- Most biological surveys of coral-reef environments are limited to depths accessible by conventional SCUBA gear; typically about 30-40 m. Over the past two decades, the emergence of so-called “Technical Diving”, which incorporates advanced diving equipment and techniques (e.g. closed-circuit rebreathers, helium-based breathing mixtures, enriched oxygen breathing gas for accelerated decompression, etc.) has allowed highly trained marine biologists access to depths of 50-150m for scientific research. Initial surveys conducted by Pyle and associates in Hawaii, the Cook Islands, Palau, Fiji, Papua New Guinea, Samoa, Taiwan, Line Islands, Vanuatu, and (most recently) across the Caroline Islands, has revealed a consistent pattern of rich biodiversity at these depths below the more traditionally-surveyed shallow coral reefs. This habitat, variously referred to as “deep coral reefs”, the “coral-reef Twilight Zone”, or “Mesophotic Coral Reef Ecosystems”, is characterized by organisms typically associated with shallow coral-reef habitats (e.g., soft corals, sponges and other reef-associated sessile invertebrates, and fishes belonging to coral-reef fish families), a high proportion of which represent undescribed species. Through these initial surveys, it has become apparent that the depth-zone traditionally surveyed with conventional SCUBA represents only the upper 30% of the overall coral-reef ecosystem; the deeper 70% of which remains mostly unexplored, and poorly understood. The non-profit Association for Marine Exploration (AME; was established specifically to facilitate scientific exploration of undersea habitats that are otherwise inaccessible to divers using conventional SCUBA. Since its inception in 2003, AME has organized or participated collaboratively in five major expeditions focused on collecting voucher specimens and tissue samples of fishes and invertebrates from coral-reef habitat at depths of 50-150m. AME divers use the most advanced electronically-controlled closedcircuit rebreathers available, and have perfected diving and collecting techniques that allow efficientspecimen acquisition with minimal impact to the reef infrastructure, within very conservative margins for safety. Each diver uses a Cis-Lunar MK-5 rebreather and carries five separate gas supplies (primary and secondary oxygen, air for blending enriched airnitrox during decompression, heliox for blending trimix, and a large-capacity trimix [helium/ nitrogen/oxygen] cylinder for emergency bailout). After more than two thousand rebreather dives by AME divers, there has never been a need for a full bailout. Nevertheless, all dives are conducted with access to a full open-circuit bailout pathway back to the surface, using different techniques depending on specific conditions and circumstances. In-water Recompression (IWR) has proven to be an extremely effective emergency response to symptoms or suspected symptoms of decompression sickness (DCI), with complete success in the very rare circumstances when it has been utilized (never during an AME project – only in the previous experience of AME divers). Fish specimens are usually collected with custom-built monofilament hand-nets, sometimes with barrier nets. Chemical anesthetics and ichthyocides are sometimes used when circumstances are appropriate and proper permits are available, but always with the utmost care to prevent bycatch or other damage to the reef habitat. Small fishes and invertebrates are collected with the aid of a custombuilt Electric Underwater Vacuum Cleaner (EUVC), which proved extremely effective during an expedition to Vanuatu in 2006. All dives and most observed species are documented by Hi-Definition video taken by one of the deep divers who is dedicated for this function. Such video has proven valuable for documenting additional species that escape collection, and for documenting the general habitat characteristics. For this project, we propose to contract with AME to acquire specimens of selected organisms (fishes and marine invertebrates) from depths of 50-150m at selected sites around Moorea. Two separate trips are planned – approximately three weeks each – to allow diving in different areas with different optimal seasons (swell conditions). Voucher specimens will be brought to the surface, photographed, and processed following preservation protocols appropriate to each kind of organism. Tissue samples will be extracted from each specimen for sequencing and analysis, and vouchers will deposited in appropriate institutional collections. AME will be responsible for all diving equipment and personnel involved with deepdiving activities, except possibly for support divers (if available locally). One deep dive per day is conducted by a team of 3-4 deep divers, with 1-2 support divers in the boat. Dives typically last from 3-7 hours each, depending on depth and duration. The maximum depth accessible by AME divers is approximately 150m, but most dives are made in the range of 100-120m, and some are focused on depths of 50-100m. Typically one of the deep-diving team is dedicated to capturing Hi-Def video of the habitat and specific organisms, while other deep divers are focused on collecting with either nets or the EUVC. Bottom times typically range from 30-60 minutes at deeper depths, and 60-90 minutes for intermediate depths; with decompression times lasting from 2-6 hours. Ideally, decompression is conducted along a reef slope or dropoff, but can also be conducted in mid-water following a line to the surface. Depending on specific circumstances, deep divers may either drag a line with afloat on the surface, or follow an underwater line or natural contour down to depth, and then deploy an inflatable surface float upon ascent. Mechanisms for communication among deep divers, and between the deep-diving team and the surface-support team depend on specific circumstances, and are established well in advanced of dive trips. All personnel involved with the deep diving activities are fully briefed on emergency protocols, including IWR.


- The protocols for fish were well established in the pilot MBP. Pyle's AME team has extensive experience archiving the voucher specimens, capturing the associated metadata, and identifying the species. A smaller tissue sampling and archival team will accompany the AME group onsite for the processing of genetic material and stabilization of the marine invertebrate component. Serge Planes will coordinate the tissue team and the biocode technicians will handle the material derived from the EUVC. All samples will be extracted at Gump and sequenced at LAB.


Two field trips will be made during 2009 (probably July and December).