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Project Summary Overview:
Evaluation of the effects of the honeybee microbiome on the honeybee epigenome.

Gut microbiomes have been shown to have significant and varied effects on host health and development (6). Honeybees provide a good model for understanding gut microbiota (7). Like humans, the honeybee gut microbiome is comprised of horizontally transferred species. Unlike humans, the system is a simple one: eight species of bacteria make up over 95% of the gut community (7). All eight of these main species can be cultured in a laboratory. Also, techniques have been developed that allow for the rearing of germ-free (GF) bees, that can be used to explore how the conventional gut community affects health (3, 7).

The bee gut microbiome has been shown to help prevent infection by upregulating immune genes, including antimicrobial peptides (3). And disturbances in the microbiome have been shown to make the host more susceptible to pathogens (5). The mechanism of regulation is unknown. The microbiome has also been shown to regulate worker development through promoting weight gain and metabolic processes (4). However, the relationship between gut microbiomes and host epigenomes has not been greatly explored.

Honeybees have also served as a model for epigenetic studies. Queen bees and worker bees share identical genomes. The developmental and behavioral differences between the two castes are epigenetically controlled, and the nurse and forager roles (or subcastes) of workers are associated with different methylation states (39). Subcastes are defined by behavioral changes that develop with age. Worker honeybees produce varying levels of antimicrobial peptides depending on their age and subsequent subcaste (28).

The epigenetic triggers that determine social caste between worker and queen has been shown to be controlled by their diet (47). Microbiome metabolites have been shown to control weight gain and hormone levels in worker bees (4). This project will explore if and how the microbiome is contributing to the epigenome through bacterial metabolites.

The growing literature on the microbiome and the proven epigenetic regulation of subcastes provide excellent control data sets to work from.

Specific aims:
1. Determine if and how DNA methylation patterns differ between bees lacking microbiomes and bees with conventional microbiomes.

2. Determine if and how Acetylation (<-- Don't capitalize.) patterns differ between bees lacking microbiomes and bees with conventional microbiomes.

3. Determine if common honeybee microbiome metabolites act as histone deacetylase inhibitors.

Significance
Honeybees are an essential species in crop pollination around the world. Since 2006, elevated rates of hive mortality in North America, termed "colony collapse disorder," have threatened the stability of the honeybee population (1).

Honeybees are both a species of interest and a model species. The economic damage colony collapse disorder inflicts could be mitigated by a better understanding of how honeybees are affected by environmental factors. The use of antibiotics in bee keeping has been shown to decrease a hive's ability to fight infection (5). Investigating the relationship between the microbiome and the epigenome can illuminate what other effects those antibiotics may have. Studying honeybees as a model for how the microbiome and epigenome interact could also provide more general insights into horizontally transferred microbiota, gene expression, and social behavior.

Innovation
This proposed project combines techniques developed to study the honeybee microbiome with those developed to study its epigenome. Given that honeybees are used as a model for inquiries into both host-microbe interactions and epigenetic differentiation, they provide a promising opportunity to combine the two fields.

The growing body of work on honeybees allows cross-disciplinary studies like the one proposed possible. The Apis mellifera genome was sequenced in 2006, and the Apis cerana genome and transcriptome was sequenced and explored in 2017 (28). Honeybee researchers have assembled and coninued to expand BeeBase, a genetic database inspired by FlyBase. Epigenetic studies of honeybee castes and subcastes consistently reveal more about honeybee gene regulation (39).

Microbiome studies have provided a large body of work that can be used to develop new experiments (3,4,5,7). Rearing honeybees in the lab without the traditional gut microbiome allows us to investigate epigenome and transcriptome differences.

Approach
A problem that you have is that you will use whole larvae. But each tissue in the larvae might respond differently. In flies, we often focus on just the larval brain because it can be easily and rapidly dissected from the body. In flies, you can also get body wall, which is muscle, epithelia and a tiny bit of PNS. Can you do this in bees? The more homogenous that you can get the better off you are.

1. Determine if and how DNA methylation patterns differ between bees lacking microbiomes and bees with conventional microbiomes.
First we will raise germ-free bees and conventional bees in laboratory settings. In order to control for the effects of lab conditions on DNA methylation patterns, we will raise the three sets of bees under nearly the same conditions.

We will collect honeybees and extract brood frames from two different hives with different queens, and therefore different genetics. We will duplicate the experiments on sets of bees from each hive. (<-- So two hives and two assays on each hive. N=4 correct? You may find that there is a statistical lack of independence here. The duplicates from a hive are not really repeats because they come form the same hive. Why not use three hives? Then you could have a real N=3.) First, we will extract larvae from the frames that are within a few days of emerging. We will separate the extracted larvae into three groups, (<-- So here you end up expanding this to an N=3 but it is not really three true biological replicates. I think that the only solution is to start with three hives.) and place them in a germ free incubation chamber with germ free pollen doused in sugar syrup. The "germ free" group will be left to mature without further interference. For one of the treatment groups, we will introduce marked worker bees that we've collected from their respective hives. We will first chill these bees, then mark them with paint in order to separate them later from the newly emerged workers. These bees will inoculate the new workers with the microbiota bacteria. A third group will be fed a defined community of bacteria via a sugar syrup solution that will be added to the pollen. This community will include all major species of the honeybee gut microbiome. In this way, we will be able to separate any effect that the interaction between newly emerged bees and workers may have from the effect that the microbiome has.

We will also look at a fourth group. For each brood frame we will ensure that a significant number of brood are left untouched. The entire frame will be kept in an observation chamber, to which we will add marked bees. This will allow a fourth group of newly emerged workers to develop as similarly to natural environments as is allowed in the lab.

We will sample across the group after three days of development. We will compare the four lab-grown groups in two ways: we will survey the DNA methylation patterns in brain and body tissue using bisulfate sequencing, and we will extract guts in order to compare the microbiome composition of each group.

Potential Pitfalls and Solutions:

Stress can cause varied and antagonistic responses in honeybees, and lab conditions can effect the phenomena we are trying to explore. We are separating into several treatment groups in order to control for those effects by subjecting each group to the same stresses. We will also attempt two different means of obtaining samples: we will chill the bees and extract their tissue while they are still alive, and we will flash freeze them. Flash freezing might remove any effect that the slow chilling will have on the transcriptome. In addition, we may also test a fifth group. We will return to the hives, collect workers, and immediately sample them. While this would not control for age, it might allow us to compare the methylation states of our lab-raised groups with the average states of workers in the hive. (<-- I still don't get it. Why not just flash freeze at liquid nitrogen temperatures? Is there any evidence that this is stressful or alters the genome? If you do both then how will you know if the slow freeze changes the transcriptome? If you can't tell then it is just going to sew confusion without provided any logical way to recognize if a problem exists. If you are just going to assume that the flash freeze is correct then ditch the slow freeze. In a grant if you put stuff in that can never ever be recognized then people like me will jump on it. ;))

2. Determine if and how Acetylation patterns differ between bees lacking microbiomes and bees with conventional microbiomes.

We will follow the same steps above, and separate the bees into the same groups, in order to determine differences in histone acetylation. When we sample the bees and extract the DNA, we will run ChIP-Seq with an antibody against an acetylated histone residue.

In your real grant (not here), you will have to expand the technique part a bit, expand how the data will be analyzed (programs, perhaps analysis pipeline, and especially how statistically distinct changes will be detected), AND most important for you, you should note that the technique has been successfully used in your parent lab. This lets you lean on them more.

Potential Pitfalls and Solutions:

Extracting larvae from brood frames can have a low success rate: in order to get an accurate picture of the acetylation of the genome we will need many samples per group. We may run this experiment in a rolling fashion, doing the same experiment with new brood frames several times, in order to increase our sample size.

<-- Why do you need to extract them from the brood frame? Why not just grind up the frame? Is it that they are all different ages? Even so, why not figure out which ones you want and leave them in the frame. A sonicator or homogenizer will make short work of them. This might be a trivial question in your field. Everyone might know the answer. But your reviewer might not know the answer. If you have a difficult manipulation that must be done then you need to account for why you can't just work around it using a method wither "percieved" higher success.

3. Determine if common honeybee microbiome metabolites act as histone deacetylase inhibitors.
We will then explore specific metabolites for epigenetic functions. Bacterial metabolites are one of the biggest contributions of the microbiome, and we will explore the common metabolites provided by the gut microbiome (4). Butyrate, one of the most common metabolites contributed by the microbiome, has been shown to have HDACi activity (48). We will confirm this and determine which common metabolites, including lactate, acetate, and succinate, could serve epigenetic functions in the honeybee.

To determine their function, we will attempt to use a honeybee cell line to test their effects in vitro using a honey bee cell line derived from from honey bee embryonic tissue (10). (<-- How do you know that they won't damage the cell line? Are there citations that show that they survive this? Also, what cells grow in this culture? I know that in flies, how you manipulate the embryonic cell line influences what cell type you get. You probably don't want a mixed culture because the compounds might differentially affect the cell types leading to the death of some. Also, will the cell type that you test be relevant to your stated interests. Do you need to test neurons, muscles, epithelia? All of them? Why or why not. Justify your choices.)

We will then extract two brood frames from two different hives. We will allow new workers to emerge in observation chambers, and feed the treatment group with metabolite rich sugar syrup. We will then sample bees from the two groups. We will extract their hemolymph to ensure the treatment group has elevated levels of the metabolite of interest, and then we will run ChIP-Seq with an antibody against an acetylated histone residue to compare the acetylation between two groups.

(Here you clearly indicate that you will feed this to animals. It should be clear in the first paragraph that both cell lines and bees will be tested. It is confusing to have one sentene about tissue culture and then a sudden switch without justification. Oddly, you don't say how you will administer it to the bees. Yes, I know it is obvious and implied by the rest of the grant. But you must not imply anything in a grant. Also, how do we know that bees won't refuse to eat butyrate, acetate, lactate, or succinate infused food. At some concentration it will become repulsive I am sure. How do you know that you can get enough into a bee to produce an effect? If there are papers then cite them.)

Potential Pitfalls and Solutions:

This set of experiments would not be determinative, but would serve as a starting point for further exploration. Adding butyrate to the sugar syrup could induce some other effects in the honeybees. In order to account for this, we will attempt different pairings of honeybee groups. If we see significant difference in acetylation patterns between germ free and conventional bees, then we will be able to use the germ free bees as a treatment group and control, and see if metabolites alter the acetylation pattern.