NOAA Office of Ocean Exploration Semi-Annual Progress Report Format

I. OVERVIEW Grant Number (if applicable) NA07OAR46000289

Amount of funding from Ocean Exploration $55,550

Project Title Operation Deep-Scope 2007: Characterization of cliff ecosystems using new technologies Area of Operation (if applicable) Bahamas (Gouldings Cay and Little San Salvador) Principle Investigator (name, address, contact information) Sönke Johnsen, Biology Dept. Duke University, Durham, NC, 27708 (919-660-7321; [email protected]) Participating Institutions Duke University, Harbor Branch Oceanographic Institution, University of Texas at Austin, University of Queensland, Ocean Research and Conservation Association (ORCA) Award Period: From _7/1/07___ To __5/31/08_ Period Covered by this Report: From _7/1/07___ To _12/31/07_

II. Evaluation: 1. Work Accomplishments:

General

Deep-Scope 2007 was extraordinarily successful. Nineteen submersible and fifteen SCUBA dives were performed, primarily at Gouldings Cay and Little San Salvador. Due to the excellent weather and now-reliable technologies, we were able to collect large amounts of data (primarily imagery) and samples, which are detailed below by section. The cruise also led to a feature news article in Nature, the world's top science journal, and to future programs produced by both NOVA and the BBC.

Eye-in-the-Sea Camera investigations

The Eye-in-the-Sea (EITS) observatory was designed to be acoustically quiet and to use far-red illumination to observe deep-sea animals unobtrusively. The EITS has clearly demonstrated the critical importance of stealth during Deep Scope 2004, 2005 and now during 2007. During the 2004 and 2005 operations in the Gulf of Mexico we concentrated on site-specific deployments near biologically rich communities of macrofauna in association with fossil hydrocarbon seeps. Our rational was to focus our very limited number of available exploratory deployments on these sites, as we hoped that these virtual deep-sea oases were likely to be frequented by large mobile predators. This rational was clearly borne out with the discovery of a large squid that is so new to science it cannot be placed in any known scientific family (Widder, 2007). In the Bahamas in 2007 the Eye-in-the-Sea was placed in proximity to cliff-face communities, which concentrate biomass in narrow vertical corridors. In this case we reasoned that such cliffs also represent biological oases likely to be frequented by large predators. Once again this rational proved sound when, during three 36 hr deployments, at 487, 548 and 694 m, at least nine species of deep-sea shark were recorded. A clear diurnal rhythm was apparent with smaller sharks such as Squalus cubensis seen during the day and larger sharks such as Hexanchus griseus at night. Most exciting was the observation of bottom- rooting by six-gill sharks, a discovery which may help account for how such inordinately large organisms manage to survive in a largely food-poor environment.

Figure 1: Hexanchus griseus bottom rooting - observed with red light illumination with Eye-in-the-Sea. Black container in foreground is bait box surrounded by large numbers of isopods.

Figure 2 : The six gills appeared to be scooping up isopods in the sediment…

Figure 3: ... and then spewing bottom sediment from their gills. Also observed for the first time were bioluminescent displays that occurred in response to a rapid repetitive flash by the electronic jellyfish.

Figure 4: Two "string of pearls" displays (labeled BL), which occurred during a rapid repetitive flash display by the electronic jellyfish.

Visual Physiology of benthic crustaceans

On this cruise, novel collecting techniques were used to collect live deep-sea benthic crustaceans. Collections were made from the HBOI Johnson-Sea-Link (JSL) submersible under red and orange illumination, making it possible to collect animals without blinding them. Electrophysiological measurements of spectral sensitivity and temporal resolution were made from the photoreceptors of the decapod shrimp Eugonatonotus crassus, and the isopod Booralana tricarinata. The spectral sensitivity data indicate that these species have blue sensitive visual pigments. The temporal resolution of all these eyes is quite low, indicating that these photoreceptors have a long integration time. The temporal resolution of Booralana tricarinata is the lowest that has even been recorded using these techniques. It is so low that it is unlikely that this isopod could track any moving object. Rather, because it lives below the depth at which downwelling light would provide much ambient light, its photoreceptors appear to be designed for finding bioluminescence that glows rather than flashes, such as detritus covered with bioluminescent bacteria. Figure 5: the decapod shrimp Eugonatonotus crassus, collected under red sub lights.

Figure 6: The isopod Booralana tricarinata, which has the slowest eyes of any known animal.

Fluorescence of pelagic and benthic species

Our main focus of the deepScope 2007 expedition was biotechnology of marine bio-fluorescence. We used SCUBA diving (including blue water), submersible collection and plankton tows to collect a variety of fluorescent organisms including yet untapped sources of fluorescent compounds such as polychaetes and fish. All the organisms were documented, their fluorescence spectra were measured and RNA isolated from the fluorescent parts. Four of these samples - pontellid copepod, two jellyfishes and a fireworm - have been already studied by means of bacterial expression libraries. Pontellid yielded eight fluorescent clones, while over 50 clones of slightly different fluorescent properties were isolated form the jellyfishes library. The fireworm library did not produce any fluorescent clones possibly because the fluorescent pigment in this organism is a novel protein that does not express easily in bacteria. To isolate such a pigment we are going to proceed via proteomics route followed by mass-spectrometry to identify its sequence. Collections were made using Johnson Sea Link submersible to enable a new project in my lab, that is going to address pathways of deep-sea biodiversity evolution focusing on living fossils and deep-sea adaptive radiations. In addition, in the vicinity of Little San Salvador island we collected enigmatic objects of obvious biogenic origin that are extremely abundant on the surface of the sediment at that particular site but, to our knowledge, have never been reported before. We will use molecular techniques to identify their origin. Figure 7: Fluorescence image of the bearded fireworm, Hermodice carunculata collected during a SCUBA off the coast of Little San Salvador. The contrasting green-red fluorescence most likely serves as a warning signal advertising the worm's unpalatability and toxic spines.

Figure 8: This species of pelagic polychaete worm from the family Alcyopidae, known for their large camera-type eyes, displays very bright fluorescence surrounding its eyeballs and also exudes a cloud of fluorescent ink when disturbed (lower portion of the photo). The function of these displays is most likely to startle a potential predator. Figure 9: green fluorescence of the eye of an unidentified Chlorophtalmid fish may function as a sexual signal.

Figure 10: The function of the bright green fluorescence in this unidentified juvenile eel is unclear.

Pelagic visual ecology and polarization vision The objectives of this expedition were to firstly gather material and data regarding the sensory capabilities and communication strategies of deep-sea and pelagic organisms and secondly to continue our observations on polarized light by taking advantage of the legendarily clear waters around the Bahamian walls. Excellent progress was made in both areas. Continued use of custom-built underwater polarising cameras in blue water situations finally gave us the results we have been looking for in all 3 Deep-scope cruises -- namely good quantifiable polarization imagery of transparent zooplankton in situ, rather than in the lab. This data is currently being analysed. Camera use in shallow waters also revealed some exciting new observations regarding the corneal characteristics of fish, in particular Hypoplectrus indigo and other fish of the serranid family. Fig.1 (not shown) demonstrates the use of the polarizing camera system to detect polarizing tissues. The polarization activity shown here suggests a solution to an old problem regarding the potential for polarization vision in fish. In the case of plankton body tissue it allows estimates of camouflage breaking potential for animals possessing polarization sensitivity. Hundreds of frames from the submersible videos were also digitized. These are being split into color channels and analyzed for crypsis.

Figure 11: The reef fish Hypoplectrus indigo – indigo hamlet, demonstrating corneal tissue that polarizes light, or is likely birefringent, the frame on the left taken through a horizontal polarizing filter and on the right vertical, as indicated by arrows.

2. Expenditures: (7/1/07-12/31/07)

Proposed Spent c) Travel Costs 1) SJ and student from Durham, North Carolina $ 433 $358 2) JM and tech from Brisbane, Australia $4500 3) MM and tech from Austin, Texas $ 500

Also travel and meeting costs for SJ, EW, and TF to OE symposium at Ocean Sciences Meeting $1500 each $400 e) Supplies 1) 24 Digital S tapes to use in JSL sub @ $45 $ 1080 2) 60 Mini DV tapes (64 min) to use in JSL sub @$8 $ 480 3) Jars for specimen samples $ 500 $315 4) Electrodes for electrophysiological recordings $ 600 $527 5) Computer storage media $ 300 6) Fluorescence and Polarization optics hardware $ 500 7) Supplies for histology $ 500 8) replacement hardware for tucker trawl $ 500 9) bait for EITS $ 60 10) mechanical bait for EITS $ 3000 11) batteries for EITS (including housing and assembly) $ 6000 12) 48 BetacamSP video tapes @$9 each $ 432 $1483

1 full time @at Sea web site manager from the HBOI media $1020 $1020 lab to post mission events for the HBOI@Sea website (Jon Saint)

1 full time @at Sea Science videographer from the HBOI media $ 5073 lab to provide real time coverage of mission events for the HBOI@Sea website and the Ocean Explorer website. (Brian Cousin)

1 full time @at Sea Science Correspondent from the HBOI media $ 3676 $3676 lab to provide real time coverage of mission events for the HBOI@Sea website and the Ocean Explorer website (Mark Schrope)

Satellite Communications costs $ 685 - ~1700 kb at $0.40/kb; for satellite transmission of mission highlights to @sea and Ocean Explorer websites;

Shipping of scientific gear from Brisbane, Durham, and Austin $ 1500 to and from Fort Pierce

Indirect costs at 55% $ 4,412

Total federal share $ 12,291

Reasons for discrepancies

We had expected to spend most of the money in the grant by 12/31/07, but were unable to due to clerical errors at both NOAA and Duke that prevented us form using any of the funds until December. So we are currently in the process of reimbursing people for their travel and supply expenses. The $5073 that was budgeted for Brian Cousin could not be used because he developed a brain aneurysm and was unable to go on the cruise. We hope to be able to use some of that balance to account for the fact that the travel cost for the two Australians was about $7,700 USD instead of the $6,000 budgeted, due to the drop of the US dollar relative to its Australian counterpart. Most of the money for the scientific meetings has not been spent yet, because the meetings are in March. 3. Results (if any at this time): a. Inventory of activities (number of submersible dives, CTD, net tows, etc.)

Submersible dives: 19 (16 for science, 1 training, 1 failed, 1 to recover lost equipment). All science dives were taped and tape copies will be sent to NOAA/OE. Dive logs have been already been sent.

SCUBA dives: 15 (8 on reef [2 at night], 7 in blue water [2 at night])

b. Inventory of samples collected

Order Decapoda, Suborder Pleocyemata, Family Eugonatonotidae, Euganatonotus crassus - collected 4

Isopod Booralana tricarinata - collected 30 – 1 to 2 inches long - sent 10 to Rachael King, at the Southeastern Regional Taxonomic Center for I.D. and to add to their collection

Isopod Bathynomus giganteus – collected 10 – 1-2 inches

Order Decapoda, Suborder Dendrobranchiata, Family Benthicymidae, Benthesicymus sp. – collected 1

Order Decapoda, Suborder Dendrobranchiata, Family Glyphocrangidae, Glyphocrangon sp. – collected 2

2 deep-sea octopus species to be investigated for retinal anatomy and polarization vision potential. Species identification underway.

White anglerfish (Lophoides beroe) eyes, wholemount retina extracted for photoreceptor counts and mapping.

Small benthic shark (Galeus area). Handed on to elasmobranch lab at UQ for sensory system mapping and comparison.

Glass sponge tissue collected for international collaborator Gert Worheide.

c. Significant findings or discoveries

Slowest eyes ever measured (found in isopod Booralana tricarinata) Extreme polarization in eyes of reef fish Hypoplectrus indigo Intense red and green fluorescence in the fireworm Hermodice carunculata Bioluminescent displays that occurred in response to a rapid repetitive flash by the electronic jellyfish. Discovery of bottom-rooting as a feeding behavior in six-gill sharks

See “accomplishments” for details on all of the above.

d. Papers or presentations submitted or planned

Frank, T. M. (2007) Sebastian River High School, Sebastian, FL.

Frank, T. M. (2008) Ocean Sciences meetings, Orlando, FL. (upcoming)

Frank, T. M. and E. A. Widder (2008). Teacher training workshop sponsored by NOAA/OE. Charleston, SC.

Frank, T. M. (2008) Smithsonian Marine Station, Ft. Pierce, FL.

Frank, T. M. (in prep) . Visual ecology of deep-sea benthic crabs: an unusual case of UV photoreception

Frank, T. M. (in prep). Temporal resolution and spectral sensitivity in deep-sea isopods and shrimp.

Johnsen, S. and N. J. Marshall (in prep). The ecology of polarization vision in the pelagic realm.

Johnsen, S. (2008). Conference on New Directions in Research on Polarization of Light Heron Island, Australia. (upcoming)

Marshall, N. J. (2008). Conference on New Directions in Research on Polarization of Light Heron Island, Australia. (upcoming)

Matz, M. M., and C. Mazel (2008). Annual Meeting of the Society for Integrative and Comparative Biology. San Antonio, TX.

Whitehill, E. A. G. and T. M. Frank (2008). Annual Meeting of the Society for Integrative and Comparative Biology. San Antonio, TX.

Widder, E.A. (2007) Sly eye for the shy guy: Peering into the depths with new sensors Oceanography. 20(4): 46-51 (invited review)

Widder E. A., Raymond, E. H., and T. T. Sutton (2008) Ocean Sciences meetings. Orlando, FL. (upcoming) e. Media coverage

NOVA: Camerman Sean Glenn accompanied us to film and interview Edie Widder for a profile on “Nova ScienceNOW”. Air date not yet set.

BBC: Cameraman Warwick Sloss accompanied us to take footage of animals recovered using the submersible. He was also able to take footage from the front the submersible during a dedicated dive. This footage will be used in the upcoming BBC series “Life”.

Nature: The journal commissioned our of our crew to write an article on deep sea vision and optics, which is primarily based on Deep-Scope work. The article is attached to this report.

Schrope, M. (2007). Lights in the Deep. Nature 450 (22 November issue): 472-475 (attached)

Prepared By: ______Signature of Principal Investigator Date