Marianna Nava
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Symbiosis
Dr. Crafts

Symbiosis between Vibrio fischeri and the Hawaiian squid, Euprymna scolopes
E. scolopes
E. scolopes

V. fisheri
V. fisheri














Introduction

Vibrio fischeri is a bacteria that lives in marine environments. In order to survive, it forms symbiotic relationships with several other species of animals. For example, this gram-negative bacteria forms a mutualistic symbiosis with a variety of species of squids, mainly Euprymna scolopes. V. fisheri is a motile bacteria that lives freely in oceans, and are also able to thrive inside host organisms. In the case of E. scolopes, the bacteria is able to proliferate inside a specific organ that is responsible for bioluminescence.
There are several proteins that are responsible for the bioluminescence in the bacteria, and they are in encoded in many different genes. This bacteria produces light in a chemical reaction, which is used by the squid in order to counter-illuminate and be able to survive in their nocturnal environments. Counter-illumination means that the squid is able to match the amount of light which is being emitted by the moon, and produces a similar amount of light through its ventral side, so it does not have a shadow in the water. Through this mechanism, the squid is able to escape from predators.
The way that bioluminescent bacteria control their metabolic functions are through a method of quorum sensing, where the bacteria are able to measure their density and figure out whether or not they need more of each other in order to produce light. This is important because single bacteria, do not bioluminescence.
Euprymna scolopes are very small squids, that inhabit the Hawaiian Islands. They are nocturnal creatures and therefore rely on V. fischeri to survive other nocturnal predators. They live in extremely shallow waters and feed during the night. E. scolopes have a very specific organ that not only houses the symbionts, but also emits the bioluminescence. During the day, these particular creatures bury themselves in the sand, to camouflage into their surroundings and escape predators. During this period of time is when they also expel 95% of the symbiont bacteria into the ocean. This behavior of expelling the V. fischeri provides the opportunity for other symbionts to populate the organ, as well as other juvenile hosts to become colonized.
The remaining 5% of the bacteria regrow in the light organ so the squid is able to produce light to protect itself. This curious fact will be the focus of this web-page, while also exploring some fascinating morphological features of this organ.

Symbiosis
E. scolopes and V. fischeri have adaptively coevolved, and now maintain an exclusive symbiotic relationship. The squid benefits from the bacteria because it produces light, and that allows it so escape predators and maintain its nocturnal behavior. The bacteria also benefit from this relationship because it has a protected environment in which to flourish. It also gains nutrients from within the crypts of the light organ. This way, both organisms benefit making this a mutualistic relationship.




The Light-Producing Organ
The only way that the squid is able to develop its light organ is if it is infected with the bioluminescent bacteria. The feasibility of E. scolopes also depends on the presence of a specific bacterium of this type. Since the light producing organ expels most of the bacteria in the morning, the 5% left of V. fischeri remains to repopulate the light organ, and the squid becomes a site for bacterial multiplication. Newly hatched squids are born without the bacterium and must acquire them from the surrounding seawaters. It is believed that the presence of V. fischeri is very minute, compared to the presence of other types of bacteria in the ocean. It is curious however, that the squid becomes infected with V. fischeri within a few minutes after it is born. Additionally, experiments have shown that bacteria that are not V.fisheri are not able to colonize the host.
The interior of the light organ is a complex structure, where V. fischeri and host cells interact. The colonization of the light organ is a very specific process, and only the non-flagellated gram-negative bacteria are able to reach the inside of the organ completely.


external image 500px-JuvScolopesDiagram.jpg

Figure 1a: The juvenile light-organ system. a. The pattern of water flow through the mantle cavity. b. A confocal micrograph showing the pores, on the surface of the organ through which the symbionts enter host tissues. Source: The Winnowing: Establishing the Squid-//Vibrio// Symbiosis




The Selectivity of the Light Organ in E. Scolopes


In the infection stage of the colonization process, free floating bacteria in the ocean induce the newly hatched squid to secrete mucus from one of its appendages in the light organ. It is believed that V. fischeri is attracted to the mucus that is secreted, as well as other bacteria. Next, V.fisheri must use its flagella to migrate from the mucus aggregation to pores in the organ. After passing through the pores, the bacteria must go into the crypts of the light organ. This is a difficult process because narrow ciliated ducts connect the pores to the crypts. This ensures that the other type of bacteria that were able to enter the light organ are “selected out”. This way, only non-flagellated gram negative V. fischeri are able to reach the end destination.





external image nrmicro957-f8.gif
Figure 2: The winnowing. This model depicts the progression of light-organ colonization as a series of steps, each more specific from symbiosis-competent Vibrio fischeri. a. In response to Gram-positive and Gram-negative bacteria ( alive or dead) the bacterial peptidoglycan signal causes the cells of the ciliated surface epithelium to secrete mucus. b. Only viable Gram-negative bacteria form dense aggregations c. Motile or non-motile V. fischeri out-compete other other Gram-negative bacteria for space and become dominant in the aggregations. d. Viable and motile V. fischeri are the only bacteria that are able to migrate through the pores and into the ducts to colonize host tissues e. Following successful colonization, symbiotic bacterial cells become non-motile and induce host epithelial cell swelling. Only bioluminescent V. fischeri will sustain long-term colonization of the crypt epithelium. Also taken from The Winnowing: Establishing the Squid-Virio Symbiosis


THE SPECIFICITY OF THE LIGHT ORGAN IN THIS SYMBIOTIC RELATIONSHIP

Specific Internal Conditions Necessary for Maximal Auto Inductions of Luminescence
Bioluminescent marine bacteria Vibrio fischeri, when cultured away from the light organ association with the squids depress their emission of light by over 1,000 fold. This is because of the underproduction of the autoinducer luciferase, which positively regulates this reaction. The autoinducer acts as a sensor that regulates the induction process, and eventually results in bioluminescence. If there are not enough bacteria, then this process obviously does not occur. This suggests that some conditions specific to the internal environment of the light organ are necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.
This proves the specificity of the light organ in the E. scolopes, which include physiological and biochemical controls of expression of luminescence. This study removed the bacterial from the light organ environment and observed the results. Immediately after the removal from the light organ, the symbionts visibly which were visibly luminous, decreased their bioluminescence substantially. This process was not reversed by culturing the bacteria on any of a variety of laboratory media. The variation in luminescence of different strains of the symbionts appeared to also be due to the different rates of autoinducer synthesis. The conclusion from this experiment proved that the bacteria and the squid both need extremely specific conditions in order to produce bioluminescence.
Source: Depressed Light Emission by Symbiotic Vibrio fischeri of the Squid Euprymna scolopes


Growth Rate and Flagellation during Initiation of the Light Organ
When the juvenile squids emerge from its egg, it is symbionts-free and it must obtain a bacterial infection from the surrounding environment, in order to survive. This study documented the process of the squid acquiring its symbionts. When placed in seawater containing only few colony-forming-units, the juvenile squid became quickly infected. “Colonization of the nascent light organ was initiated with as few as 1 to 10 bacteria, which rapidly began to grow at an exponential rate until they reached a population size of approximately 105 cells by 12h after the initial infection.” The number of bacteria was maintained.
They also studied whether or not the bacteria produced flagella. The V. fischeri strain is usually flagellated and motile, however once the symbiosis was established, these bacteria did not develop these characteristics. Once they were removed from the light organ however, they quickly were able to develop these appendages. “Thus, two important biological characteristics, growth rate and flagellation were modulated during establishment of the association, perhaps as part of a coordinated series of symbiotic responses.” This proves that this relationship not only needs specific environmental conditions, but also needs the bacteria to have certain morphological characteristics as well.
Source: http://www.medmicro.wisc.edu/labs/mcfall_ruby_papers/pdf/1993/Ruby_Asato_1993_ArchMicrobiol.pdf


Bacterial Toxins Govern Organ Development
The toxin, cytotoxin is known to cause serious infections and health problems in humans and other animals. In the symbiotic relationship with squids however, it promotes the development of the light organ. This is important because it may cause all scientists to rethink the role of bacteria in the world. One of them states: “it is all context dependent. It has to be that we have mechanisms to use these molecules in different ways. Until now, molecules of a virulent nature have not been recognized as having essential roles in their development” says McFall-Ngai.
In the Hawaiian bobtail squid, the toxin was found to promote development of the light organ, which is responsible for bioluminescence. When the squid are born, they are about the size of a grain of rice and they must acquire toxin-producing bacteria from their ocean environments. “Specialized features on the surface of the squid’s nascent light organ facilitate colonization by the bacteria, which in turn, promote the development of the functioning light organ. If you deprive the animal of those microbes, the system simply doesn’t develop.” This is further evidence of the specificity of the light organ in this interesting symbiotic relationship.
Source:
http://www.eurekalert.org/pub_releases/2004-11/uow-iat110904.php


OTHER INTERESTING SOURCES:
http://web.uconn.edu/mcbstaff/graf/VfEs/VfEssym.htm
- Paper talking about the specific conditions in the light organ necessary for the genes to be transcribed and translated
http://www.medmicro.wisc.edu/labs/mcfall_ruby_papers/pdf/1996/Ruby_1996_AnnuRevMicrobiol.pdf
-Paper arguing that growth rate and flagenation were modulated during the establishment of the symbiotic relationship. Suggests regulations of both physiological and morphological structures