Alpha Diversity: Diversity of species within the same community or habitat.

It focuses on the
number of species and their abundances within a particular area.
MEASURES  Richness (total numb. Of species) and evenness(how evenly individuals
are distributed among species)
= focuses on the diversity within a single habitat

Beta Diversity: Represents the change in composition or diversity between different habitats
or ecosystems. It measures the turnover of replacement of species from one habitat to
another.
MEASURES  Similarities and Dissimilarities of species composition between
different locations.
- For example, when you compare the plant species in two different forest, beta
diversity measures how much the species composition differs between the two
forests.
= measures the turnover or change in species composition between habitats

Gamma Diversity: Represents the overall diversity across a large geographic region, covers
multiple ecosystems or habitats. Provides comprehensive view of the total species richness
in broader area.
MEASURES  Cumulative diversity of all local habitants within a regional or
landscape scale.
= covers the overall diversity across a larger geographical scale, considering multiple
habitats.

Relative abundance: Proportion or percentage of a particular species or taxonomic group in

relation to the total abundance of all species in a given ecological community or sample.
( abundance of a specie/ total abundance of all species) * 100 )

Symbiosis: Biological interaction between different species that lives in close physical
association with each other. It can be beneficial, harmful or neutral for the organisms that
are involved. Symbiosis is important for in terms of shaping the ecosystems and evolution of
species.
a. Mutualism: Symbiotic relationship that is beneficial for both individuals that are
participating. (+ + )
b. Commensalism: Symbiotic relationship that is beneficial for one participant and
neutral for the other participant ( + 0 )
c. Parasitism: Symbiotic relationship that is beneficial for the parasite and often harmful
for the host (+ -)
d. Amensalism: Type of symbiosis in which one participant is unaffected while the other
is harmed ( 0 – )
 Symbiotic relationships between human-animal and their microbiota, especially in
the gut, represents forms of mutualism and comensalism. Microbiota aid in
digestion, absorbtion and immune system development.
  EXAMPLES GIVEN IN THE LECTURE:
1. Mutualistic symbiosis between the tube worms and chemosynthetic bacteria
- These chemosynthetic bacteria lives within the trophosome of the worm and use the
chemicals from the vent to produce organic compounds through chemosynthesis.
- In this case, worms provide the bacteria a habitat to live in and in return, bacteria
supply the worms with nutrients
2. Mutualistic symbiosis between the anglerfish and bioluminescent bacteria
- The anglerfish has a specialized organ called esca which acts as a lure and
responsible for emitting light. So the anglerfish harbors the bioluminiscent bacteria,
vibrio fischeri, through the esca. Bioluminiscent bacteria has a light emitting pigment,
LUCIFERIN, and an enzyme called LUCIFERASE. When oxygen interacts with luciferin
with the presence of luciferase, it produces light **LUCIFERIN LUCIFERASE
ACTIVITY**
- The importance of this symbiosis for the anglerfish is actually for it’s hunting strategy
in deep seas where visibility is limited. For the bacteria, in turn, it receives a habitat
and nutrients from the anglerfish. The esca of the anglerfish provides a protected
environment and anglerfish’s tissues supply the necessary nutrients for the bacteria’s
metabolic processes.
3. Mutualistic symbiosis between Coral Reefs and Zooxanthellae
- Zooxanthellae is a photosynthetic algae.
- While corals provide them a protected habitat within their tissues, the algae in return
provides oxygen and organic compounds through photosynthesis

 Lynn Margulis is considered as the head of symbiosis revolution due to her work on the
endosymbiotic theory. She proposed that certain organelles within eukaryotic cells, such
as mitochondria and chloroplast, originated from free living bacteria through symbiotic
relationships with ancestral cells. Margulis’s theory revolutionized our understanding of
evolution, highlighting the importance of symbiosis in the development of complex life
forms.

 Almost all animals and plants are happened to be in a symbiotic relationship, which is
discovered nearly last 10 years. But why did it take so long to understand the prevalance
of microbial symbiosis?

 Microbes are extremely abundant and diverse. But it is not always possible to grow all of
the organisms in culture media (VIABLE NONCULTURABLE), It is a technical difficulty that
blocked symbiotic studies. Also some organisms look alike, that is why it was hard to
distinguish them. By the sequencing revolution, it allowed researchers to explore
microbial communities more comprehensively which eventually led to the discovery of
previously unknown symbiotic relationships.

16S rRNA Sequencing:

- Due to the difficulties mentioned above, 16S rrna sequencing was a groundbreaking
development since it is a very conserved region for bacteria and by that we are able to
distinguish the genus or the species.
- The work of Norm pace and Carl woese revolutionarize our view on biosphere by allowing us
to through visual analysis to molecular analysis.
 What does Woese and Pace’s revolution mean for symbiosis?
o Now we are able to identify microbes and determine the relationships among them
whether or not they are culturable.
 Human Metabolic Map comes from sequencing. Thanks to it, we know how genes are
connected with each other. On the map, we are able to see lactate production but it does not
mean that we can ferment lactose but it refers to some particular cells that is able to use
lactose under certain conditions, muscle cells while you do sports.
 With the sequencing, we are observing the microbes and relative abundance but low
abundance does not mean lower importance! Sequencing by itself is not enough. We need:
o Abundance
 vast majority of microbe-plant interaction runs by bacteria of <0.1 relative
abundance),
o Activity
 because it can be dormant or active,
 In environmental context it can depend on light, nutrients and abiotic factors.
o Correlation
 that is different from causation.
 Wolbachia is fundamental for mosquitos ecology and reproduction
o Wolbachia lives inside the ovaries of mosquitos. During the studies of wolbachia,
scientists found out that if FEMALE has it, in any case progeny is affected by
wolbachia but id MALE has it and WOLBACHIA not, all the offsprings are dead, no
progeny.
o It means that the fitness of Culex pipiens depends of wolbachia!
 They took 2 gene of bacteriophage and put in the mosquito, the mosquito with the gens
form bacteriophage cause incompatibility. Is not Wolbachia affect mosquito but
bacteriophage. Before this discover they think is Wolbachia that cause incompatibility
Human microbiome: Collection of all microorganisms included with their DNA that is in
association with the human body.
We are holobionts! :
- Holism: Interconnectedness and interdependence of elements within a system.
- Holobiont: Host organism and all of the symbiotic organisms that are living in or on it,
forming a single ecological unit.

Importance of gut microbiota for host development and biology :

1. Immune system development
2. Gut function
3. Exclusion of pathogens
4. Gut tissue development
5. Nutrient acquisition
6. Host metabolism

Characterization of microbiome:
1. Composition
2. Function **
Gnotobiology : Scientific discipline that involves the study of organisms living in a KNOWN and
CONTROLLED microbial environment. Organisms are raised in environments where the microbial
composition is precisely defined, allowing researchers to investigate the influence of specific
microorganisms on the host organism’s physiology, development and immune response.
- In terms of microbes, we are getting poor, less diverse and the changes in the gut microbiota
causes some diseases as well. And microbiome related diseases are reaching to epidemic
proportions!
Unfunctionalities and diseases caused by the alterations in gut microbiota:
1. Heart disease
2. Obesity
3. Chronic inflamation
4. Neurological and mental disorders
5. Colon cancer
6. Diabetes
7. Allergies
8. Autoimmune diseases

What are the consequences of the lose of microbiota?

- In the paper, it suggests that the consequences of loss can vary depending on the specific
situation and context. In the case of the disappearence of H.pylori, there are potential
physiological consequences for the host as well as the potential changes in the microbiota
composition and interactions within the host body. For host, it can lead to some diseases and
changes in immue system, cytokine traffic in the stomach potentially affecting the host’s
ability to fight with infections. Also it can create a void in the gastric niche which can be filled
with other mo that may be more virulent and harmful for host. The factors that may be
contributing to the decrease in microbiota:
- The widespread use of antibiotics,
- Changes in human ecology such as increased use of pre-term antibiotics, reduced
breastfeeding and smaller family sizes
- Increased use of antimicrobial products such as soaps, mercury-amalgam dental fillings
- These changes in human ecology may be disrupting the equilibrium between the human
microbiota and their hosts leading to the decrease in microbiota diversity and potentially
impacting human health.
- The paper emphasizes the idea that humans and our microbiota have evolved together and
formed a complex relationship. The microbiota helps in extracting energy from food,
providing nutrients and vitamins, promoting tissue development and stimulating both innate
and adaptive human immune system. These functions are crucial for sustaining human life,
and without microbiota, humans would not be able to survive. Disappearing microbiota
hypothesis suggests that modern global ecological changes have destabilized the equilibrium
between humans and our microbiota, potentially leading to the rise in various health
conditions such as obesity and asthma. This further supports the idea that humans can not
functionally survive wo microbiota, as any disruptions or loss of certain bacterial species can
have detrimental effects on human health.
The old friencds hypothesis: Lack of exposure to certain microorganisms during early childhood may
contribute to the increased prevalence of inflamatory and autoimmune diseases. With the
improvements in sanitation, hygiene and antibiotic use, there has been a decrease in exposure to
certain beneficial mo that were once common in the environment associated with humans. Exposure
to these old friends during early development is believed to have a crucial role in training the immune
system and promoting regulatory mechanisms.
Environmental factors that are known to distrupt the gut microbiome associate with a higher
incidence of chronic diseases:
- C section
- Formula feeding
- Early life antibiotics
- Low fiber diet

What are the evidences for the role of the gut microbiota in disease?
DYSBIOSIS: Altered composition of gut microbiota, alteration in diversity (alpha and beta : quantity
and type)
- Studies have consistently observed changes in the composition and diversity of the gut
microbiota in individuals with various diseases such as IBD, IBS, obesity, diabetes etc.
ANIMAL STUDIES:
- Germ free or Gnotobiotic ( raised in sterile env.) have demonstrated the impact of the gut
microbiota on host physiology and immune function.
- Transplanting microbiota from diseased animal to healthy ones can induce disease-like
conditions.
MICROBIOME-ASSOCIATED STUDIES:
- The gut microbiota produces a range of metabolites through the fermentation of dietary
fibers and other substrates.
- SCFA’s, Secondary bile acids and other microbial metabolites have been implicated
influencing the host metabolism, inflamation and immune responses.
ASSOCIATION WITH METABOLIC DISORDERS:
- Dysbiosis has been associated with metabolic disorders such as obesity and type 2 diabetes.
Changes in the gut microbiota composition can affect energy extraction from the diet,
influence the adipose tissue metabolism and contribute to the insulin resistance.
IMPACT ON NEUROLOGICAL FUNCTION:
- The gut-brain axis which is a bidirectional communication system, is thought to play a role in
mood disorders, anxiety, neurodegenerative diseases and neurological conditions.
MICROBIAL SIGNATURES IN DISEASES:
- Specific microbial signatures or patterns have been identified in various diseases.
- Certain bacterial taxa may be more prevalent or less abundant in individuals with specific
conditions. These microbial signatures may serve as potential biomarkers.
THEURAPEUTIC INTERVENTIONS:
- Interventions aimed at modulating the gut microbiota such as probiotics, prebiotics and fecal
transplantation (FMT) have shown promise in the management or improvement of certain
diseases.

Mechanisms by which the microbiota influences human health and contributes to the
pathophysiology of chronic diseases:

1. Microbes Short Chain Fatty Acids

- Microbes in the gut ferments dietary fibers and produce SCFA’s such as acetate, propionate
and butyrate which are bioactive molecules that can influence various physiological
processes. They have been associated with maintaining gut health, regulating immune
response and impacting host metabolism.
- Absence of SCFA can lead to leaky gut.
2. Host and Microbes  Bile Acids and Trimethylamine
- The interaction between host and gut microbiota also involves the metabolism of bile acids
and the production of trimethylamine.
 - Bile acids are synthesized by the liver and modified by the gut microbiota, play a role in lipid
digestion and absorbtion.
- Trimethylamine is a microbial metabolite derived from dietary components and further
processing by the host can lead to the production of TMAO which is implicated in
cardiovascular disease.
3. Host Gut Hormones, Inflamatory Cytokines, Mediators of Intestinal Barrier Function
- The host influences the gut environment through it’s own physiological processes. This
includes the release of gut hormones such as peptide YY and glucagon like peptide 1, which
can impact appetite and energy metabolism.
- Inflamatory cytokines, signalling molecules involved in immune responses are also influenced
by the host and can affect systemic inflammation.
- The host contributes to the regulation of the intestinal barrier function which is crucial for
preventing the passage of harmful substances from the gut into the bloodstream.

SHORT CHAIN FATTY ACIDS IMPORTANCE:

- SCFA’s are the organic compounds that are produced by gut bacteria during the fermentation

brain communicatiopn
SCFAs from gut microbiota in gut
According to the paper “The role of
of dietary fibers.
- They are the main metabolites produced in the colon by bacterial fermentation of dietary
fibers and resistant starch. Following their production, SCFAs are absorbed by colonocytes,
mainly via Hydrogen dependent or sodium dependent monocarboxylate transporters(MCTs
and SMCTs). SCFAs that are not metabolized in the colonocytes are transported into the
portal circulation and are used as an energy substrate for hepatocytes, except for acetate
that is not oxidized in the liver.
- SCFAs improve the gut health with the maintenance of intestinal barrier integrity, mucus
production, protection against inflammation to reduction of the risk of colorectal cancer.
- SCFAs binding to G protein-coupled receptors.
- They are used as an energy source for intestinal cells.
- They are maintaining gut health by producing mucus and supporting the integrity of gut
lining.
- Butyrate possesses anti-inflamatory effects, reducing gut inflammation and helping with IBD
- SCFA’s has metabolic effects by affecting:
o Improving insuline sensitivity which is crucial for blood sugar regulation
o Appetite regulation
- SCFA’s effects neurotransmitters production and by that it regulates mood and behavior.
- Epigenetic regulation
o Histone acetylation: Butyrate in particular is a potent histone deacetylase (HDAC)
inhibitor. HDAC are the enzymes that remove acetyl groups from histone proteins,
leading to more condenced chromatin structure and decreased gene expression By
inhibiting HDACs, butyrate increases histone acetylation, promating more open
chromatin structure and facilitating gene transcription.
o DNA methylation: By promoting SAM which is a methy donor, they can effect DNA
methylation.
o Regulation of Epigenetic Enzymes: SCFAs can influence the expression and activity of
epigenetic enzymes such as DNA methyl transferase (DNMTs) and histone
acetyltransferase (HATs). The modulation of these enzymes can affect the epigenetic
landscape.
POTENTIAL FOR THEURAPEUTIC AND NUTRITIONAL INTERVENTIONS:
1. Drugs and drug-like theurapeutics:
a. Live Biotheurapeutics
 LBP’s are biological products containing live mo’s such as bacteria or yeast,
intended to provide health benefits when administered to humans. LBP’s
 commonly known as probiotics and they work positively influencing the gut
microbiota.
b. Fecal Transplantation
 FMT involves the transfer of fecal material from a healthy donor to a
recipient to restore a balanced and healthy gut microbiota. It aims to renew
the beneficial mo’s in recipient’s gut especially after disruptions such as
antibiotic treatments.
c. Phages/Genetic Approaches
 Phages can selectively target harmful bacteria, offering a precise approach
to microbiota modulation and genetic approaches aim to enhance or modify
the functions of specific microbes.
2. Food Additives/Supplements
a. Probiotics
 Live organisms that confer healthy benefits when consumed in adequate
amounts. They contribute to gut health by promoting a balanced
microbiome, producing beneficial metabolites and modulating immune
responses.
b. Prebiotics/Fiber/MACs
 Non-digestible substances, often fibers or resistant starches that selectively
promote the growth of beneficial mo’s in the gut. They provide a substrate
for the growth of beneficial bacteria.
 The prebiotic concept has evolved over the years. Initially, prebiotics were

comprehensive concept for prebiotics.”

According to the paper “towards a more
defined as nondigestible food ingredients that selectively stimulate the
growth of beneficial bacteria in the colon. However the definition has
expanded to include other body sites and not just the colon, and the term
“improves host health” has been changed to “beneficial physiological
effects”.
 Non digestible compounds that have been shown to have a beneficial effect
on the gut microbiota and ultimately lead to improved health and wellbeing.
 They are selectively fermented by mo’s in the gut and are not affected by
digestion in the upper GI tract.
 Prebiotics are able to stimulate the growth and activity of beneficial bacteria
in the gut, while inhibiting the growth of potentially harmful bacteria.
 They also influence the balance of gut microbiota, leading to a more diverse
and beneficial microbiome.
 Improved digestion, immune function, nutrient absorption, lower cholesterol
levels and regulate blood sugar.
 Examples: FOS (fructooligosaccharides), GOS (galactooligosaccharides),
inulin, lactulose and human milk oligosaccharides.
c. Synbiotics
 Combination of both probiotics and prebiotics that are designed to work
synergistically to enhance the survival and activity of beneficial mo’s. It aims
to maximize the positive effects on gut microbiome by providing both live
mo’s (probiotics) and necessary nutrients (prebiotics) .
3. Diet/Nutrition
 Foods rich in dietary fiber, omega-3 fatty acids, low in sugar and saturated
fats, phytochemicals etc.

Comparison of Microbiome Prudent Diet and Western Diet in terms of gut health:
Microbiome prudent diet is designed to support health and diversity of the gut microbiome. It
emphasizes a diverse range of plant based foods including fruits, veggies, whole grains and legumes.
These foods provide fiber and a variety of nutrients which supports the microbiome. In terms of
introducing beneficial probiotic bacteria to the gut; it involves fermented foods such as yogurt, kefir
etc. With the probiotics, also prebiotic rich foods such as garlic, leeks and asparagus serves as fuel for
beneficial gut bacteria. Also healthy fats, particularly omega-3 fatty acids are incorparated via fish,
flaxseed and walnuts. Nutrient intake is balanced and on the other hand, processed foods are limited
which can negatively impact gut microbiome.
On the other hand, Western Diet basically tends to be low in dietary fiber due to a high intake
of processed food and refined carbs and often high in saturated fats. It involves less fermented foods
which results in lower intake of beneficial probiotics. Also the highness of processed foods that
contain artificial additives and preservatives. Since it includes excessive sugar intake which can
contribute to the growth of less desirable gut bacteria. It lacks the adequate prebiotic-rich food which
impacts the growth of beneficial bacteria in the gut and unlikely to prudent diet, it is not balanced in
terms of macro and micronutrients combined with low fiber intake which leads to dysbiosis.
SUMMARIZE: The microbiome prudent diet focuses on promoting a diverse and balanced gut
microbiome through the consumption of nutrient-dense, fiber-rich and minimally processed food. In
contrast, the Western Diet characterized by its high intake of processed foods, saturated fats and
lower fiber content, may contribute to an imbalance in the guts microbiome potentially leading to
unfavorable health outcomes. Adopting a microbiome-prudent diet is associated with better gut
health and overall well-being.

How is the diet-microbiome-host interrelationship related to health?

This interrelationship plays a crucial role in determining overall health and well-being.
- Microbiome composition is effected by dietary influences. The composition of the diet
significantly influences the diversity and abundance of mo’s in the gut. Different dietary
components serve as substrates for specific bacteria, shaping the microbiome.
- Microbial metabolism is effected by dietary substrates because the food we consume
provides substrates such as fiber, carbs, proteins that are metabolized by gut bacteria.
Microbial metabolism produces various metabolites including SCFAs which are pretty
important for overall well being and other bioactive compounds.
- SCFA’s involved in health impact. They are produced during the fermentation of dietary fibers
by gut bacteria and play a key role in maintaining gut health. They also contribute to energy
metabolism, support the integrity of the gut barrier by promoting mucus synthesis and have
anti-inflammatory effects.
- Immune function is also influenced by microbiome. A balanced and diversed microbiome
contributes to the development and regulation of the immune response.
- Dietary factors plays a crucial role in inflammation and disease. Certain diets, such as
western diet that is high in processed foods, saturated fats, and low in fiber may contribute
to inflammation. Chronic inflammation is associated with various diseases such as metabolic
disorders, cardivascular diseases and autoimmune conditions.
- The gut and brain communicate bi-directionally through the gut brain axis and the gut
microbiome influences the axis, potentially impacting mental health mood and cognitive
function.
- Diet-microbiome connection effects metabolic health. The diet influences the gut microbiome
and alterations in the microbiome composition have been linked to metabolic disorders such
as obesity and type 2 diabetes.
- Microbial contribution effects nutrient absorbtion. Gut bacteria contribute to the digestion
and absorption of certain nutrients. The microbiome can enhance the bioavailability of
nutrients and influence energy metabolism.
- Dysbiosis is associated with various diseases including IBD and IBS.

How does low carb diets effect health in short and long term?
Low carb diets typically restrict the intake of carbs and increase the consumption of fats and proteins.
Short Term Effects:
 1. Rapid weight loss
2. Improved blood sugar control
3. Reduced bloating and water retention
4. Appetite suppression
o Increased intake of proteins and fats may contribute to increased satiety.
5. Changes in energy levels
o Fluctuations in energy levels during adaptation phase with potential initial feelings of
fatigue or low carb flu is often temporary
Long Term Effects:
1. Weight maintenance
2. Improved metabolic health
o Some research suggests that low carb diets may contribute to improvements in
metabolic markers including blood lipids and triglyceride levels
3. Cardiovascular risk factors
4. Nutrient deficiency risk
o Depending on the food intake there may be a risk of nutrient deficiencies espeially if
the diet lacks a variety of veggies, fruits and whole grains.
5. Ketosis and kidney health
o Extended periods of ketosis, a state in with the body uses ketones for energy, may
impact kidney function

Bubble boy: David Vetter, born with severe combined immunodeficiency (SCID) and germ free into
the isolator. Due to hic vulnerable immune system, he lived in a sterile environments, including a
plastic bubble to protect him from infections.

Microbial Conditions Of An Organism:

1. Conventional:
- Non germ free
- Has natural and diverse microbiota
- Host-microbe interactions are dynamic and complex
- Microbiome vary between individuals and can be influenced by factors such as genetics,
environment and diet.
2. Axenic
- Germ free
- Devoid of any detectable mo’s
- Raised and maintained in a completely sterile environment
- Lacks a naturally occurring microbiota
- Achieved through cesarian section delivery and subsequent isolation in sterile conditions
- Useful for studying the effects of specific microbes on the host wo interference from the
natural microbiota
3. Gnotobiotic
- Known and defined microbial community that might be composed of specific strains or
species of mo’s
- Researchers can introduce a controlled set of mo’s to an axenic host
- The microbial community can be manipulated and studied to understand the specific effects
of individual microbes or combinations on the host.
- Allows for controlled experiments to explore the roles of microbes in health and disease.

Germ free and gnotobiotic breeding today:

 Isalators, gnotocages and systems like the Tecniplast IsoCage play crucial roles in both germ-
free and gnotobiotic breeding. Isolators provide a barrier against external contaminants,
 gnotocages are the units within isolators designed for specific breeding conditions and
advanced systems like IsoCage ensure a controlled environment for precise research in
microbiology and immunology.
 Uses in germ-free breeding:
o Isolators involves maintaining animals in a completely sterile environment. Isolators
are enclosed systems that prevent the entry of mo’s from external environment.
These isolators are designed to maintain a germ-free state, providing a controlled
environment for breeding and housing animals.
o Gnotocages as a term could refer to a cage or housing unit within an isolator
specifically designed for gnotobiotic conditions. In germ-free breeding, these cages
aim to ensure that animals remain free from any detectable mo’s throughout their
lives.
o Tecniplast isocage system is an isolator sytem that may be used in germ-free
breeding. It incorporates isolators designed to maintain sterility, advanced
ventilation and filtration systems and specialized cages within the isolators to house
animals. The system ensures a controlled and sterile environment for germ-free
conditions.
 Uses in gnotobiotic breeding:
o Isolators in gnotobiotic breeding involves introducing a known set of mo’s to germ
free animals to study the effects of specific microbes on the host. Isolators are crucial
to maintain controlled conditions required for gnotobiotic research.
o Gnotocages are similar to germ-free breeding, this cage allows the controlled
introduction of specific mo’s to germ-free animals.
o The tecniplast isocage system may be adapted for gnotobiotic breeding. The isolator
system can be configured to introduce a defined microbiota to animals within
controlled environment.

Sterility Checking Methods:

1. Cultivating methods:
- It involves plating samples on media to detect microbial growth. This allows identification of
a broad range of mo’s.
- For axenic animals, it can be useful for monitoring potential contaminants.
- For gnotobiotic animals, culturing is essential for confirming the presence of the intended
mo’s in gnotobiotic animals
 CON’S  Some slow-growing or fastidious mo’s may be missed and the time required for
culture results can delay detection.
2. Polymerase Chain Reaction:
- PCR based methods offer high sensitivity and specificity, enabling detection of specific
microbial DNA. They can be faster than culturing.
- For gnotobiotic animals, PCR is used to ensure that only intended mo’s are present.
 CON’S  PCR may not differentiate between live and dead mo’s and it requires knowledge of
specific target sequences
3. Gram Staining:
- It can be used for bacterial detection and initial characterization.
- It provides rapid information on bacterial morphology
 CON’S  It is limited to bacterial detection and provides limited information on the identity
of mo’s and is not as informative as molecular methods.

How to make an axenic animal?

1. Antibiotic treatment:
- Inexpensive and applicable to any genotype and does not require specialized equipment
 - CON’S
o Some bacteria still remains other mo’s are still present.
o Also it may effect eukaryotic cells which can potentially influence the host organism’s
physiology.
o Prolonged antibiotic use may lead to the selection of antibiotic resistant strains
among the microbial population.
o Antibiotic treatment can disrupt the balance of microbial communities, potentially
allowing opportunistic fungi to proliferate.

2. Germ free conditions:

- Germ free conditions provide a completely sterile environment, free of bacteria, viruses, fungi
etc.
- Researchers have precise control over the microbial environment allowing for exclusive
colonization with specific mo’s
- CON’S
o Expensive and requires specialized equipments and skilled personnel.
o Introducing new genetic backgrounds or strains often requires re-derivation in germ
free conditions which can be time consuming and complex. ,
GERM FREE MICE PRODUCTION :
- To create a germ-free mouse, an embryo is created through in vitro fertilization and then
transplanted into a germ free mother. BUT if this method is not available, a mouse can be
born through cesarean birth (higher risk of contamination). This process uses a non-germ free
mother which is sacrificed and sterilized before the birth.
- After the c-section the pup’s must be delivered to a sterile incubator with a germ-free mother
for feeding anf growth. These methods are required for the generation of a germ-free mouse
line.
- Once line is created, all progeny will be germ free unless contaminated.
- Typically for the experiments, each mouse is housed seperately in a sterile isolator to prevent
cross-contamination between mice.
- The mice are provided with sterilized food and water to prevent contamination.
- CONFIRMATION OF GERM FREE STATUS:
o Routine plating
o 16s rRNA seq
o Serology
** Real germ-free animals achieved when we use larvaeas instead of adult ones. Because for the
adults, only thing you can do is antibiotic treatment which is not completely promising in terms of
achieving fully germ-free animal.

Why mice for biomedical research?

- Genetic similarity and common ancestor
- Short reproductive cycle
o It allows for the generation of multiple generations in a relatively short period.
- Ease of handling and housing due to its size.
- Isogenic mouse strains
o İsogenic refers to a state where two or more individuals or organisms have identical
or nearly identical genetic compositions ; particularly in terms of their alleles at
specific genetic loci. ESSENTIALLY GENETICALLY IDENTICAL OR HIGHLY SIMILAR.
- Availability of tools and resources
o Genetic databases, antibodies, molecular tools are available for mice which
facilitates experimental design and analysis.
 - Axenic breeding.

Comparison of mouse vs human microbiota:

- Overall composition:
o Predominantly composed of bacteria with smaller proportions of viruses,fungi and
archaea BUT specific microbial species and their relative abundances can vary
significantly
- Bacterial diversity:
o Even if they exhibit high bacterial diversity with numerous taxa contributing to
overall composition, the specific bacterial genera and species present in gut
microbiota may differ
 For instance, certain bacteria that are prominent in mice may be less
abundant in human vice versa.
- Firmicutes and Bacteriodetes ratio:
o Firmicutes and Bacteriodetes phyla are prominent in both. But, the ratio can vary
between species and is influenced by diet and genetics.
- Microbial metabolism:
o They contribute to the metabolism of dietary components, producing SCFA’s and
influencing host energy balance but specific metabolic pathways and the efficiency of
microbial metabolism may differ between them.
- Immune interactions:
o Both mouse and human microbiota interact with the host immune system,
influencing immune development and homeostasis. But the details of this interaction,
including the types of immune responses elicited can vary.

Comparison of mouse vs human intestinal tract:

- Anatomy:
o Both have similar basic structure of the tract that consists of small intestine and
colon. But the size and the length of the intestines vary between species. Mice have
relatively shorter intestine compared to humans.
- Microbial composition:
o Predominantly composed of bacteria with smaller proportions of viruses,fungi and
archaea BUT specific microbial species and their relative abundances can vary
significantly
- Cell types and function:
o Both species have a similar array of cell types in the intestinal epithelium including
absorptive enterocytes, goblet cells and enteroendocrine cells. Relative abundance
and distribution of certain cell types and distrubutions of them may differ. For
instance Paneth cells are more prominent in the small intestines of mice.
- Digestive enzymes:
 o They produce many of the same digestive enzymes to break down carbs proteins and
fats. But the activity levels and efficiency of nutrient absorption varies.
- Physiological Parameters:
o Basic physiological processess such as peristalsis, nutrient absorption and water
reabsorption are common in both. But the rate of these processes may vary

Power of non-model organisms:

- A researcher utilized a whole island as the ecosystem for studies on wild mice that are living
there. This utilization of a whole island as an ecosystem for studying wild mice exemplifies
the power and relevance of non model organisms in ecological research. Non-model
organisms refer to species that are not traditionally used in lab settings, unlike model
organisms that are commonly employed due to their genetic tractability and ease of
manipulation. The island serves as a natural and uncontrolled environment, providing a
realistic and complex habitat for the wild mice. Most of the time, the things that happen in
lab does not happen in real life.
- **This approach offers ecological realism, allowing for the exploration of natural behaviors,
adaptations and population dynamics in a dynamic and diverse habitat. The island ecosystem
provides a holistic understanding of ecological interactions, genetic diversity, and behavioral
adaptations that may not be captured in controlled lab settings. Research of non model
organisms in their natural environments has practical applications in conservation and
ecosystem management, contributing valuable insights to broader ecological and wildlife
biology contexts.

How anatomy affects gut microbiome composition?

1. Variation in physicochemical conditions:
a. pH levels:
 The stomach has an acidic environment, affecting the types of microbes that
can survive, while the colon has a more neutral pH, influencing different
microbial communities.
b. Oxygen availability:
 Variations in oxygen levels along the GI create niches for both aerobic and
anaerobic ones.
2. Length and surface area:
a. Length of GI:
 Longer intestines provide more surface area for microbial colonization and
diversity.
 Herbivores, with longer intestines, facilitate prolonged microbial
fermentation for cellulose digestion. On the other hand, carnivores, with
shorter instestines have a faster transit time.
b. Surface area of organs:
 Different organs have distinct surface characteristics influencing the
available habitat for microbes
  Distinct surface structures of the small intestine like villi and microvilli
provides a larger surface area for microbial colonization compared to the
relatively smoother colon.
3. Transit time:
 The time it takes for food to pass through different gut sections influences
microbial residence and interaction.
 Rapid transit time in the small intestine limits the duration of microbial
exposure, while the slower transit in the colon allows for more extensive
microbial interaction.
4. Epithelial structures:
a. Microbial niches:
 Unique structures like villi and crypts (deep invaginations) in the gut
epithelium create specific niches for microbial attachment and colonization
b. Mucus layer:
 The thickness and composition of the mucosal layer act as a physical barrier,
influencing which microbes can adhere to the epithelium.
5. Physical changes during development:
a. Embriyonic to adult transition:
 The shift from a sterile environment in the embryo to exposure to microbial
communities postnatally influences microbiome development.
b. Maturation with age:
 Changes in a diet and environmental exposures during different life stages
contribute to the maturation of the gut microbiome
6. Sex hormones and immune responses:
a. Hormonal influence
 Sex hormones can impact the gut environment
 Estrogen and progesterone fluctuations in females during the menstural
cycle can impact the gut microbiome composition.
b. Sex-specific immune responses
 Estrogen and testosterone can modulate immune responses.
 Estrogen has been associated with enhanced immune function while
testosterone may have immunosuppressive effects.

#Last slide examples and topics explained#

 In the insect gut, redox potential exhibits spatial variations with oxic conditions in anterior
regions such as foregut and midgut while hypoxic conditions are prominent in posterior
hindgut. The oxic zones support aerobic mo’s adapted to oxygen rich environments, engaging
in aerobic respiration. Conversely, the hypoxic hindgut fosters anaerobic mo’s that thrive in
low or absent oxygen conditions and contribute to microbial fermentation. This redox
gradient influences microbial metabolism, with aerobic respiration dominating in oxic regions
and anaerobic fermentation prevailing in hypoxic zones. These microbial activities contribute
to the reduction and oxidation of substrates, generating diverse metabolites, including SCFAs.
The redox dynamics along the insect gut shape the structure of the microbial community,
impacting insect digestion, nutrient absorption and overall host physiology.
 In the mammalian gut, the redox potential creates a dynamic environment that varies along
the GI. The lumen of the mammalian gut experiences diverse redox conditions, with the
proximal regions, such as small intestine, being more oxic due to the exposure to atmospheric
oxygen. This oxic environment favors the growth of anaerobic and facultative anaerobic
mo’s. As we move towards the distal regions such as the colon, the gut becomes increasingly
hypoxic, providing a niche for anaerobic mo’s that engage in fermentation process. The redox
 potential in the mammalian gut plays a crucial role in shaping the composition and metabolic
activities of the gut microbiota, influencing host-microbe interactions, nutrient metabolism
and overall gut homeostasis.
 In gut microbiota, in the absence of oxygen, dietary fibers (complex carbs) are converted to
SCFAs !!!! with the presence, fermentation to co2

In terms of physical barrier, there is an example given by using hydra in the slides:
 Complex microbial community interactions in hydra. Hydra has two layers (inner,outer). Inner
layer consists of ectodermal epithelial cells that is the barrier which prodouces vast amount
of AMPs (antimicobial peptides) which plays crucial role in the organisms innate immune
response. They can inhibit the growth or kill mo’s such as bacteria and fungi.In hydra, these
peptides acts as a defence mechanism against potential pathogens. When the organism is
exposed to harmful mo’s, the production of AMPs is upregulated to combat and neutralize
the invading pathogens. This contributes to the maintenance of a symbiotic relationship
between hydra and it’s associated microbiota while also protecting the organism from
potential infections. Outer layer consists of glycoprotein and polysaccharides which together
refers to glycocalyx. It is a mucus like layer and serves various functions including protection
to the cells, playing role in cell recognition and adhesion and participating in interactions with
the external environment. This gel-like layer helps in preventing foreign particles from coming
into direct contact with the epithelial cells and contributes to the overall integrity and
functionality of hydra’s epithelial layer. Most importantly, it is involved in proesses such as
host-microbe interactionsan is an essential component for maintaining hydra’s symbiotic
associations with mo’s. In this complex system microbes may interact with each other in
multiple ways. For the species involved interactions can have a positive, negative or no
impact.
 Glycocalyx is a habitat for symbionts!
o They can be cooperating  syntrophy ( ++)
o One of them is predating  food chain with waste product inhibition
o Competing  Substrate competition (--)
o Amensalism  waste product inhibiton (0-)
o Commensalism  food chain (0+)
o No interaction  no common metabolites(00)
 In insects,both septate junctions and peritrophic membrane plays vital roles in shaping
dynamics of the gut microbiota. Septate junctions act as specialized barriers between
epithelial cells, preventing the invasion of microbes from the gut lumen into the insects body
cavity. This seperation ensures a controlled interaction between the host and its gut
microbiota. On the other hand, the peritrophic membrane, a chitinuous structure lining in the
gut lumen, forms a physical barrier around food particles. It creates a microenvironment for
microbial activities, protecting the gut epithelium from abrasive particles and potential
pathogens. Together, these structures contribute to maintaining gut homeostasis by
regulating interactions between the host and its microbiota.

How to identify the influence of host-genotype on microbiota composition?

Techniques that are used to understand how our genes are affecting microbiota composition:
1. GWAS (Genome wide association studies)
 o Powerful approach used to identify associations between genetic variations and
specific traits or phenotypes
 SNP’s and microbiota identifying sequences.
o In the context of micobiota composition, GWAS can be employed to identify genetic
factors that influence the abundance and diversity of microbial species in the gut.
o The sequencing of the whole genome is followed by the statistical matching.
o For example, a presence of a certain SNP is found to be related with the presence of
Lactobacilli. And this is statistically relevant within a population.
2. QTL (Quantitative Trait locus)
o Genomic region that contains genes or DNA sequences associated with the variation
of a quantitative trait, which is a trait that exhibits continuous phenotypic variation
such as height, weight, blood pressure etc. Rather than discrete categories.
o In QTL, we are collecting phenotype data that could be gathered from metabolome
or from RNA sequencing. The purpose of crossing individuals in QTL is to detach
genetic sequence from the neighboring landscape which has a regulatory effect in
gene expression.
o QTL tries to match different traits and the presence of specific microbe.
o CORRELATION BETWEEN GENETIC TRAIT AND A SPECIFIC PHENOTYPE!
3. 16S rRNA sequencing

 Combining GWAS, QTL and 16s rRNA seq allows researchers to connect genetic variations
with specific microbial taxa, providing a more comprehensive understanding of the host
microbiota relationship
 Additionally functional metagenomic approaches can be employed to investigate the
functional capacities of microbial communities influenced by host genetics.

Paper: Genetic mapping of microbial and host traits reveals production of immunomodulatory
lipids by Akkermansia municiphilia in the marine gut

- QTL mapping identified marine genomic regions associated with variations in bacterial taxa.
Then, they delved into metabolomics by focusing on lipidomics and they found the majority of
the lipids were unknown but they found a huge variability between the profile of mice.
- THE CORRELATION HIGHLIGHTED:
o Everytime there was a specific sequence, there was also Akkermansia municiphilia
and a particular lipid that is called Ornithine Lipid (OL). OL was high when there is a
presence of Akkermansia. Also they observed the same correlation in human gut too.
o So the study revealed that A.municiphila is the major source for OL in the gut.
 OL has immunomodulatory effects
 OL helps with reducing the susceptibility to antibiotics, so the bacteria is
protected and this is also beneficial for the host since this bacteria is
beneficial in terms of their anti-inflammatory properties.

- Studies on the lactase gene showed that if we have bacteria that carries lactase gene, we can
metabolize lactose, otherwise we can’t
- Bifidum and lactose :
o If you have lactase gene, you have less bifidum and if you don’t have the gene, you
have more bifidum.
Drosophila paper:
 Gut associated bacteria are transmitted to offspring through contamination of eggshels.
Recent studies have shown that wild populations of Drosophila have a greater diversity of gut
ass. Bacteria compared to lab flies.
 Investigation of microbial diversity associated with Drosophile carried out via both culture
based and molecular based techniques to identify the microbes present. The most common
phyla found in these studies are PROTEOBACTERIA, FIRMICUTES and BACTERIODETES. Some
of the most common genera identified, LACTOBACILLUS, ACETOBACTER, ENTEROCOCCUS.
Lactobacillus and acetobacter is most common and enterococcus faecalis is specific to lab
stocks.
 The microbiota in the gut plays crucial role in the host immune system by stimulating the
production of antimicrobial peptides and ROS, which helps to eliminate potential pathogens.
 The way Drosophila melanogaster is cultivated in the lab can have a significant impact on it’s
microbiome. The use of antimicrobials in fly medium reduces the diversity of bacteria that
can associate with the flies. Additionally, the transfer of flies onto fresh and sterile medium
can result in the same bacteria being present in both the flies and the medium.
 Diet, environmental and host factors effects gut microbiota. Host factors are the immune
system also plays role in controlling the density and composition of gut microbiota. But still
there is a lot to learn about the specific interactions between fly and gut microbiome.
 The gut microbiotas composition changes with developmental stages and as the flies age.
Bacterial density in the gut increases throughout the larval stage and reaches plateau in
third-instar wandering larvae. There is a sharp decrease in bacterial density during
metamorphosis but increases again after 48 hours. This change is related with the alterations
in host gene expression.
 Yeasts are major food source for drosophila. It provides nutrients necessary for their
metamorphosis and reproduction. Drosophila reflects the type of substrates the host utilizes.
The interaction between fly and yeast is mutualistic.
 The gut microbiota plays role in protecting against pathogenic microbes and promotes host’s
survival. It also impacts host growth and metabolism by contributing enzymatic activities that
break down non-digestible carbs and producing SCFA’s. Lactobacillus planarum promotes
larval growth by enhancing amino acid assimilation and activating TOR-dependent host
nutrient sensing system.
 The gut of Drosophila has a local antimicrobial response mediated by lmd pathway which is
activated upon the detection of certain types of peptidoglycan. Negative regulators such as
Pirk and dUSP36 downregulates the lmd pathway while transcription factors like caudal
restrict the expression of antimicrobial peptides to specific regions of the gut. These
mechanisms help maintain a balance between immune activation and the presence of
beneficial gut microbes.
 The researchers found that the microbiota can influence several host traits such as larval
growth and mating preference by altering sex pheromones.
  Gut microbiota influences various aspects of host physiology including NUTRITION
ABSORPTION, ENERGY METABOLISM, IMMUNE FUNCTION, PROLIFERATION AND
DIFFERENTIATION OF INTESTINAL STEM CELLS THAT ARE RESPONSIBLE FOR MAINTAINING
THE INTEGRITY OF THE GUT EPITHELIUM, HOMEOSTASIS.
 Early studies showed that Drosophila can live in aseptic conditions and yeast is an important
component of their diet.
 Drosophila melanogaster is attracted to specific fruits that are high in fermentative yeasts
and low in bacteria.

State of the art: Highlighting the latest research and knowledge on the gut-associated microbial
symbionts of Drosophila melanogaster with a specific focus on bacteria and yeast. It discusses the
history and importance of this research as well as current advancements in understanding the
diversity of microbial communities and the factors that influence their interactions with the host.
1. Microbiota of Drosophila and it’s effect on host metabolism and phenotypic expression of
mutations.
2. Diversity of microbial communities associated with both lab and wild type populations.
3. Genetic and environmental factors that influence these interactions
4. How ecological context plays a role.
5. Importance of yeast in flies microbiome.
6. Role of yeast in nutrition and it’s contributions to the ecology and the evolutionary
biology of Drosophila.
7. Impact of ephemeral resource pathces on animal-microbe interactions with a particular
focus on dead organic matter and associated mo’s.
8. Role of food-derived odors and microbiota in assortative mating.

The gap of knowledge : Lack of understanding of the interactions between drosophila and their
associated microbiota in their natural environment. This includes unanswered questions about the
role of bacteria found in the gut of the fly and whether they are strictly residents or dietary microbes
as well as the mechanism involved in drosophila-microbiota interactions. Limited attention given in
the topic of role of the yeast in drosophila microbiome especially in the comparison to bacteria.
Biological question:
- Is there a direct relationship between bacteria in the substrate(food source) and those in the
gut and how different environmental and host factors shape the drosophila microbiome?
- How does the diet effects the bacterial composition of drosophila?
- What is the potential role of microbiota in Drosophila behavior and mating?
Aim of the study :
1. To analyze and understand the diversity of bacteria associated with Drosophila
melanogaster, both in lab stocks and wild-caught flies.
2. It also aims to determine the extent to which the association between drosophila and
bacteria is controlled or random.
3. The study seeks to investigate the role of these bacteria in host physiology, particularly in
digestive and immune functions.
4. Exploring the potential of drosophila as a model for studying host-microbe interactions and
the impact of these interactions on host health in immune defenses
5. Understanding the ecological context drosophila microbiota, including the role of the
environment and food sources in shaping the microbiota and it’s impact on drosophila
biology and evolution.
Methods:
 1. Chemically defined medium to demonstrate the role of yeasts as a major food source for
drosophila species.
o This medium was shown to provide nutrients such as aa, sterols, vitamin B and fatty
acids that are not generally found in decaying plant manner.
2. Isolation of yeasts from adult crop by culturing on defined medium and identifying isolates by
traditional morphological methods.
o This method was used to characterize the diversity of yeasts associated with
drosophila
3. Culture based methods to detect culturable bacteria from homogenized whole flies.
o This method was used to study bacterial transmission and persistance during the
drosophila life cycle. It was found that bacterial counts are initially low in young
adults but increase significantly in older flies.
4. Contamination of eggshells which are ingested by young larvae.
o This method was used in a past study
Results
1. Identification of relatively low number of taxa in lab stocks of the fly, primarily from the
genera Acetobacter and Lactobacillus and presence of lab-specific bacteria such as
Enterococcus faecalis and Gluconobacter morbifer
2. Identification of bacteria from the family Acetobacteriaceae and the order Lactobacillales as
dominant in the microbiota of lab-reared flies.
3. Selection of rotting fruits over poor nutritional sources or pathogens by wild-caught
drosophila, potentially promoting growth and avoiding over-populated substrates or
nutrient-deficient environments.
4. Impact of host factors such as antimicrobial peptides on gut microbiome composition.
5. Differences in the gut associated bacteria between lab-stocks even fed on identical lab-
defined food sources.
6. Role of microbiota in promoting male courtship and potential involvement in assortative
mating.
7. Importance of understanding the environment-drosophila-microbiota interaction for
evolutionary studies.