
Peterli (cut in small pieces)
Basilicum (cut in small pieces)
Krauseminze (cut in small pieces)
100g bacon (cut in small pieces)
2 Landjäger (cut in small pieces)
1 Salsiz (cut in small pieces)
75 g Sbrinz, grated
Mix flour, eggs and milk to a nice dough and let it stand at room temperature for 30min. Add spices, herbs and meat. Mix well. Take a tea spoon full and throw it on a leaf of Capuns. Pack it up nicely.
Cook up a big pan of water and add the packages for 7min. Take them carefully out and let them dry a bit. Put them in an oven form and add melted butter and graded cheese. Ready to eat.
Table 1: The influence of treatment, maternal, paternal, maternal x paternal effects, and bacterial diversity on brown trout embryo survival.
a = Five main logistic mixed effect models were compared to a reference model (in bold) to test if treatment ‘T’, dam ‘D’, sire ‘S’, and bacterial alpha diversity (phylogenetic distance, for its calculation see Material and methods) on the eggs explain a significant part of the variance in embryo mortality of brown trout. Alpha diversity was measured on the eggs before fertilization ‘A1’ or 14 days after treatment with nutrient broth ‘A2’. Two additional models were fitted to investigate the interaction of treatment with dam and sire effects. These models were fitted using the combined data of all three treatments (controls, nutrient broth at a dilution of 1:1000 and 1:500 in the wells).
I will write this blog better sooner than never!
Many fish are external fertilizers. That means that females lay their eggs when they think it is time to reproduce and males add their sperm, finished. Little embryos will then develop inside the eggs, out in the wild. Some eggs are buried in the gravel of a river, as it is common for brown trout (Salmo trutta) or grayling (Thymallus thymallus), and others are freely distributed on the ground, as it is common in whitefish (Coregnous spp.). After many days or weeks, these little embryos will hatch and start feeding and swimming around but in the meantime they are exposed to whatever visits them. Some of these embryos will be eaten by other fish or fungi, and most definitely all of them will be colonized by bacteria.
Bacteria do not have to be pathogens, some of them might start to collaborate with the developing embryos and many of them are most probably just there, do their daily business and are not even noticed.
In the Wedekind group there have been many brave PhD students who collected eggs and sperm from wild, adult fish and bred them in the laboratory. Fish embryos can be raised in little wells with a tiny amount of oxygenated water. This experimental set up allows manipulating bacterial communities on the eggs or adding specific bacteria to the eggs.
When I started my PhD there were a few very exciting projects going on in our coldrooms. Emily could for example show, that adding an oomycete in combination with a pathogen to developing whitefish eggs would mitigate the detrimental effects of the pathogen. [Oomycetes = fungus-like microorganisms]
Beat and Emily also showed nicely, that fish embryos do not just wait in their eggs until they can hatch and swim away.
Embryos interact with their pathogens. Depending on who the parents were of these baby fish, some are more susceptible to pathogens and others less. If most of the offspring of a specific mother survive better than others, that can be explained by the mother putting some compounds to her eggs before spawning that help her offspring during pathogen attacks. If the baby fish from the same father show better performance during a stress experiment, the situation is even more complicated, but also more beautiful. Since we assume that fathers usually only contribute sperm to their offspring, these paternal effects on embryo performance must be genetic. Genetic means that the offspring inherited some fancy immune genes from their father that make them better fighters.
This is about how much I understood when I started my PhD in this group. I was and I am still fascinated by the idea that there are bacteria growing on the eggs of developing fish embryos.
For me this looks like a micro-ecosystem. A little container with a developing fish embryo inside its egg. If you could zoom in you would most probably see hundreds of bacteria and other microbes. Interacting with each other and with their fish embryo host. Trying to make a living. Who knows, maybe there have been bacteria since hundreds of generations that specifically adapted to fish eggs. A little town of bacteria on each single egg.
When it was time for me to do my first research I wanted to go out and screen these communities of bacteria developing on fish eggs. I wanted to travel to these little cities to see who is there, what are they doing, are they different among different fish host species, do they change during the development of their fish embryo hosts, do they correlate with environmental factors and how do they interact with their host? Early during a PhD you think you can answer all your questions and maybe even some more. Four years seem to be an eternity and everything is possible. But soon I realized that I had to go step by step and answer one small question after the other. So I started with diving. I had the following first goals:
i) Describe which bacteria are growing on naturally-spawned whitefish eggs
ii) Compare these bacterial communities with the bacteria in the surrounding water
iii) Look for changes in bacterial composition during embryo development
iv) Find variables that correlate with the bacterial composition and bacterial diversity on the eggs
I dove in all bigger lakes of Switzerland where I could find fertilized whitefish eggs. At Christmas time.
I wanted to collect as many different populations of whitefish eggs at different developmental stages. Fish inspectors but mostly fishermen pointed me in the right directions. At each location I also collected water samples in order to compare my little egg cities to the universe. Marianne taught me how to dive in cold waters.
Beat accepted the challenge and traveled west again. Many friends and colleagues helped me but in the end it was Grégory who did not get tired of me and of diving in super cold sub-alpine lakes. Grég never gave up.
And then he saved my life. After we sunk a boat. And that’s how we ended up with a decent sample size of wild whitefish eggs.
What then followed were months of tedious lab work and bioinformatics analyses. In order to characterize as many bacteria as possible on the eggs, we extracted all the DNA we could find on the eggs and amplified a bacteria-specific gene. Using an online reference database that is continuously being updated, we could compare our amplified genes and find out which bacteria there are.
We found bacterial communities on early embryos to be very diverse and to resemble the bacterial composition of the surrounding water environment. Bacterial communities on late embryos were significantly less diverse than on early embryos and displayed a clear shift in taxonomic composition that corresponded poorly with the bacterial composition of the surrounding water environment. Besides this main finding I did an extensive literature search and discussed the putative relationships of specific bacterial species that we could only find on fish eggs but not in the surrounding water. Supported by previous findings from the group’s coldroom experiments we concluded that natural symbiotic bacterial communities become more specialized during embryogenesis because of specific interactions with their embryo host.
This was the first chapter of my doctoral thesis and it just got accepted in a scientific journal (Aquatic Sciences).
A bit sleep deprived. Too much work. But soon, very soon, I want to elaborate on this: