They saw the light: Euprymna scolopes and Vibrio fischeri symbiotic relationship
Reasons to Study this Symbiotic Relationship:
1.) Each organism can be grown/live separately.
2.) Colonization is easy to detect by luminescence.
3.) E. scolopes (Hawaiian bobtail squid) is small, grows rapidly, is available year round, and has a short lifespan. These features make it a suitable model organism.
What is unique about this particular cephalopod is the relationship between its unusual light organ and the bacterium Vibrio fischeri. The bacteria are housed within epithelial-lined crypts, or cavities, of this light organ, encouraged to produce light through bacteria regulated quorum sensing and oxygen provided by the squid. The squid uses this light organ in counterillumination, a technique that camouflages the squid within the environment. Since the squid is active at night, the light from the moon and stars can penetrate the shallow water and illuminate the squid’s presence to its prey or predators. Through sensors on the squid’s back, the squid is able to match the amount of ambient light by manipulating the amount of light given off by the light organ with its ink sac. Thus, the squid does not have a shadow. Every morning, the squid expels about 90% of the bacteria from the light organ, allowing a fresh culture for the remaining bacteria to flourish in as the squid buries itself in the sand for the day. (Lee 2009)
1.) Core of 3 branching epithelial crypts that house the symbionts (Vibrio fischeri)
3.) Diverticula of Ink sac (blind- ended tubes that dynamically control luminescence intensity)
5.) Yellow filters that serve to shift the light’s wavelength to match environment
A squid hatchling’s light organ is dramatically different than an adult’s light organ. This fact paired with the sterility of the hatchling organ implies that infection of the crypts causes post-embryonic development in the squid.
Pre-Symbiont Development of the Light Organ
In a newly hatched squid, the light organ is poised for inoculation with Vibrio fischeri. At this point, the light organ is a bilobed organ partially embedded in the ventral surface of the ink sac. The 3 bilaterally symmetrical pairs of crypts have developed but in various sizes. The largest crypt, situated between the two smaller crypts, developed first, and therefore had the longest time to grow. The smallest crypt is the most anterior while the medium-sized crypt is located the most posterior. The size of each crypt is related to the time it developed.
There are 2 types of simple columnar epithelial crypt cells at this point: non-ciliated but microvillous cells that eventually interact directly with the symbiont and ciliated cells that form the duct connecting the crypts to the surface pore. The pores, located near the base of the appendages, are about 5-15 micrometers in diameter and each pore is connected to a different crypt.
There are two pairs of appendages, one situated more anterior and one more posterior. These appendages are ciliated to direct symbionts towards the pores. There is a pronounced ciliated ridge with elongated cilia along the base of the appendages that emphasizes the boundary between ciliated and non-ciliated surface cells.
The light organ also is beginning to incorporate the ink sac (simple squamous epithelium) and it also includes the reflector tissue, composed of iridosomal platelets. The other accessory tissues, like the lens and filters, have not developed yet. The lens, which is derived from muscle, begins to differentiate 7 to 10 days post-hatch, which is well after V. fischeri colonizes the light organ. (Montgomery 1993)
As previously stated, the ciliated epithelium, including the ciliated appendages are constructed to facilitate colonization by Vibrio fischeri. In experiments where the cilia movement has been arrested, inoculation has not occurred.
Ambient seawater passes across light organ during normal ventilatory processes. The mantle cavity expands when seawater is drawn in and then collapses, pushing water past the light organ, as the seawater is expulsed through the funnel. On the surface of the light organ, mucus is secreted by the ciliated appendages that catches the bacteria for harvesting.
The symbionts then enter the pores and travel down the ciliated ducts to the blind-ended crypts. In the crypts, the symbionts are provided with essential amino acids and oxygen. The squid is able to control the bacterial population size by regulating the amount of oxygen. (McFall-Ngai, 1998)
It is remarkable that out of the multitudes of bacteria in ocean, only V. fischeri populates the light organ. This specific interaction is a consequence of bacterial mannose receptors called adhesins reacting with mannose on the squid’s surface and crypt epithelium. Mannose is the most common glycan expressed on the surface of these epithelial cells.
There is also evidence that the light organ might be inhospitable to other species of bacteria. The crypts contain high levels of halide peroxidase, which synthesizes toxic hypochlorous acid. Coincidentally, halide peroxidase activity is significantly lower in colonized squids than in aposymbiotic (uncolonized) squids. It is hypothesized that V. fischeri has a peroxidase that competes with the host peroxidase for its hydrogen peroxide substrate. (Claes, 2000)
Nitric oxide is hypothesized to be a participant in the specific interaction between Vibrio fischeri and Euprymna scolopes. In 2004, Davidson et al. established that nitric oxide and nitric oxide synthase are present at high levels in the ciliated appendages, ducts, crypts, and mucus in the squid’s light organ. Bacteria that utilize nitric oxide aggregated near the pores but only V. fischeri colonized the light organ. In addition, V. fischeri attenuated nitric oxide and nitric oxide synthase production. In the bacteria, nitric oxide prompts flavohemoglobin (Hmp) transcription. Hmp prevents nitric oxide’s normal role in inhibiting aerobic respiration and a Hmp mutant does not colonize efficiently. (Wang, 2010)
In a natural environment, newly hatched squids are infected with Vibrio fischeri cells within 24 hours. The bacteria’s effect upon the light organ is obvious soon after they populate the crypts.
The ciliated appendages regress within days of inoculation. Through observations of pycnotic nuclei, indicators of apoptosis (programmed cell death), it was determined that the appendages are diminished by massive cell death. When treated with antibiotics at various time points, it was discovered that the bacteria only need to be present for at most 8 hours to initiate the apoptosis program, implying that the bacteria provide a transient and irreversible signal to the host cells. It is also significant to note that the bacteria do not stay in contact with the tissue that regresses.
Utilizations of mass spectrometry and fractionation revealed that the bacterial signal is tracheal cytotoxin (TCT), a disaccharide-tetrapeptide monomer of the bacterial surface molecule peptidoglycan. At first, it was thought that the cell death was triggered by lipopolysaccharide (LPS) and peptidoglycan (PGN) working together in some concentration but experimentation demonstrated that this combination was not consistent and did not elicit the same level of apoptosis. However, squids treated with only TCT, even without LPS, prompted the normal response. (Koropatnick, 2004)
This TCT generated cell death first causes the elongated ciliated ridge to regress and be replaced with non-ciliated cells. The number of dying cells within the light organ peaks at 14 hours post-inoculation but cells continue to die for the first 5 days. Within 3 days in unfiltered seawater, the posterior appendage is completely absent and the anterior appendage is markedly reduced in size. In sterile seawater, and still infected, the appendages are only slightly decreased in size. (Montgomery 1994)
TCT provokes an accumulation of blood cells (hemocytes) in the ciliated fields that act like macrophages, digesting the soon- to- be dying epithelial cells. The toxin then induces the apoptosis program The mucus on the surface cells is also shed, since there is no longer a need to trap bacteria flowing through the ventilation system. (Koropatnick, 2004)
It is not certain that TCT specifically elicits the rest of the morphology changes though they are highly correlated with the presence of the bacteria. These modifications include an increase in crypt cell volume to create cuboidal epithelial cells as well as a momentous increase of microvilli density in the crypt’s ciliated border.
Through western blotting, this large-scale cell death activated by TCT has been shown to utilize p53 signaling between cells. P53 signaling is a known tumor suppressor gene. In the absence of a stimulating signal, p53 forms a complex with MDM2 in the nucleus, causing a move into the cytoplasm, where it gets ubiquinated and degraded. When the proper signal does come along, p53 is stabilized through phosphorylation, blocking MDM2 binding. The p53 protein then is able to build up in the nucleus and performs its role as a transcription factor, leading to apoptosis.
The western blot of aposymbiotic organisms showed a higher concentration of p53 in the cytoplasm whereas organisms infected with V. fischeri had a notable higher concentration of p53 in the nucleus instead. (Goodson, 2006)
TCT is also associated with the NOS/NO signaling. It has been shown that TCT attenuates NO production within the host. NO production is typically an immunological response against MAMPs (microbe associated molecular patterns). This interaction with the immune system is extremely interesting and opens up a whole new realm of research that could be conducted concerning this symbiotic relationship. (Altura, 2011)
After the initial cell massacre, the additional accessory tissues begin to develop, including the 2 yellow filters (which mature near the ducts along the anterior/posterior axis), the muscle-derived lens, and the diverticula of the ink sac.
Another major change associated with the presence of the Vibrio fischeri is the extensive branching of the epithelial crypts. There are still 3 separate crypts but it is harder to distinguish due to the branching. The branching also causes the modification of the ducts connecting each crypt to their separate pores. Now, the crypts connect to one 5-branched duct, which leads to the surface to one pore. There is one branch to each crypt, one branch to the anterior yellow filter, and one branch to the posterior yellow filter. In aposymbiotic squid, branching still occurs but it is much slower and not as elaborate. (Montgomery, 1998)
The individual duct cells are also altered with the bacterial colonization. At this point, these cells contain a large number of cytoplasmic vesicles, hypothesized to have a secretory role in the daily expulsion of the Vibrio fischeri cells. In aposymbiotic duct cells, there are no inclusions and the cells are more heterogenous in appearance.
In addition, the crypts are wrapped in dynamic diverticula, which are blind tubes derived from the ink sac described earlier. These tubes wrap around the light organ, expanding and contracting in either an anterior/posterior direction or a medial/lateral direction. Again, diverticula still develop in aposymbiotic organisms but it is a slower process and there are less tubes covering the crypts. (Claes, 2000)
Another piece of evidence demonstrating the symbiotic relationship’s effect upon Euprymna scolopes’ development is the change in the squid’s proteome after being infected. Judith Lemus showed that Vibrio fischeri infection caused a significant change in the squid proteome within the first 96 hours, which correlates with the morphological changes. When compared to age-related changes, it was found that the symbiosis actually sped up the age-related changes, further implying the symbiosis impacts maturation. This adaptation of the protein composition within the cell provides a more solid link between the effect of the bacteria on the physiological changes and on individual cells. (Lemus, 2000)
Induction of Luminescence
Besides being able to control population size through expulsion and oxygen levels, the squid has very little control over the bacteria and the amount of luminescence the bacteria exude. Fortunately for the squid, they control the one aspect that can indirectly influence the amount of light produced.
The bacteria decide when to produce light by communicating with each other through quorum sensing. Quorum sensing uses the molecule N-homoserine lactone (acyl-HSL) for communication and at high cell density, the concentration of acyl-HSL is high in direct relation. Only at high concentrations are the lux genes, which code for the light-producing luciferase enzyme, transcribed.
The two proteins synthesizing acyl-HSL are AinS and LuxI. These two proteins monitor and control quorum sensing and luminescence. At low cell densities, as can be seen in Figure 6 of Lupp’s 2003 paper, transcription of LuxO results in inhibition of the other Lux genes. However, as cell density increases, AinS arrives and inhibits LuxO, allowing a little light production. At the high cell densities found in the light organ, LuxI is transcribed because AinS inhibited LuxO and then LuxI promoted further Lux gene transcription through a transcription factor.
The host squid can also tell by an unknown mechanism if the bacteria colonizing its light organ is producing light. If a non-luminous mutant does populate the light organ, the host responds by reducing its oxygen transport to the crypts, where the symbionts are housed. (Lupp, 2003)
For example, LuxA and LuxR mutants cannot produce normal luminescence levels. These mutants are unable to maintain high cell numbers in the light organ fully and fail to induce the crypt cell swelling. One hypothesis is that a diminished oxygen consumption by the luciferase enzyme and thus a lower oxygen depletion by the population can be detected by the squid, who then reduces oxygen and blood flow to the light organ until a suitable bacterial population has arrived. (Visick, 2000)
This relationship has been studied since the early 1990’s and as more information has been discovered, the light organ’s story has become more interesting, involved, and comparable to other systems. In conclusion, the light organ of Euprymna scolopes is selectively colonized by the bacteria Vibrio fischeri a few hours after hatching. The bacteria then causes the maturation of the light organ through massive cell death and cross-signaling. Luminescence is induced in the bacteria through quorum sensing and utilized by the squid in a counter-illumination techinique to assist in catching prey.
Altura, M. , Stabb, E. , Goldman, W. , Apicella, M. , & McFall-Ngai, M. (2011). Attenuation of Host NO Production by MAMPs Potentiates Development of the Host in the Squid-vibrio Symbiosis. Cellular Microbiology, 13(4), 527-537.
Chun, C. , Troll, J. , Koroieva, I. , Brown, B. , Manzella, L. , et al. (2008). Effects of Colonization, Luminescence, and Autoinducer on Host Transcription During Development of the Squid-vibrio Association. Proceedings of the National Academy of Sciences of the United States of America, 105(32), 11323-11328.
Claes, M. , & Dunlap, P. (2000). Aposymbiotic Culture of the Sepiolid Squid Euprymna Scolopes: Role of the Symbiotic Bacterium Vibrio Fischeri in Host Animal Growth, Development, and Light Organ Morphogenesis. Journal of Experimental Zoology, 286(3), 280-296.
Davidson, S. K., Koropatnick, T. A., Kossmehl, R., Sycuro, L. and McFall-Ngai, M. J. (2004), NO means ‘yes’ in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association. Cellular Microbiology, 6: 1139–1151. doi: 10.1111/j.1462-5822.2004.00429.x
Goodson, M. , Crookes-Goodson, W. , Kimbell, J. , & McFall-Ngai, M. (2006). Characterization and Role of P53 Family Members in the Symbiont-Induced Morphogenesis of the Euprymna Scolopes Light Organ. Biological Bulletin, 211(1), 7-17.
Lee, P., McFall-Ngai, M., Callaerts, P., & de Couet, H. G. (2009). The Hawaiian Bobtail Squid (Euprymna scolopes): A Model to Study the Molecular basis of Eukaryote-Prokaryote Mutualism and the Development and Evolution of Morphological Novelties of Cephalopods. Emerging Model Organisms, 45, n/a.
Lemus, J. , & McFall-Ngai, M. (2000). Alterations in the Proteome of the Euprymna Scolopes Light Organ in Response to Symbiotic Vibrio Fischeri.Applied and Environmental Microbiology,66(9), 4091-4097.
Lupp, C. , Urbanowski, M. , Greenberg, E. , & Ruby, E. (2003). The Vibrio Fischeri Quorum-sensing Systems Ain and Lux Sequentially Induce Luminescence Gene Expression and Are Important for Persistence in the Squid Host. Molecular Microbiology, 50(1), 319-331.
Troll, J. , Bent, E. , Pacquette, N. , Wier, A. , Goldman, W. , et al. (2010). Taming the Symbiont for Coexistence: A Host PGRP Neutralizes a Bacterial Symbiont Toxin. Environmental Microbiology, 12(8), 2190-2203.
Wang, Y. , Dunn, A. , Wilneff, J. , McFall-Ngai, M. , Spiro, S. , et al. (2010). Vibrio Fischeri Flavohaemoglobin Protects Against Nitric Oxide During Initiation of the Squid- Vibrio Symbiosis. Molecular Microbiology, 78(4), 903-915.
Wollenberg, M. , & Ruby, E. (2009). Population Structure of Vibrio Fischeri Within the Light Organs of Euprymna Scolopes Squid from Two Oahu (Hawaii) Populations. Applied & Environmental Microbiology, 75(1), 193-202.
Vang, Chue. “Bobtail Squid.” UW Eye Research Institute. University of Wisconsin- Madison, 20 Nov. 2009. Web. 31 Mar. 2011. <http://vision.wisc.edu/news_fall09.html>.
Visick, K. , Foster, J. , Doino, J. , McFall-Ngai, M. , & Ruby, E. (2000). Vibrio Fischeri Lux Genes Play an Important Role in Colonization and Development of the Host Light Organ. Journal of Bacteriology, 182(16), 4578.