The Shrinking Styrofoam Cup

Image1: Foam cup attached to ROV

How much impact could a small Styrofoam cup have? For many people, when styrofoam and the ocean are associated with each other it is through images of plastic pollution littering beaches and the water. For Deep Sea researchers the styrofoam cup has become a tool to visualize the incredible conditions that are present in the dark depths they explore.1 Attached to the frame of a submersible, a normal 8 oz. foam cup will shrink to the size of a thimble after experiencing the weight of hundreds or even thousands of meters of ocean water.2 Explaining why this happens, its impact on the cup, on the equipment researchers use, and the animals they study has become a tool for involving and educating young scientists and the  public in deep sea research. So why does the cup shrink?

Foam cups are made from polystyrene, a synthetic hydrocarbon polymer made from many small styrene units. These small “Lego” like styrene units allow polystyrene materials to take on many different forms.3 Starting off as small beads, they are typically heated with a pentane gas that allows the beads to expand to 40 to 50 times their original size. The beads will cool and then are reheated, compressed, and molded into large blocks.This Expanded Polystyrene (EPS) block is approximately 98% air making it an extremely light and durable product.4 Qualities that make EPS an excellent packing and building material also mean that it is not readily biodegradable, and it is often manufactured for single use. The amount of air is ultimately what is important to shrinking the foam cups our deep-sea researchers place on their abyssal vessels.

Image 2: SEM photograph of normal (left) and compressed (right) EPS  

When submersibles, like a Remotely Operated Vehicles (ROV’s), descend into the deep sea they experience an increase of 10 atm for every 100 meters.1 When traveling to depths of > 1000 meters, the air bubbles in the foam cups are compressed and eventually collapse. The foam cup retains its shape because pressure is exerted evenly on all sides of the cup. ROV’s have many sensitive electronics and tools that are integrated into the vehicle and need to be engineered to withstand these intense pressures.2 Other materials such as steel, aluminum, glass, and even other plastics like PVC contain less air and therefore are less compressible when under deep ocean pressures. This allows researchers to send down ROV’s with sophisticated instruments such as flow cytometers, respirometry systems, and even laser spectroscopy tools.5 These tools have given us better insight into the oceanographic conditions that define the deep sea and the biological organisms that have adapted to this extreme environment.

Image 3: A hadal snailfish, N. kermadecensis, photographed at 7,199m

All respiring organisms have some air in them due to the requirement of oxygen for metabolic processes. Yet, how do deep feeding or deep dwelling organisms not become crushed by the intense pressures like the styrofoam cup? Mammals, which are osmoregulators, must go through a set of physiological changes called a dive reflex. These deep diving feeders, such as the Sperm Whales, Elephant Seals, and Cuvier’s Beaked Whales have an increase in hemoglobin content and blood volume which carries more oxygen and increases metabolic transport of oxygen. They dive with no air in their lungs allowing them to collapse, their heart rate slows, and their blood vessels constrict.6 The record of deepest living macro-organism goes to the Snailfish genus, though. Teleost’s, ray-finned fishes, are osmoconformers; and their lower depth limit may also be regulated by pressure. Biochemically, hydrostatic pressure can have detrimental effects on the proper function of proteins in metabolic pathways. A set of organic compounds called neutral amino acids, or TMAO, are not only essential to deep sea protein function, but as they accumulate and reach an osmotic disequilibrium, and the imbalance of intracellular solutes disrupt physiological function at depths greater than 8,400 meters.7 This shift in TMAO content may have larger implications for other deep dwelling taxa. This warrants further investigation which will be spearheaded by kids learning about the deep sea, drawing on styrofoam cups, and sending them to researchers.

                The styrofoam cup is an easy and effective method of educating the world about one of the most dangerous and fascinating aspects of the deep ocean, pressure. Twitter images and blog sites from deep sea oceanographers show off their cup collections from previous expeditions, like little souvenirs from the deep. Hoards of small hand-drawn cups from elementary students are sent to institutions so they too can become a part of the science and have their own deep-sea treasure.1,8 Rarely, complex interdisciplinary fields such as oceanography and deep-sea biology have easily understandable tools and concepts, due to the alien world they work in. The Styrofoam cup lends a window into that hostile underwater world, but also acts as a symbol. Plastic doesn’t have to be a symbol of pollution. It is a durable material that if used properly can be an effective tool. It is our job as scientists to effectively communicate the sophisticated tools, the clever inventions, and even the common, every day, household, items that we use to accomplish our research goals. Sometimes it is the most common mundane instruments that have the greatest power to reach into the community and inspire curiosity, creativity, and conservation.

Image Credits

Image 1: Styrofoam cup mount: https://nautiluslive.org/blog/2014/08/21/shrinking-cups-deep

Image 2: Scanning electron microscope styrofoam structure:https://www.sciencefriday.com/educational-resources/high-pressure-in-the-deep-ocean/

Image 3: Hadal snailfish: Jamieson AJ, et al. (2011) Bait-attending fauna of the Kermadec Trench, SW Pacific Ocean: Evidence for an ecotone across the abyssal-hadal transition zone. Deep Sea Res Part I Oceanogr Res Pap 58(1):49–62.

Resources

1 Griffies, S. 2017. The styrofoam cup experiment & aspects of ocean mass, pressure, and heat. DynOPO Cruise 2017. https://dynopocruise2017.blogspot.com/2017/04/the-styrofoam-cup-experiment-aspects-of.html.

2Hinchey, E.K., Adams, J.M., Rose, C.M., Nestlerode, J.A., Patterson, M.R. 2013. The Incredible Shrinking Cup Lab: Connecting with Ocean and Great Lakes Scientists to Investigate the Effect of Depth and Water Pressure on Polystyrene. Science Activities, 50: 1-8. DOI: 10.1080/00368121.2012.727754.

3Wypych, G. (2012). Handbook of Polymers: PS polystyrene. pp. 541–7. doi:10.1016/B978-1-895198-47-8.50162-4. ISBN 978-1-895198-47-8.

4 Expanded Polystyrene Australia Inc. 2014. How is EPS made? http://epsa.org.au/about-eps/what-is-eps/how-is-eps-made/

5Zych, A, Rayner, R. 2016. High pressure in the deep Ocean. Science Friday. https://www.sciencefriday.com/educational-resources/high-pressure-in-the-deep-ocean/

6Panneton, W. M. (2013). The mammalian diving response: an enigmatic reflex to preserve life?. Physiology (Bethesda, Md.), 28(5), 284–297. doi:10.1152/physiol.00020.2013

7Yancey, P.H., Gerringer, M.E., Drazen, J.C., Rowden, A.A., Jamieson, A. 2014. Marine fish may be biochemically constrained from inhabiting the deepest ocean depths. PNAS. Proceedings of the National Academy of Sciences, 111 (12) 4461-4465. DOI: 10.1073/pnas.1322003111

8 Totten Expedition and Killara Primary School: Armand, L. 2014. Pressure. Totten Expedition. https://sites.google.com/site/tottenexpedition/home

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Vampire Squid from Hell

“Current-borne, eternally drifting, driven by the gross might of the ocean, Vampyroteuthis infernalis lingers in the bathyal abyss. The light never illuminating, the dark ever encompassing. Borne, flung, tugged from anywhere, to nowhere, for in the deep sea there is no compass nearer or farther, only higher and lower, the vampire squid hangs and sways; fins, arms, tentacles move daedal and dynamic to bear it, to feed it, to defend it as the vast diurnal pulses beat in the deep dark sea. Hanging, swaying, preying, the most cryptic and apocryphal creature, it has for its defense the malice and horror of the deep, to which it has committed its essence, its cunning, its resolve.” ~ adapted from Ursula K. Le Guin, Lathe of Heaven

chun_1903

Figure 1: Carl Chun’s original depiction of V. infernalis

Vampyroteuthis infernalis, the vampire squid, is equal parts enigmatic and horrifying. Enigmatic in its ability to represent a duality in phylogeny, morphology, and evolutionary history; horrifying in the biological structures and physiological adaptations this solitary Order of Vampyromorphida has developed to survive and thrive in one of the most hostile zones in the deep sea (Young 2019) Since the discovery of V. infernalis during the Valdivia Expedition in 1898 by Carl Chun, a celebrated German zoologist and deep-sea researcher, this organism has mystified the philosophical and scientific community, alike (Chun 1903; Flusser & Bec 1987). Recently, deep-sea biologists and oceanographers have collaborated to delve into the depths and further investigate this cryptic cephalopod in its natural habitat (Hoving et al. 2014). Deep in the sea, they uncovered an array of V. infernalis peculiarities: repeatable reproductive cycles (Schwarz et al. 2018), detritivore feeding (Hoving et al. 2015), light production (Robinson et al, 2003), oligoaerobic activity (Siebel et al. 1999), and even longer life (Hoving & Robinson, 2012).

The vampire squid is taxonomically classified in the Domain Eukaryota, Kingdom Animalia, Phylum Mollusca, Class Cephalopoda, Order Vampyromorphida, Family Vampyroteuthidae, Genus Vampyroteuthis, Species infernalis (Young 2019). In summary, this describes a multicellular, heterotrophic, respiring, sexually reproducing, neurologically advanced, invertebrate cephalopod that is the oldest living species of its kind (Hoving et al. 2014). Evolutionary and Phylogenetic scientists have utilized morphological (Sutton et al. 2016), mitochondrial (Yokobori et al. 2007), multi-loci (Strugnell et al. 2005), and transcriptomic data (Lindgren et al 2018) to resolve the neocoleoid cephalopod tree. These studies repeatedly place V. infernalis at the base of the cephalopod tree with the Nautilus, classifying it as a living fossil. Although appearing to have some morphological and genetic geographical variation, all vampire squid found in the ocean are classified as a single species (Hoving et al. 2014). While V. infernalis shares many features with its shallow-water ancestors, but since retreating to the deep sea the vampire squid has developed unique set of characteristics that allow it to survive in the bathyal depths.

tree

Figure 2: Phylogenetic tree of the neocoleoid cephalopods created from transcriptomic data, Yokobori et al. 2007.

Vampire squid are found in tropical and temperate oceans at a depth range of 500m to 1500m (Young, 2019). This meso-bathypelagic habitat can be defined by a transition from photic to aphotic conditions, low oxygen concentrations (<0.5 ml L-1), a rain of dissolved/particulate organic material (DOM/POM) called marine snow and contains the deep scattering layer (Mann & Lazier 2006). Aphotic conditions decrease the primary productive capacity of this zone to nearly zero, and the resident organisms here are primarily heterotrophic primary/secondary consumers. Low oxygen conditions create a region called the oxygen minimum zone (OMZ), where extended stay by organisms with high metabolic turnover and higher respiratory rates become physiologically stressed and may die. The persistent rain of marine snow creates a steady but sparse food source for nekton; while the deep scattering layer, a layer of diel migrating organisms, brings more POM to the bathyal zone (Mann & Lazier 2006). It in in this “hell” like zone that V. infernalis calls home.

vamp-filament4f-350

Figure 3: MBARI ROV photo of feeding filaments

The morphological and physiological adaptations that V. infernalis utilizes are unique to cephalopods. Typically, cephalopods are semelparous only having one reproductive cycle before they undergo planned senescence. The vampire squid is iteroparous, having multiple reproductive cycles with large broods (Hoving et al. 2015). They also have a longer life span than most cephalopods reaching maturity in 3 -5 years, affected by the cooler temperatures and higher pressures at depth (Schwarz et al. 2018). Light production as a prey lure, defense strategy, and communication device is emitted from four light emitting organs in the arms, the fins, small epidermal dots scattered over the body, and nodules just above the eye. Fluorescent luminous clouds can also be formed by extruding a viscous mixture of proteins, specifically luciferase, coelenterazine, and other chemical units (Robinson et al. 2003). Until 2012, the feeding habits of V. infernalis were largely unknown due to the soft-body and difficulty of retrieving whole samples. By recording feeding interactions via MBARI’s remotely operated vehicles (ROVS), Hoving and Robinson were able to describe the two long thin retractable and extensible filaments used to collect marine snow. Making the vampire squid is the only known cephalopod detritivore. Lastly, the oxygen binding capacity of hemocyanin in vampire squid blood, gill diffusion capacity, and metabolic balancing of Citrate synthase and Octopine allow for maximum efficiency in low oxygen environments (Siebel et al. 1999).  Even though V. infernalis is a “living fossil”, it has developed and adapted to the deep-sea environment in a novel fashion.

Vampyroteuthis infernalis

Figure 4: Illustration of V. infernalis with photophores and light organs. Credit: Richard Ellis, “Deep Atlantic”

Pursuits to understand V. infernalis are met with two opposing mysteries. One is borne of darkness, pressure, cold temperatures, lack of oxygen, but can be overcome by leveraging robotic, ocular, and mechanical engineering. Allowing us to peer into depths where we can never truly exist. The other is borne of molecules, electrostatic interactions, transcription, translation, creation, destruction, but can be understood through physiological, genomic, and molecular methods. Allowing us to alter our scope of existence to an unseen world that governs our biological realities. The exceptional, weird, and extraordinary life of the vampire squid continues to mystify researchers, generate novel exciting research questions, and represent a bewildering and terrifying place. The deep dark abysses of the Ocean.

References

Chun, Carl (1903). Aus den Tiefen des Weltmeeres. Jena: Gustav Fischer. pp. V, 12. doi:10.18452/2.

Flusser, V., Bec, L. 1987. Vampyroteuthis Infernalis: A treatise, with a report by the Institut Scientifique de Recherche Paranaturaliste. Univ. Minnesota Press. 1-50.

Hoving HJT, Robison BH. 2012. Vampire squid: Detritivores in the oxygen minimum zone. Proc. R. Soc. B Biol. Sci. 279:4559–4567. doi:10.1098/rspb.2012.1357.

Hoving HJT, Laptikhovsky V V., Robison BH. 2015. Vampire squid reproductive strategy is unique among coleoid cephalopods. Curr. Biol. 25:R322–R323. doi:10.1016/j.cub.2015.02.018.

Hoving HJT, Perez JAA, Bolstad KSR, Braid HE, Evans AB, Fuchs D, Judkins H, Kelly JT, Marian JEAR, Nakajima R, et al. 2014. The study of deep-sea cephalopods. Adv. Mar. Bio. 67:235-359. doi: 10.1016/B978-0-12-800287-2.00003-2.

Lindgren AR, Anderson FE. 2018. Assessing the utility of transcriptome data for inferring phylogenetic relationships among coleoid cephalopods. Mol. Phylogenet. Evol. 118:330–342. doi:10.1016/j.ympev.2017.10.004.

Mann, KH, Lazier, JRN. 2006. Dynamics of Marine Ecosystems. Blackwell Pub. 3rd Edition.

Robison BH, Reisenbichler KR, Hunt JC, Haddock SHD. 2003. Light Production by the Arm Tips of the Deep-Sea Cephalopod Vampyroteuthis infernalis. Biol. Bull. 205:102–109. doi:10.2307/1543231.

Schwarz R, Piatkowski U, Hoving H. 2018. Impact of environmental temperature on the lifespan of octopods. Mar. Ecol. Prog. Ser. 605:151–164. doi:10.3354/meps12749.

Seibel BA, Chausson F, Lallier FH, Zal F, Childress JJ. 1999. Vampire blood: respiratory physiology of the vampire squid (Cephalopoda: Vampyromorpha) in relation to the oxygen minimum layer. Exp. Biol. Online 4:1–10. doi:10.1007/s00898-999-0001-2.

Strugnell J, Norman M, Jackson J, Drummond AJ, Cooper A. 2005. Molecular phylogeny of coleoid cephalopods (Mollusca: Cephalopoda) using a multigene approach; the effect of data partitioning on resolving phylogenies in a Bayesian framework. Mol. Phylogenet. Evol. 37:426–441. doi:10.1016/j.ympev.2005.03.020.

Sutton M, Perales-Raya C, Gilbert I. 2016. A phylogeny of fossil and living neocoleoid cephalopods. Cladistics 32:297–307. doi:10.1111/cla.12131.

Yokobori S ichi, Lindsay DJ, Yoshida M, Tsuchiya K, Yamagishi A, Maruyama T, Oshima T. 2007. Mitochondrial genome structure and evolution in the living fossil vampire squid, Vampyroteuthis infernalis, and extant cephalopods. Mol. Phylogenet. Evol. 44:898–910. doi:10.1016/j.ympev.2007.05.009.

Young, Richard E. 2019. Vampyroteuthidae Thiele, in Chun, 1915. Vampyroteuthis infernalis Chun, 1903. The Vampire Squid. Version 26 March 2019. http://tolweb.org/Vampyroteuthis_infernalis/20084/2019.03.26 in The Tree of Life Web Project, http://tolweb.org/.

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First Research Fellowship

It has been a long time coming. Nine years of college from 2009 – 2018. I have many mixed emotions about it. Excited to be done. Fearful of debt. Ready to take the next step. Reluctant to move away. Most of all I am proud of what I have accomplished. I am the first person in my family to receive a BS from a four-year university. As I walked onto that stage, nine long years of struggle, mistakes, long-nights, longer work days, even longer research days, and the memories of all the people who I have been fortunate enough to be taught by and to teach came rushing through me. That rush was of the past. The things that have happened. Now onto what will happen.

20180519_131054

In 2017 I was awarded the Sally Casanova Pre-Doctoral Scholarship (SCPDS) meant to help low-income, minority, and marginalized students realize their scholastic goals of becoming a Ph.D. Getting to travel to Oregon State University to meet Dr. Becky Vega and visit the lovely city of Corvallis was something I never dreamed of doing, especially without breaking my checking account. Attending the Western Society of Naturalists conference in Pasadena and getting to present my research on the nuances of Transcriptome Assembly alongside my peers was unforgettable. Visiting the University of Rhode Island (URI) during a Noreaster to get a taste of New England weather, and of course, the oysters was an absolute blast with my adventure buddy. Little did I know these were just the first instances where the SCPDS would shape my path.

January 2018. I have a research proposal due. I had met Dr. Jonathan Puritz at WSN as he took an interest in my poster and I liked his talk. During the conference, we got to talking about bioinformatics and his work on exome sequencing which seemed to be a much more efficient and cheaper compared to the traditional RNA-Seq method. He seemed like a biological tinkerer, looking for methodologies that could enhance this process on the wet-lab side but also on the bioinformatical side. Also, he had a brand new lab with room to grow at URI. Our conversation walking down the streets of Pasadena encouraged me to look at my science from a different perspective, one that could exist outside of California. You have to realize that due to being born and raised in the Central Valley of California, going to UC Davis,  transferring to Santa Barbara City College, and then graduating from CSU Monterey Bay I thought I would be staying here. That would all change with this proposal.

We wrote the proposal fairly quickly. It is pretty dense but I will attach my side of the proposal here: Green_Jacob_SCPDS_summer_research_application_essay The committee would be SCPDS administration and the pool was only open to SCPDS scholars who had completed all the necessary work to stay as scholars. We got it! In fact, all four SCPDS scholars at CSUMB got it! You see I was excited because I had chosen to put off applying to graduate school for one year, but I still needed to continue to develop as a researcher. This opportunity would put me in a pool of my own and hopefully prepare and propel me towards a larger national fellowship. On top of that, I was going to be doing work in a fantastic place at the University of Rhode Island with an important research question in a brand new lab, all paid for by the SCPDS! What more could I ask for?

If you are looking for things to turn sour, they haven’t. I am currently in the lab growing little baby oysters from tiny embryo stages up to trochophore larvae. We will then filter them out of the water column and freeze them with liquid nitrogen to later figure out if we can use these new types of extraction kits to get DNA and RNA from samples in one fail swoop. At the same time, the BinPacker program is running on the server; putting little pieces of exome reads together in ways that I have yet to analyze. Three assembly programs have already finished, but I have many more to go. Rhode Island is beautiful, full of good beer, good food, great people, and some of the most wonderfully green scenery this California boy has ever seen during the summer season (California is all brown in the late spring, all summer, and most of the fall). The things I miss are my girlfriend, my family, my friends, my dog, working at A Taste of Monterey, SCUBA diving, spearfishing, and my favorite restaurants. But that’s what this is all about. Getting to figure out how I could become a Ph.D. candidate at URI, while also reminding myself of the things back home that I cherish and love.

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How do we measure pH? Shifting pKw values across Monterey Bay

In the following presentation, we explored how pKw shifts across Monterey Bay utilizing buoy data, excel macros, and ArcGIS. Since pH is a measurement of the concentration of [H+] ions in solutions measuring pKw helps to contextualize changes in pH. We found that pKw does not always equal 14, which is what we are taught in general chemistry courses. Near-shore environments typically have depressed pKw values averaging less than 14 while off-shore environments have elevated pKw values greater than 14 on average. pKw_powerpoint.pptx

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Myripristis berndti: the Squirrel, the Soldier, and Bigscales

What a cute little fish.

myrip_school

The following presentation is an overview of the M. berndti pan-tropical species of fishIt goes by many names, as indicated above, but it is an extremely successful fish thanks to long pelagic larval development period that allows it young to travel far and wide, spreading their influence all over the Pacific and Indian Oceans. In this presentation, there is also an overview of the genetic structuring of this species from the coral triangle and the red sea. These areas contain many endemic species and are areas rich in genetic diversity. These rich areas may act as sources of genetic variation and allow these species to be resilient to changes in the oceans predicted by the most conservative climate change models. We hope to explore the genetic diversity of this fish using analysis in the C. striatus presentation, Dongsha a source of Ctenochaetus striatus in the coral triangle, and will be asking similar questions of how can we protect these species now to ensure their future.

 

Presentation: Myripristis berndti the Squirrel, the Soldier, and Bigeyes

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Abstract for Transcriptome Project

FINDING THE TOOLS: USING MULTIPLE ASSEMBLERS TO GENERATE A COMPLETE TRANSCRIPTOME FROM A NON-MODEL LARVAL MARINE FISH, SEBASTES AURICULATUS

Green, J.M*1, Cline, A.J.1, Bernardi, G.2, Jue, N.K.1, Logan, C.A.1

1 School of Natural Sciences, California State University Monterey Bay, Seaside, CA, USA

 2 Department of Ecology & Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA, USA

RNA sequencing is transforming the ability for ecologists and evolutionary biologists to study population genomics and gene expression in non-model, ecologically important species. A major challenge, however, is building a robust de novo transcriptome assembly in the absence of complete, well-annotated genome. Assemblies are frequently built using only a single assembly program, such as Trinity, yet each program has unique biases. Here, we use the non-model brown rockfish (genus Sebastes) as a case study to build and compare multiple transcriptome assemblies. We tested three assemblers (Trinity, Transabyss, and Oases) using a coverage of either 30 or 100. We also built Transabyss and Oases assemblies using k-mer sizes ranging from 19 to 49. Assemblies with the highest coverage and k-mer sizes were merged into a master assembly. We assessed all 24 assemblies for completeness, coverage, and annotation rates. We found that assemblies built with high k-mer sizes and coverages produced more complete transcriptomes (BUSCO > 94% complete orthologs) and that Trinity assemblies had the highest annotation rates. According to our assessment criteria, no single assembly stood out as best. Future work will examine the effect of assembly choice on downstream RNAseq gene expression analysis.

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The Importance of Reflection

Being able to reflect on your circumstances is a cornerstone of any good scientific process. It is literally built into the fabric of how we view our lives, science, and the world around us. This past summer I was given the opportunity to be a Professional Development Mentor for the Undergraduate Research Opportunity Center Summer Program which included 60 students from the UROC, REU, and NOAA EPP programs. I felt it was imperative that we as a class have a discussion about what it means to reflect in daily life and why it is important to us as scientists, people, and the communities we inhabit.

Presentation: Importance_Reflection

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