Award: OCE-1130494

The hadal zone, primarily in tectonic trenches, is the deepest marine habitat (6000-11,000m). Much of hadal knowledge comes from 1950s trawling expeditions, yielding an initial catalogue of species. Importantly, these and later explorations found invertebrates (e.g., amphipods, holothurians) to 11,000m, but never found fish below 8,340m. However, sampling was limited and nets did not yield ecological information (densities, distributions, behaviors, food chains, species uncaught). Few studies examined adaptations to crucial factors such as hydrostatic pressure. The development of Nereus and free-falling landers (Fig. 1A,B,C) prompted our international team (USA, UK, New Zealand; Japan) to form HADES (Hadal Ecosystem Studies). Our goals: explore types and distributions of hadal organisms; behaviors; and effects of pressure, food supply, metabolism, and topography. This report covers Whitman College research by me and undergraduates on pressure adaptations and whether those confer limits to some species. Pressure reaches 1101 atm in Mariana Trench's Challenger Deep (~11,000m). Pressure, while not imploding organisms lacking air chambers, forces water molecules into proteins (e.g., enzymes), and induces non-functional protein aggregates, impairing cellular functions. Work began with fish (Kermadec Trench snailfish, 7000m, last caught 1952) collected from NIWA's Kaharoa [2011; NIWA, Aberdeen, JAMSTEC, Whitman (undergraduate Mackenzie Gerringer) researchers]. Doug Bartlett (Scripps) collected amphipods from the Challenger Deep for us on James Cameron's DEEPSEA CHALLENGE project (2012). Full-team NSF-HADES exploration occurred on R/V Thompson (landers and Nereus), Kermadec Trench between 4000m and 10,000m (spring 2014). We also leveraged NSF funding for a Schmidt Ocean Institute lander expedition to Mariana Trench (late 2014). See Fig. 2 for some species caught for analysis. Intellectual Merit How proteins work under high pressure, and whether pressure limits some organisms, remain major deep-sea questions. Many proteins have evolved pressure resistance. However mechanisms are poorly understood and often incomplete, with some proteins from deep species exhibiting inhibition in pressure chambers. Prior to HADES, we discovered (1996-2007) that trimethylamine oxide (TMAO) increases linearly with depth (as pressure does) in bony fish, at least to 5,000m. TMAO, long known as an osmolyte (solute that reduces cellular water loss due to ocean salt), is the source of marine fishy odor. I found (1970s) that TMAO stabilizes shark proteins against the waste product urea, yielding biotechnology and medical applications. Later (1996-2004) we found TMAO is a "piezolyte" (pressure solute), protecting proteins by preventing pressure-forcing of water molecules into them. Extrapolating TMAO levels, I noted fish osmolalities (solute concentrations) could exceed seawater at ~8400m, drawing excess water inwards. This suggested that fish could not go deeper: bony fishes lose water due to lower osmolalities than seawater, with gills and kidneys adapted to retain water; no fully marine fish can cope with overhydration. Invertebrates, though, are isosmotic with seawater using osmolytes like taurine. Findings: A. The first evidence that pressure may be an ecological limit: 1) No fish were seen or trapped by Nereus or landers below 8145m (one fish there; soaring amphipod densities below ~8000m without fish predation); 2) Fish TMAO (Fig. 3A) and consequently osmolalities (Fig. 3B) increase linearly to ~8,000m, strongly supporting piezolyte and 8400m-limit hypotheses. B. Novel pressure adaptations; the deepest animal enzymes yet tested: 1) Amphipods too show depth increases in TMAO, but unlike fish, replace taurine with glycerophosphocholine (GPC) and other osmolytes (Figs. 4A,B,C). GPC protects mammalian kidneys from urea, so may help with pressure; 2) Both taxa show depth increases in scyllo-inositol (Figs. 5A,B), a polyol that breaks up maladaptive protein aggregates better than TMAO. 3) One hadal fish enzyme is activated by pressure, a novel discovery (Fig. 6A); but another requires TMAO to function (Fig. 6B). Broader Impacts Pressure is used to sterilize food, but some bacteria survive by accumulating piezolytes. Scyllo-inositol is being tested to treat Alzheimer's Disease, as it breaks up protein aggregates correlated with that disease. Pressure and piezolytes are used by biophysicists as tools to probe water's roles in protein folding and function. Understanding how nature uses piezolytes has impacted these applications. Undergraduates engaged had their career prospects enhanced by emersion in cutting-edge marine research: Mackenzie Gerringer (2011-12), now Ph.D. candidate in HADES, University of Hawai'i; Gemma Wallace (2013-14), now applying to graduate programs; Anna Downing (2015-16) and Chloe Weinstock (2016-17), both planning on marine graduate work. Results were broadly distributed to the scientific community through HADES (www.whoi.edu/hades) and NOAA Ocean Explorer websites, numerous scientific presentations including a hadal session at the 2015 Deep-Sea Biology Symposium, and our metadata BCO-DMO repository (http://www.bcodmo.org/project/536452). Outreach impacted wider society. Press releases garnered widespread media attention (millions of webpage hits, ~200 published articles in ~23 countries, numerous radio interviews, shipboard blogs) that substantially advanced the public?s awareness of hadal habitats, the challenges of studying them, and instilled wonder at the marvelous inhabitants of this realm. BBC is perusing HADES videos for their Oceans miniseries. Last Modified: 11/10/2016 Submitted by: Paul H Yancey