HAWAIIAN BOBTAIL SQUID

quourum sensing in a gram neg bacteria

remember v fischeri is a gram neg bacteria in this bacteria, it is run by a simple feedback loop describe photo -bacteria cell -enzyme luxI autoinducers - signaling molecules - makes AHL The dim luminescence of ES114 in culture is caused by - the low production of the AHL (3-O-C6-HSL)

mj1 vs es114

researchers found that mji did not colonize well inside the squid - it was a mutant strain nonnative to the squid so the introduction of es114 strain came forward. the es114 strain of v. fischeri detects luminescence, which is light emission per cell. naturally es114, when it is colonized within the squid, it increases a magnitude of 2 to 3 orders. however, when researchers cultured the es114 strain, it only emitted 1/10th of the luminescence. strange.

light organ

resides within the mantle cavity is bilobed, kidney shaped develops from the same set of tissues that give rise to the inc sac develops around ~10 between fertilization and hatching of the egg. there is epithelial tissue surrounded by accessory tissues that serve to modify the light depending on the squids behavior the light organ has a thick reflector that directs the luminescence out of the ventral aspect, the belly of the squid it is important to note that 95% of the bacteria is expelled daily, while only 5% remains and continues to colonize and grow. the 95% of bacteria that is expelled is in the ocean, available for when the eggs hatch, the hatchlings/juvenile squids can then be infected/form their symbiotic relationship with the bacteria.

this presentation will feature

1) Hawaiian Bobtail Squid 2) Vibrio fischeri 3) Quorum Sensing -transcription/translation of lux operon -bacterial cell to cell communication 4) The mutualistic relationship & interaction

Chromatophores

1) are pigment filled cells within their skin that are surrounded by muscle fibers- can expand and contract to expose or hide spots of color 2) these cells consist of proteins called reflectins - gives the bobtail squid the ability to control how it reflects the sunlight 3) color change is beneficial and also extremely crucial -aids in protection from predators -it is expressed during courtship -form of cellular communication 3) bioluminescent - can shine light to conceal it's shadow.

Reproduction

1. initiated by the male. they use their chromatophores to reflect light to attract the females.. this courtship could pressume throughout the day 2. once the female and male attach, the males place the spermatophore (their sperm) in the females mantle within their ink sac which acts as a protective shell for the eggs. it is important to note that male bobtail squids have 1 shorter arm, this is used to place the sperm 3. Females spawn their eggs in the third research article I will be presenting later in the presentation, while they were observing the female - she layed eggs during daylight hours, she layed the clutch on one side of a dead coral, and layed each egg down in one minute intervals, between laying each egg, she then used her tentacles to cover the egg with sand to protect the egg. They found it took 3 hours for a female to lay 200 eggs. 4) Once the female finishes, she departs the eggs leaving the offspring to fend for themselves for development. there is very little parental investment and protection.

Quorum Sensing

3 types of quorum sensing signals are used in v. fischeri to chemically communicate. the first to are acyl homoserine lactone signals, they are autoinducers. N-3-oxo-hexanoyl-homoserine lactone N-octanoyl-homoserine- lactone these 3 signals Coordinate the regulation of specific genes in a cell-density-dependent manner

So why is this important?

Among the Vibrio species that are capable of causing human disease, there are two main groups: Vibrio cholerae infection and noncholera Vibrio infections. Vibrio infections are often characterized as a foodborne disease due to consumption of contaminated seafood, exposure of wounds to contaminated seawater, or injury caused by shark and Currently, researchers are trying to find the critical time points during which bacterial signaling occurs. gene expression brought about by interaction with Vibrio fischeri. Once potential genes have been identified, they will then conduct further research concerning the timing and location of gene expression in colonized host tissues. it is the modulation of the genes that describes the outcome of the relationship.

So now I have covered detail regarding our friend bob, the bobtail squid, it's symbiont friend vibrio fischeri. i've explained quorum sensing and how bacteria uses cell to cell communication last, i will go through the interaction between the two organisms. and explain why this mutulaistic relationship is important for future studies.

Come day time, bob tail squids bury themselves beneath the sand so they can sleep in the sand. As the daylight changes into night time, the bobtail squids, emerge in search for food the bobtail squids will their two tentacles to bury themselves out of the sand and begin swimming for crustaceans fish, crustaceans (shrimp), crabs, krill, and barnacles. but remember, these bobtail squids size, about 2 inches leave them very vulnerable when you're living in an aquatic ecosystem filled with many marine animals so what helps protect them so they can get their meal without being someone else's meal? their best friend, the the bacteria vibrio fischeri. vibrio fischeri colonize immediately after the squids hatches in order to keep the squid safe because remember, their parents make no contribution or investment in their care. so once the hawaiian bobtail squid and the bacteria vibrio fischeri join together because of -water movement -motility in the flagella -cilliated tissue in the duct of the squid the vibrio fischeri begin to thrive and divide while being protected and fed nutrients of the bobtail squid. once the vibrio fischeri reach a high cell density, the luxI induced an autodincer - AHL, which binds to luxR prtoein to regulate transcription, and then lux operon begins transcirbing the lux genes CDABEG , which then translates emit biluminescence. that light is on the ventral aspect of the squid, and this creates a flashlight, a camouflage cloak. so night time, moon, casts a shawdow of their light body - so when bigger predators are below them, they can see their shawdow and hunt - but with biluminescence, no shadow. so once the night ends, right before day time, they expell 95% of the bacteria out, which is now free living for their hatchlings and they begin to find a place to bury themselves to sleep.

Development

Development of a bobtail squid, after copulation/spawning, their is an 18-26 day embryonic period. hatchlings emerge from the egg and are initially aposymbiotic meaning they do not use their light organ immediately and weight about 0.005 grams. symbionic relationship begins to develop immediately after hatching from the egg. after a few days, the hatchlings will develop into a planktonic paralava meaning they can PARTIALLY use their light organ - and then they enter a juvenile stage at about 10 days where they are mature enough to travel into shallower waters.

Geography

Geography Habitat the bobtail squid thrive in high concentrations of salt water, and so does the bacteria. Day versus Night time

Chromatophores Image

Here I wanted to show you some really neat visuals captured, that I found on google. The pigment filled cells are extremely visible whenever the muscle fiber is relaxed. When the muscle contracts, they conceal the pigment so it reflects light. a great way to blend in with their surroundings or whenever they're going to sleep in the day time.

Slide 1

Light Emitting Bacteria Featuring the Hawaiian bobtail squid and it's symbiotic partner vibrio fischeri

Ink sac

Made of simple squamous epithelium Helps form the light organ - which is beneath it, on the ventral side of the bobtail squid. Develops during embryogenesis, changes shape during post-hatch and during maturation of the light development Highly reflective so that the ink is camouflaged and not visible through the mantle (Arnold et al., 1974) Ink Sac - releases an amount of ink when they sense a stimuli, indicating their threatened. The pool of ink resembles the shape of their body.

symbiosis

Remember the light organ is partially embedded in the ventral surface of the ink sac in the research study newly hatched juvenile squid revealed a presence of 3 pairs of sacculate crypts, noted in blue red and green. each crypt joined to a pore by a ciliated duct The crypts become populated with free living bacterial fischeri within hours after the juvenile squid hatch and begin to populate Cavities are lined with Ciliated epithelial cells sort of extend and grow to facilitate infection, v fischeri are also motile so their flagella helps swim/move into the light organ - V. fischeri has six flagellin genes flaABCDE flaF. once in the light organ, The bacteria decrease in size, lose their flagella, and emit light. One of the first known processes to occur is the secretion by the squidl of an external mucus web that is used to trap bacteria and that shows selectivity for V. fischeri. where the bacteria grow, divide, and, when a critical cell density is reached, produce luminescence. the onset of daylight signals the end of the normal nocturnal activity of the squid if you're wondering how these new hatchling juvenile squids get infected? EXPULSION : 95% of the bacteria is expelled out each morning - is now free living. for the new hatchlings while 5% those bacteria remain within the light organ must proliferate to a high cell density within the next 12 h or until night time.

Cephalopoda

Size ~ 2 inches long weigh about 0.09oz very small squids (IMAGE TALK) - in labs lifespan -grow rapidly -living in hawaii, they thrive year round -however they have a short lifespain of about 12-18 months Some important features of the bobtail squid that I will be going into depth will be their chromatophores reproduction embryonic development their light organ ink sac and their metabolism

Taxonomy

The bobtail squid comes from the animalia kingdom it is a part of the cephalopoda class it's genus name is euprymna and it's species name is scolopes

Physical Characteristics

The cephalopoda class consists of marine animals such as octopuses, cuttlefish, and squids some physical characteristics of a bobtail squid they are 1) an invertebrate - w/o bones 2) bilateral body symmetry 3) the largest structure on their body is their mantle which houses their lightorgan and ink sac 4)2 fins to help propel them in the water 5) 8 arms with suckers attached to help grip 6) 2 longer tentacles used to grab food and sand 7) they also have a funnel tube which allows water movement and air flow which aids in blow sand off of them whenever they are digging out from the ground.

Metabolism

Their position in the food chain is determined by their size Ultimately, squids are predators, and they help to maintain the ecosystem- they eat fish, crustaceans (shrimp), crabs, krill. They catch their prey with their two feeding tentacles. However, E. scolopes are fairly small, so they're also counted as prey for other creatures (sharks, seals, whales, dolphins, seabirds, fishes) - this is where chromatophors and their light organ play big roles in their defense mechanism.

In the research article

Upon discovering V. fischeri - initially MJ1 strain was used in labs to understand the luminescence regulation. MJ1 strain tested for . . -signal molecule -cloning -sequencing -functional characterization of the lux region/lux operon Experimental Limitation this particular strain could not be studied under the natural conditions in which V. fischeri produces light.

So now I have introduced to you the important features of bob, the bobtail squid. Now I will be featuring bob's best friend, it's symbiotic partner, virbio fischeri.

Vibrio ficheri is a part of the bacteria kingdom note, there has been a name change to this bacteria, its genus name is now noted as Allivibrio - however for my presentation, all of my references/primary sources, used vibrio as it's genus name - so Just a clarification.

definitions

define terms. imagine these teal squares are the bacteria cells, vibrio fischeri . well bacteria cells, produce an autoinducer. remember how i mentioned that light output is cell-specific. At low cell density, there aren't many autoinducers being created. these inducers are dilute molecules that will just diffuse because it's not being used up - there isn't enough action to create a reaction so in a high cell density enviro, like inside the light organ, there is a constant rate of autoinducers formed. if you have high cell density, meaning a lot of bacteria cells in one place and light output is dependent on cells - it's a positive correlation, more cells, more autoinducers, more light, or brighter light. the bacteria will sense a molecule signal change and in unison, they begin to undergo gene expression.

Eye development photo

e - eye develops during initial embryogenesis

bioluminesnce

encoded by 6 genes luxICDABEG regulated by the luxI and luxR the genes luxA, luxB genes encode the alpha and beta subunits of luciferase luxC, luxD, luxE genes encode a fatty acid reductase luxG is involved in flavin mononucleotide biosynthesis .

image

image of each signal pathway

Research Experiment 1

in the research article, researchers found an Anatomical, biochemical, molecular, and physiological similarity that exist between the eye and light organ. Similarities 1) A lens with crystalline protein 2) Reflectin proteins 3) Genes and proteins involved in photo transduction 4) physiological ability to respond to light Purpose of the experiment : they examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated inside the light organ by symbiosis.

Light organ development

indicated by is. develops towards the end of embryogenesis, just before the hatch.

image

luciferin, a substrate, is oxidized by molecular oxygenthis reaction ix catalyzed by the enzyme luciferase Lux A and lux B encode the two subunits of the enzyme luciferase Luciferase oxidizes (FMNH2) + aldehyde + oxygen an organic compound long chain aliphatic aldehyde, reduced flavomononucleotide to produce carboxylic acid, and oxidized flavmononucleotide FMN, water, and light lux C and D E encode a fatty acid reductase complex, is involved in recycling aliphatic acid to aldehyde, lux G and a protein reduces FMN to FMNH using NADH

Research Experiment 1

method they used for this experiment was PCR - to obtain dna sequences ISH - in situ hydrization - to localize gene transcripts in developing embryos, then compared their pattern of expression in the two organs. their result found that -each of the 4 genes is expressed in both organs - even though they're the same, they're also different. 1. they not homolougous - development from ectoderm/mesoderm 2. the organs develop at different stages during embryogenesis 3. light stimuli for the eye and light organ come from 2 different sources, environmental light - eye luminous bacterial symbiont - light organ ______ Light stimulus is important in the development of both tissues. Environmental light contributes to the maturation of the vertebrates eye Endogenous (internal) light serves as a critical cue for morphogenesis of the light organ

vibrio fischeri

oxidase-positive, Gram-negative bacteria they are motile, as you can see from this illustration, and might i add this bacteria cell is not to scale. but it is a great depiction of what the bacteria cell looks like Virbio produced light in a circadian rhythm, meaning it produces more light at night time than during the day. -it's a biluminescent bacteria - which is controlled by a small set of genes known as the lux operon The bacterium emits a blue-green light at the frequency of 490nm (nanometers) they are found free living without constantly emitting light and whenever their in a host, they emit light.

explain the photo

so here we meet with our friend bob, well inside his mantle is the light organ that is housing hundreds of v. fischeri. they have an enzyme known as luxI that is producing autoinducers. 3Oc6 and C8-HSL lactone. this autoinducer binds to luxR - a regulatory protein that has a promoter that will begin transcribing the lux genes. translation begins and eventually creates bioluminescence, light molecules.

discovering v fischeri within e. scolopes

so the researchers questioned. Q: How is it that the light within the host is so powerful yet cultured v. fischeri with the exact strain cannot produce to the same power? it was found that Light output is cell-specific. to understand, let's talk about quorum sensing

results

the researchers found that in v. fischeri - in the process of the lux operon - is really was dependent on the strain used it is crucial to include cell density as a variable es114 had metabolic means to produce higher luminescence - bc of their symbiotic relationship with the squid - the squid provides vibrio fischeri with nutrients and of course a shelter - confined space, creates high cell density. there was also a second regulator found in the es114 strain versus the Mj1 -ainS regulator which binds to an autoinducer known as c8-HSL

understanding lux operons

we're all familiar with the lac operon, an operon required for the transport/ translation and metabolism of lactose in Escherichia coli and other enteric bacteria. well, in a similar kind of way, the lux operon transcripts and translates, and encodes genes for biluminesence. for light expression this helped me understand the lux operon system. these systems are not the same but they are similar in small ways.

quorum sensing

well, its controlled by the luxI-luxR system luxI - inducer luxR- regulator this system is one part of the lux operon there are enzymes that catalyze the bioluminescence reaction is encoded by 6 genes lux CDA BEG it is important to note that luxI is cotranscribed with this set of genes.