Mckayla Burns Samantha Jackson The Diel Rhythm that underlies the symbiotic relationship between Euprymna scolopes and Vibrio fischeri Mckayla Burns Samantha Jackson

Key Words Diel- A 24 hour period Cytoskeleton- A structural support system made of microtubules and microfilaments Microarray-A technique used for biochemical, genetic analysis Chitin-a nitrogen-containing polysaccharide G3P-is a chemical compound used for the anaerobic respiration of glycerol Blebbing- a blistering or swelling that occurs in the cytoskeleton

About the Authors Andrew M. Wier Associate professor at Pace University Uses Euprymna scolopes and Vibrio fischeri as a general model to study the effects of beneficial bacteria on animal host tissues. Spencer V. Nyholm Previous research: Dominance of Vibrio fischeri in secreted mucus outside the light organ of Euprymna scolopes: the first site of symbiont specificity. Appl. Environ. Microbiol., 69(7), 3932-3937. Professor at the University of Connecticut Also studies the symbiosis between tubeworm and sulfur oxidizing bacteria in hydrothermal vents

Background Information Horizontal Transmission: Every generation of Euprymna scolopes and Vibrio fischeri must establish the symbiosis, which is different from vertical transmission where the symbiosis is passed from parent to offspring Mutualistic Relationship: The squid provides nutrients for the bacteria and the bacteria provides the squid with bioluminescence which is used for the squid’s protection

Background Information A: Shows the position of the light organ within the squid, the expulsion of bacteria from the light organ, and crypt where bacteria symbionts resides B: The blue line symbolizes the population of the bacteria throughout a 24 hour period. Bacteria’s populations are the highest when the nocturnal host is the most active. - During expulsion the squid releases 95% of it’s bacteria symbionts, keeping only 5% to regenerate There is an underlying diel rhythm to bioluminescence Figure 1: The diel cycle of the squid/Vibrio symbiosis

General Gene Expression The greatest changes in gene expression of the host cells and symbiont cells occurred before and after dawn The highest up regulation of host genes occurred before dawn The highest percentage of up regulated genes in the symbionts occurred after dawn During some intervals both partners changed the expression of genes specifically used for signaling and metabolism REFER TO GRAPH C ON THE LAST SLIDE Periods of dynamic interaction -expulsion -bioluminescence

Purpose To describe the transcriptional, metabolic, and physiological aspects of the symbiosis between Euprymna scolopes and Vibrio fischeri and determine if they follow a diel pattern

Physiological Methods: Used TEM (Transmission Electron Microscopy) Examined the epithelium cells within the light organ of Euprymna scolopes over 4 different time periods within 24 hours Results: Major change in the morphology of the epithelial cells Directly after dawn, the epithelial cells of the light organ are obliterated Following this there was an increase in the number of host vesicles anticipating the creation of new bacterial symbionts A few hours later the microvillar border is restored within the epithelial cells Next, the light organ is colonized by new bacteria cells and it is ready for night fall and bioluminescence

How do the Bacteria Repopulate the light organ? Metabolize the lipid vesicles the host cells creates after dawn Host changes its tissue organization in order to provide the symbiont with glycerol and fatty acids Bacteria incorporate fatty acids from host lipids into its membrane Fatty Acid Composition of Bacterial Symbionts

Regulation of cytoskeleton genes Up regulated just before dawn Down regulated just after down TEMS of the host crypt epithelia B- Picture of Light Organ Crypt from late morning to evening Crypt Epithelium tissue is healthy looking through out the course of the day. Bacterial Population is right next to Microvilli of host epithelia Microvilli is in tact High pop of bacteria (left side of image) C- Picture of Light Organ Crypt near dawn Microvilli of host cells no longer in tact (middle section of picture) Apical Surfaces of host cells move into crypt spaces that previously held bacteria (right side of picture) Low pop of bacteria

Transcriptional Analysis Reveals Patterns in Symbiont Metabolism Methods: Examined changes in bacterial gene expression during the day and night Microarray analysis of the RNA in the central cores of the E. scolopes light organs Results: Diel Pattern of Symbiont Metabolism Just before expulsion there is an increase in the expression of genes for the fermentation for chitin Just after dawn there is an increase in the expression of genes for the anaerobic respiration of glycerol

Why do the symbionts shift their metabolism? At night during bioluminescence symbionts use chitin fermentation to generate ATP After dawn and throughout the day the symbionts use anaerobic respiration of glycerol to generate ATP The form of anaerobic metabolism may be dependent on the substrates available at the time When chitin is available 12% more energy is produced using fermentation When glycerol is available more energy is produced using anaerobic respiration Reduces sugars and carbohydrates into carbon dioxide and alcohol through the application of enzymes in the cytoplasm Fermentation- No electron acceptor Anaerobic Respiration Electron Acceptor from an external source Nitrate Respiration Electron Transport Chain

Evidence of a diel pattern of symbiont metabolism. Growth rates per mole of carbon Growth Advantages Wier A M et al. PNAS 2010;107:2259-2264 ©2010 by National Academy of Sciences

Conclusions: There is a transformation of host tissue anatomy There is a change in gene expression by both partners These changes are controlled by a daily rhythm Bioluminescence is maintained by daily interactions between the squid and the bacteria

Strengths/Weaknesses Results for anatomical changes are consistent with diel rhythm hypothesis Results support metabolic changes occur on a diel rhythm Weaknesses: The source of the nitrate for anaerobic respiration is still unknown

Further Study: 1. “Although the nutritional conditions within the light organ remain unknown, taken together, these calculations support the possibility that over the course of a 24 hour period, the host provides different substrates to its symbionts to optimize their performance under changing physiological conditions present in its tissue over the day-night cycle.” ^(Page 2262) 2. Do changes in the anatomy and gene expression present in other mammalian systems follow a diel rhythm? Tissues in Mouse Colon are believed to follow a circadian rhythm 3. Parallels between beneficial and pathogenic bacteria induced effacement

References: -http://www.pnas.org/content/107/5/2259.figures-only -Wier, Andrew M., and Spencer V. Nyholm. "Transcriptiona Patterns in Both Host and Bacterium Underlie a Daily Rhythm of Anatomical and Metabolic Change in a Beneficial Symbiosis." PNAS 107.5 (2010): 2259-264. Print.