Symbiotic organs shaped by distinct modes of genome evolution in cephalopods

Last week I was whining about gaps in our understanding of evolutionary processes in the ocean. The universe heard me, and today I am satisfied to write about the published genome of Euprymna scolopes – the Hawaiian bobtail squid and the implications this genome has for the study of (marine) animal-microbial symbioses.

The Hawaiian bobtail squid
Chris Frazee and Margaret McFall-Ngai
via Wikimedia Commons

E. scolopes presents a very potent model species for understanding how symbioses between animals and bacteria form and evolve (Nyholm and McFall-Ngai 2004). It has two specialized organs to host bacterial symbionts, the light organ and the accessory nidamental gland. The former maintains a monoculture of bioluminescent bacteria (Vibrio fischeri). During the night when the squid is active, it can glow with the help of its symbiont bacteria. From below, its glowing silhouette will disappear in the surrounding moon light and this provides camouflage from predators. Check out a video on nature’s cutest symbiosis here! (This is a must see.) The latter specialized organ is important for reproduction and only found in females (Collins et al. 2012; Kerwin and Nyholm 2017). It hosts a microbial consortium that has been hypothesized to protect developing embryos from fouling and infection. Colonization of these two symbiotic organs has been studied in great detail leveraging a plethora of articles on microscopic structures in the host tissue, biochemical pathways, microbial genomics, gene expression patterns, and ultimately the evolution of symbiosis in this system. Microbial comparative genomics has been especially helpful in identifying elements important for the bacterial symbiont. For example, microbial biofilm formation, which was eloquently summarized by Julian Jackson. Yet, so far, most insight about the evolutionary trajectory of this symbiosis came from studies of the bacterial symbiont. Now, with the public availability of the host genome, we can learn about the evolutionary history from the host’s perspective. Once more, the charismatic little shallow water creature from Hawai’i is opening new avenues for exciting research.

The article I am referring to was published yesterday in PNAS by Mahdi Belcaid et al.

They present the most comprehensive cephalopod genome assembly up to date (Belcaid et al. 2019). It was produced by a hybrid approach of shotgun and long-range linkage sequencing. Genome annotation was guided by sequencing transcriptomic libraries from different host tissues and life stages (light organ, accessory nidamental gland, gills, brain, skin, eyes), which resulted in the recovery of almost 30,000 expressed genes. The relatively large genome is characterized by a substantial proportion of repetitive elements, mostly long interspersed nuclear elements (LINEs).

Comparative genomics

Belcaid et al. performed a comprehensive comparative genomic analysis between E. scolopes and the octopus O. bimaculoides, a closely related cephalopod that lacks both specialized symbiotic organs, to discover differences at the genome level that are important for becoming an animal host. Inferring gene families from the most common recent ancestor did not provide any evidence for genome duplication or horizontal gene transfer events. The large genome size of E. scolopes seems to have resulted mostly from repetitive element expansion (LINE content). There were a few common features of both genomes, such as numerous local gene linkages (microsynteny) that have not been reported previously in other animals. This included genes in the central nervous system as well as in the symbiotic organs of E. scolopes and the testes of O. bimaculoides. These common gene linkages indicate a large genomic reorganization that must have taken place in the cephalopod ancestor. Both genomes also harbored long, gene-free regions surrounding their hox genes. Hox genes code for transcription factors and are very important for the development of embryos into complex multicellular animals. For example, in the embryonal development of insects, hox genes determine whether a body segment will develop extremities and what type of extremity (e.g. antenna, legs, wings). Hox genes are usually highly conserved. A fly can function perfectly with a chicken hox protein in place of its own (Lutz et al. 1996)! The non-conventional architecture of hox genes in both species studied here might have contributed to innovations in the cephalopod general body plan.

How to become an animal host

To learn about the evolution of symbiosis, genes expressed in symbiotic organs of the bobtail squid were compared to genes expressed in nonsymbiotic organs within its genome. Specifically, the authors looked for genes that underwent a duplication event and compared paralogous pairs of genes in which one gene had tissue-specific expression and the second did not. For the two symbiotic organs, the genetic distances were relatively small (130 Million years ago after calibration), whereas pairs in other organs, for example the eye or brain, were much larger (270 Mya). Read this for more information on how they calibrated molecular evolution. This finding supports a scenario of relatively recent innovation within the bobtail lineage that allowed hosting microbes.

Light organ

The light organ showed significantly more tandemly duplicated genes compared to other tissues. This underlines the importance of gene duplication for evolutionary innovation. Genes exclusively expressed in the light organ, eyes or the accessory nidamental gland were typically found in tandem clusters of paralogs located on single scaffolds. Many duplications were E. scolopes-specific and happened after the octopus-squid split. A great example of such genes were reflectins, which can be found in the eyes of other cephalopod species. Reflectin genes in the light organ form a monophyletic group. The comparison of these genes to reflectin genes in the eye revealed a split of 30 Million years ago or less. 

Accessory nidamental gland

The accessory nidamental gland (ANG) is more broadly distributed than the light organ; i.e., it is present in many other squid and cuttlefish species. The ANG had the highest proportion of E. scolopes specific transcripts among all tissues when compared to cephalopod, molluscan, bilaterian, metazoan and premetazoan transcripts. Moreover, regions up and downstream of these bobtail squid specific genes showed a high proportion of repetitive element content. This points to high evolutionary turnover in regulatory regions. Aha! another mechanism that can lead to evolutionary innovation.

The vibrio-squid symbiosis has become its own field of research during the last 25 years. This small stubby squid provided enough material and excitement for several researchers to establish and maintain successful research groups. Many studies have excelled at moving from patterns to process, linking observations in the field to an understanding at the molecular basis. I am impressed. This paper was authored by 22 people and funded by five different grants. I want to point out that, besides host animal evolution, there are still many exciting questions unanswered at the microbial front. For example, I hope that this system will help us resolve the importance of strain variability for the evolution of animal-microbial symbioses.

References

Belcaid, Mahdi, Giorgio Casaburi, Sarah J. McAnulty, Hannah Schmidbaur, Andrea M. Suria, Silvia Moriano-Gutierrez, M. Sabrina Pankey, et al. 2019. “Symbiotic Organs Shaped by Distinct Modes of Genome Evolution in Cephalopods.” Proceedings of the National Academy of Sciences of the United States of America, 201817322. https://doi.org/10.1073/pnas.1817322116.

Collins, Andrew J., Brenna A. LaBarre, Brian S. Wong Won, Monica V. Shah, Steven Heng, Momena H. Choudhury, Shahela A. Haydar, Jose Santiago, and Spencer V. Nyholm. 2012. “Diversity and Partitioning of Bacterial Populations within the Accessory Nidamental Gland of the Squid Euprymna Scolopes.” Applied and Environmental Microbiology 78 (12): 4200–4208. https://doi.org/10.1128/AEM.07437-11.

Kerwin, Allison H., and Spencer V. Nyholm. 2017. “Symbiotic Bacteria Associated with a Bobtail Squid Reproductive System Are Detectable in the Environment, and Stable in the Host and Developing Eggs.” Environmental Microbiology 19 (4): 1463–75. https://doi.org/10.1111/1462-2920.13665.

Lutz, B., H. C. Lu, G. Eichele, D. Miller, and T. C. Kaufman. 1996. “Rescue of Drosophila Labial Null Mutant by the Chicken Ortholog Hoxb-1 Demonstrates That the Function of Hox Genes Is Phylogenetically Conserved.” Genes & Development 10 (2): 176–84. https://doi.org/10.1101/gad.10.2.176.

Nyholm, Spencer V., and Margaret J. McFall-Ngai. 2004. “The Winnowing: Establishing the Squid-Vibrio Symbiosis.” Nature Reviews. Microbiology 2 (8): 632–42. https://doi.org/10.1038/nrmicro957.

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