All that Glitters: Making it Big Inside a Squid

By: Diana Choi '19 and Caty Levecque '19

Off the coast of Hawaii, Euprymna scolopes, otherwise known as the Hawaiian bobtail squid, illuminates its light organ. Potential predators look on, now believing that the glow they can see above is simply sunlight reflecting off the water and not a delicious mollusk dinner. Having no glowing abilities themselves, E. scolopes can’t pull off this disappearing act alone. Bioluminescent Vibrio fischeri bacteria inhabit their light organs, creating an awe-inspiring blue-green glow in exchange for food and a place to stay. Later, the squid will expel nearly all of the bacteria in its light organ and hide itself away in the sand (Figure 1). When it’s time, the squid will welcome new V. fischeri cells into its light organ, and the colonization and glowing process will begin again. The bacteria will once more “set up shop” inside E. scolopes through the critical process of biofilm aggregation.

This mutualism has captured the attention of biologists for decades. Its study has contributed much to the field of ecology, illustrating the complexities of the interspecies mingling we see every day. The symbiosis has far-reaching implications outside of the ocean, too, as scientists begin to examine the similarity between human ciliated tissue, squid ciliated tissue, and the bacteria that inhabit them. Despite all of the flashy scientific history, however, new research about these two organisms continues today. What researchers are finding now is that V. fischeri are not all created equal in forging this symbiotic relationship and that difference may lie within the bacterial genome.

Figure 1. Euprymna scolopes burying itself in sand. Gif courtesy of Spotmydive.

For several years now, microbiologists interested in V. fischeri have understood there to be two overarching phenotypic categories of competition within the bacterial species (Bongrand et al., 2016). These phenotypic categories are referred to as “strains” and they describe the slight differences that exist between members of the same V. fischeri species. The so-called “D” strains are dominant, outcompeting the aptly named sharing “S” strains. If a single D type strain makes its way into the E. scolopes light organ first, all other subsequent strains will fail to colonize the space. These strains are so efficient at colonization that even other D types will find themselves unable to inhabit the organ once one strain has taken hold. In the absence of any D strains, however, S strains have been known to co-colonize the light organ, allowing other S strains to share the space with them. While these dynamics have been well-established, what has been missing from this area of research is the mechanism behind it all. How can two bacteria of the same species look so different in action? A new study by Koehler et al. has put forth a new idea: key genes seem to affect each strain’s ability to form aggregates.

The research team first investigated the exact nature of the differences in aggregation between D and S strains. Fluorescently tagged V. fischeri were introduced into E. scolopes, and the light organs were examined using confocal microscopy, allowing the researchers to distinguish the speed and size of aggregation for the two strain types. The team’s findings were striking: the D type strains exhibited up to 250 times the average number of cells per aggregate and took about a quarter of the amount of time to form the aggregates compared to S type strains (Koehler et al. 2018). Given the speed of formation and robustness of these aggregates, D type strains were able to colonize the light organ extensively (Figure 2).

Figure 2. A confocal microscopy image of the squid’s light organ. The label “aa” refers to the anterior appendage of the organ, whereas “pa” is the posterior appendage. The Vibrio fischeri cells are shown in red, the host’s cilia in green, and the host’s mucus in blue. This image shows the overall structure of the light organ, as well as the interaction between the cilia and the bacteria. Image courtesy of Koehler et al. 2018.

These disparities do not simply affect the bacteria’s ability to thrive in the light organ. They also seem to impact the host. Specifically, Koehler et al. noted the significant disfigurement of the squid cilia, finding that D types were demonstrating extensive warping of the structure, even more so than S types (Figure 3).

Figure 3. A high magnification image of the squid’s light organ cilia after interaction with Vibrio fischeri MB13B2, a D strain. The tips of the cilia have swollen and twisted into small balls, indicating significant effects on the squid’s morphology through its symbiosis with V. fischeri. Image courtesy of Koehler et al. 2018.

The mechanism resulting in this morphological change is still unclear, though past research has suggested the effect may be a biomechanical one, as a result of tensile stress (Nawroth et al. 2017). What is clear from Koehler et al.’s work, however, is that the extent of this effect directly correlates with bacterial cell aggregate size, which is itself influenced by strain type.

Now we understand that the D-strain can be dominant because of their hyperaggregating behavior on the host organ’s surface. But what dictates the superiority of the D-type strain over S-type bacteria for the hyperaggregation of biofilms? Genes! There are two known genes responsible for bacterial biofilm formation: rscS and sypQ. The researchers found out that the hyperaggregating strain (D-strain) is actually syp-dependent. To find out which of the two genes is mainly responsible for the hyperaggregation in the D-strain, the researchers conducted an experiment in static liquid cultures, which mimic the conditions that they might encounter in their natural environment. They found out that a deletion of sypQ gene almost completely eliminated the aggregating phenotype with no pellicle formation, while a deletion of the gene encoding RscS only partially reduced the level of aggregation with some pellicle remaining (Figure 4). This finding shows that the sypQ gene is a more essential gene in regulating the hyperaggregation in D-strain bacteria, and therefore plays a more important role in determining the dominance of a bacterial strain. The fact that the RSC mutant retained the ability to form a pellicle in the liquid culture, suggests that rscS gene is necessary, but not sufficient for the hyperaggregation phenotype in the D-strain bacteria.

It turns out, while both strains of V. fischeri maintain the syp-biofilm formation gene, the D-strain bacteria are better at forming syp-dependent biofilms, relative to the S strains. D-type bacteria formed an extensive pellicle in liquid culture while S-type strain formed only a thin pellicle that could be easily suspended. This tells us that the main reason D-strain can take over the host squid is that the D-type bacteria can form syp-dependent biofilms that are larger than those of the S-type strains during the symbiosis initiation.

Figure 4. D-type strain (MB13B2) and S-type strain (ES114) in static liquid culture. When rscS gene is overexpressed in the S-strain (ii), pellicle forms. The sypQ mutant D-strain does not form pellicle. Image courtesy of Koehler et al. 2018.

The mutually beneficial relationship between the Hawaiian bobtail squid and its luminous symbiont bacterium, Vibrio fischeri, is important for the squid, as well as for the bacteria. Thanks to the study, we now understand that the host-symbiont mechanism is not only species-specific, but also strain specific. The study reveals that bacterial strain variation could be a major determinant in the initial stages of the host-symbiont association.

In summary, the impact of bacterial strain variation on the initiation of the squid-vibrio symbiosis is clear. The two bacterial strains, “D-type” and “S-type” are different in their relative level of biofilm formation, in that the strain that forms larger biofilms have a colonization advantage over those that form smaller biofilms. Therefore, the D-type strain of V. fischeri has a colonization advantage over S-type strain, for it appeared and aggregated more quickly and formed larger aggregations than the S-type strain. The amount of aggregation of the bacteria seems to be an important factor that affects the host-cilia interaction with D-type bacterial cells since the structural modification of the cilia tips were more extensive in the areas of hyperaggregating D-strain. The study also finds that sypQ gene is required for the hyperaggregation behavior of the D-type bacteria, although important questions remain concerning the genetic mechanisms and the pathway the hyperaggregation strain uses to overexpress the sypQ biofilm formation genes.

The selection for the specific partnering bacteria occurs with a highly complicated mechanism. While we have come to understand that there exist genetic differences between the normal and hyperaggregating strains, the genes responsible for biofilm synthesis only partly control the hyperaggregation phenotype of a D-type strain. There are certainly other regulatory inputs, in addition to RscS, that control the hyperproduction of biofilm by D-type strain. Furthermore, the ability to aggregate on the light-organ surface is necessary, but not sufficient, step in initiating symbiosis, which encourages the need for further studies to fully understand the symbiotic mechanism.

There remain unresolved questions behind the special association between the Bobtail squid and their bacterial partner. Nevertheless, the information presented in this study provides an underlying mechanism of the strain and species-specific relationship between the bacteria and the host organism. Because V. fischeri symbiont is easily grown in culture, molecular genetic techniques have been developed to analyze the role of specific genes in the dynamics of the symbiosis. This has allowed scientists to study how a symbiotic association between a bacterium and an organism is established and maintained. The squid-vibrio model system could be promising for directing and designing an experiment of ciliated tissues in human organ culture, as the authors claim.


Literature Cited

Bongrand, C., Koch, E.J., Moriano-Gutierrez, S., Cordero, O. X., McFall-Ngai, M., Polz, M.F., and Ruby, E.G. (2016) A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior. ISME J 10: 2907–2917.

Koehler, S., Gaedeke, R., Thompson, C., Bongrand, C., Visick, K.L., Ruby, E., and McFall-Ngai, M. (2018) The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement. Environmental Microbiology 00: 1-15.

Nawroth, J.C., Guo, H., Koch, E., Heath-Heckman, E.A.C., Hermanson, J.C., Ruby, E.G., et al. (2017) Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome. Proc Natl Acad Sci USA 114: 9510–9516.


About the Authors:


Diana Choi '19 is a senior at Mount Holyoke College studying Biology and Italian. She will be attending veterinary school in the fall. She loves traveling, Italian films, and Korean food. Her favorite microbe is Paenibacillus vortex.


Caty Levecque '19 is a Biological Sciences major and a senior at Mount Holyoke. After graduation, she hopes to attend medical school to pursue a career in pediatrics. In her free time, Caty enjoys long walks with her two pet dogs and obscure Netflix documentaries.