CMarZ - Ronald H. Brown cruise report

This report was prepared by Peter Wiebe, Larry Madin, Francesc Pagès, Dhugal Lindsay, Hege Øverbø Hansen, Saramma Panampunnayil, Martin Angel, Hiroyuki Matsuura, Mikiko Kuriyama, Astrid Cornils, Russ Hopcroft, Colomban de Vargas, Silvia Watanabe, Yurika Ujiie, Hui Liu, Barbara Costas, Tracey Sutton, C. B. Lalithambika Devi, Rob Jennings, Paola Batta Lona, Brian Ortman, Ebru Unal, Leo Blanco Bercial, Nancy Copley, Chaolun Li, Joe Catron, and Dicky Allison.

This was the first major CMarZ cruise in the Western North Atlantic. The success of the cruise was due to the collective efforts of Captain, Officers, Crew, and all members of the Scientific Party. Throughout the cruise there was a camaraderie and friendliness among all the participants that made this expedition a great pleasure. This cruise was supported by the NOAA Ocean Exploration Program.

Sitting Row 1 (L-R):Chaolun Li, Saramma Panampunnayil, Silvia Watanabe, Hui Liu, Francesc Pagès, Paola Batta Lona, Tracey Sutton, Martin Angel, Dicky Allison.

Standing Row 1 (L-R): Mikiko Kuriyama, Lalithambika Devi, Brian Ortman, Russ Hopcroft, Nancy Copley, Ebru Unal, Joe Catron.

Standing Row 2 (L-R): Yurika Ujiie, Leo Blanco Bercial, Hiroyuki Matsuura, Larry Madin, Barbara Costas, Hege Øverbø Hansen.

Back Row 3: Colomban de Vargas, Astrid Cornils, Erich Horgan, Dhugal Lindsay, Rob Jennings, Peter Wiebe.

The focus of the CMarZ program is on the development of a taxonomically comprehensive assessment of biodiversity of animal plankton throughout the world ocean. The project goal is to produce accurate and complete information on zooplankton species diversity, biomass, biogeographical distribution, genetic diversity, and community structure by 2010. Our taxonomic focus is the animals that drift with ocean currents throughout their lives (i.e., the holozooplankton). This assemblage currently includes ~6,800 described species in fifteen phyla; our expectation is that at least that many new species will be discovered as a result of our efforts. The Census of Marine Zooplankton (CMarZ) program is an ocean realm field project of the Census of Marine Life (CoML).

On this cruise, the focus was the tropical/subtropical waters of the Atlantic Ocean west of the mid-Atlantic ridge. The objective was to collect and identify the zooplankton distributed throughout the entire water column, with a particular focus on the under-sampled mesopelagic, bathypelagic, abyssopelagic zones, and then to sequence them genetically at sea. Thus, the scientific participants on this cruise include CMarZ researchers, expert taxonomists, molecular specialists, staff, and students. Sampling was conducted along a transect extending from the northern Sargasso Sea to the equatorial waters northeast of Brazil (Figure 1). At five primary stations, environmental data and zooplankton samples were collected using three Multiple Opening/Closing Nets and Environmental Sensing Systems (MOCNESS). One was a large opening/closing trawl and two were smaller multiple net systems (See MOCNESS Sampling section below for more detail). Other samples were collected with ring nets and water bottles, and by blue water SCUBA diving.

Samples were analyzed at sea using traditional taxonomic approaches and molecular systematic analysis, including DNA sequencing of a target gene portion for each species. After the cruise, follow-up molecular analysis, species counts, and expert taxonomic evaluation and description of any putative new or undescribed species will be done in association with the CMarZ Taxonomic Network. In addition to the intensive sampling of the water column, a series of lectures and workshops were conducted as part of the at-sea training in zooplankton morphological and molecular systematic approaches.

10 to 12 April 2006: The cruise got underway at 1400 on 10 April when we left the port of Charleston, South Carolina after four days of intense set-up of the equipment and laboratory spaces needed for the work at sea. Shortly after leaving the dock, the ship spent time in Charleston Harbor while a calibration of the Robertson Navigation system took place, which had undergone repairs while the ship was in port.

The R/V Ron Brown finally left the harbor and got underway for the first station located in the Northern Sargasso Sea about 1800. During the early hours of the night of the 10^th, the ship’s ride was comfortable, but the winds picked up to around 20 kts towards the morning of the 11th as we steamed out across the continental shelf and the Gulf Stream, and the motion of the vessel made many of the scientific party a bit seasick. The moderately rough weather continued into daylight on the 11^th of April and the loading of nets onto the three Multiple Opening/Closing Nets and Environmental Sensing Systems (MOCNESS) and other work in the laboratories went slowly. The initial abandon ship and the fire and boat drill took place around 1030.

Problems with the operation of the trawl winch with 0.68" conducting cable, which was the mainstay of our sampling program, had been identified a couple of weeks before the start of the cruise and most of the repairs had taken place by the time the ship left the dock. On the evening of the 11^th, 8000 meters of trawl wire was streamed to test the ability of the winch to carry out the deep MOCNESS tows. Although the winch had no difficulty in paying the cable out, there was significant difficulty bringing the wire back on board at a reasonable haul in speed. During the test, a load cell burnt out and it was replaced. A second test took place on the evening of the 12^th. This test indicated that the winch could be used to tow the MOCNESS trawl to bathy- and abyssopelagic depths.

Several science meetings took place during the steaming to Station 1. On the evening of the 11^th, there was an introductory meeting to discuss the cruise objectives and to review plans for work at the stations. On the afternoon of the 12^th there were two additional meetings. The first was to work out the final details of the sampling at a station and the protocols for how each of the zooplankton samples was to be processed. The second involved a number of the officers and crew and was focused on the methods of deployment of the over-the-side equipment and who would be in charge of coordinating them.

During the afternoon of the 11^th, a ½-m diameter ring net was deployed to 100 m to provide a collection of zooplankton that could be used by the taxonomic experts on board to begin their work on this cruise. A number of species of copepods, ostracods, gelatinous animals, formaminifera, and other groups were picked live from the sample and a number of the species were identified. Some of these were then selected for genetic sequencing on the 12^th. Earlier in this day, a first occurred when a zooplankton species (a copepod, Disseta grandis, caught on a previous cruise) was sequenced at sea. As far as we know, this is the first time a gene sequencer has produced a gene sequence at sea. This is a very significant early milestone for this cruise.

13/14 April 2006: Finally, waiting for arrival at the first station ended with a phone call from the bridge saying that we were on station at 0500 on 13 April. The seas were moderate and the winds were in the 10 to 15 kt range, fine for work on station. The air was about 17°C, cooler than the sea water, which had a temperature around 19°C. The science watch for the midnight to noon period was awake quickly and the first event was a 1-m MOCNESS tow. The net system was a bit late in getting into the water because the deployment through the stern A-frame required some experimentation to see how best to get it over the 10-m MOCNESS, which was positioned within the stern A-frame area. The tow went well with Larry Madin taking the lead on flying the net for the first part of the tow and Peter Wiebe taking the last part while Larry coordinated preparation of the SCUBA dive. There was lots of excitement when the net system came on board around 0930 and a flurry of activity as the samples were moved from the deck in buckets chilled with ice packs to keep the samples cool to the walk-in refrigerator. They were stored there until they could be photographed and picked for large organisms in the wet lab, and then split into fractions for additional live picking of smaller animals or preservation in either alcohol or formalin.

A blue-water dive was next up and Larry Madin, Erich Horgan, James Brinkley, and Keegan Plaskon (latter two from the ship) went out in the RHIB (Rigid Hull Inflatable Boat) for about two hours of collecting of the more fragile zooplankton that are usually destroyed by collection with nets. The boat, with the divers, boat operator, and gear already on board, was launched from the 02 deck by an automated launch and recovery system that made the operation safe and efficient.

The first tow with the 1/4-m MOCNESS in the early afternoon of the 13^th was not successful. A problem with the battery power in the underwater unit resulted in a loss of signal about a third of the way through the deployment. Only a single sample was obtained, but it also proved to have a number of microzooplankton species of interest.

The final bit of rigging of the 10-m MOCNESS trawl system was completed and by 1600 it had been launched off the stern and was going down to 5000 m. It took about 4 hours to deploy it to the maximum depth and it was a very smooth deployment. Once at depth, the ship’s speed was increased to get the angle of attack of the net frame up to the normal towing angle of about 45 degrees and to let the net system rise up with the increased velocity. When the winch began to haul in the towing wire at the slow rate of 10 m/min, the winch started up and then stopped. There was a lot of activity for awhile to try to determine what caused the winch problem. In the end, the winch was enabled by the ship’s engineers so that it could haul in at 6 m/min. With 8000+ meters of wire out, it appeared it would take 22 hours to haul the net back in. In reality the wire was hauled in less time because the speed of retrieval increased as more wire came on board, and the net system reached the surface at 0740 on the 14^th of April some 16 hours after the tow was begun. The recovery of the frame went very smoothly with a combination of the A-frame and air-tuggers pulling the frame on board and down onto a pair of stanchions used to secure the top portion of the trawl frame. Once the frame was secure, a number of individuals from the morning watch pulled in the nets, which only took about 10 minutes. The newly designed trawl nets fabricated from very fine 335 µm nylon mesh proved successful and were in fine shape after the tow. This tow also had some flaws. One cod-end bucket that did not have its fasteners taped or rubber-banded was lost (Net 2) and the tab on net bar 3 broke off during the tow. The cable and swaged fitting from this net bar was found in the cod-end of net 4. This meant that net 1 fished from 5000 to 4000, net 2 fished from 4000 to 3000, and net 4 fished from 3000 to 1000. When net 2 was closed, net 3 closed with it and never was open. When net 3 was supposed to be released, the cable and swaged fitting plus part of the tab fell into the open net 4. Because of the length of the tow and the repairs needed to get the trawl back in service, the second tow at station #1 with this system was scrubbed.

The MOC-10 catches, while not spectacular in terms of biomass caught, were very interesting in terms of the species collected. While water collection with a 30 liter Niskin bottle was being conducted, the taxonomists were bunched in the ship’s wet lab processing the MOC-10 samples. It was clear that a number of animals were caught in the deep nets that had not been seen in the shallower MOC-1 tow, especially the net that sampled the 3000 to 1000 m zone. There were many shrimp and gelatinous animals including ctenophores. Martin Angel found at least two new species of ostracod, one he knew about and one he had not seen before. Tracey Sutton found a male anglerfish in very good condition - this specimen was from a group that is only known to the generic level. He also found other specimens of fish species that are rarely ever caught.

The afternoon of the 14th went quickly in calm seas, clear skies, and bright sunlight. The 1/4-m MOCNESS haul came first around 1330. The underwater unit that had failed during the first tow was replaced with the one from the MOC-10, and it and the options case with the fluorometer attached worked very well. This tow was a bit of a bear because it was difficult to keep on the intended course and the bridge had to keep changing speed to keep the towing wire clear of the ship’s starboard quarter. The net system came on board around 1600 and the catch again was of great interest to a large group of people, especially those working on the microplankton. Right after the 1/4-m MOC came on board, Russ Hopcroft put his Reeve net in for a deep (500 m) vertical tow during the dinner period to collect larvaceans and other live animals for identification and photography. Larvaceans are particularly fragile animals and rarely come up intact in the MOCNESS. The Reeve net with its very large cod-end allows the fragile animals to be collected more effectively.

A 1-m MOCNESS night tow was started about 1800 and this tow was completed in under 3 hours, which kept us on schedule and made it possible for the divers to leave the ship in choppy seas by 2130 for their night dive. They arrived back about 2315. Both the MOCNESS and the divers’ collections provided additional animals that had not appeared in the earlier tows and dives.

The last activity for the first station was another 1/4-m MOCNESS tow and it also was an ill-fated tow. With about 600 m of cable out and the net at 290 m depth, the deck unit lost signal with the underwater unit and data collection ceased. Something catastrophic had happened and the only recourse was to retrieve the system. With the winds picking up sharply since early evening (they were about 17 kts around midnight, up from 8 kts before the divers went out), the decision was made to call it quits at this station and set sail for Station #2.

During this two day transit, there was tremendous activity in the forward biochemistry lab where the Applied Biosystems Hitachi gene sequencer was located. The “UCONN Team DNA” was working around the clock to prepare and sequence animals. A number of species identified by the taxonomic experts on board were placed in a queue for sequencing and a dozen or more were successfully sequenced. Of particular note is the fact that Martin Angel had identified a number of ostracods from the samples acquired so far and some of these were prepared for sequencing. These were the first sequences ever produced for marine planktonic ostracods.

15 April 2006: In the early morning of the 15^th of April, there was bright sun coming through the port holes and mix of sun and clouds. The winds were from west southwest at about 25 kts. We were in the southern fringe of the gale that had been forecast for just north of our station #1 for today and there were lots of white caps on the starboard bow as we steamed south. Fortunately we were steaming away from the gale as we headed for Station #2 at 30°N; 70°W.

During the day, the main laboratory was the center of activity with the taxonomic specialists sorting through the samples collected at Station #1, identifying zooplankton species, and working with Team DNA to prepare the specimens for sequencing.

In mid-afternoon, we crossed paths with the lead boat in the Volvo Ocean Race. The leg five leader, ABN AMRO ONE was off on our starboard about 4.5 nm and was headed for Baltimore. There were seven boats in the race. In addition to ABN AMRO ONE, there was movistar, Pirates of the Caribbean, Brasil 1, Ericsson Racing Team, ABN AMRO TWO, and Brunel.

The work at Station #2 began about 1800 with the deployment of the 1/4-m MOC. It did not go well. The same problem that occurred at the previous station re-occurred. With 600m of wire out and the nets down bout 250 m, the deck unit again lost the underwater signal and it could not be re-established. There was no indication of a problem up to the time the system failed. After several hours of testing, two of the underwater units were unable to work on the CTD cable. In addition, one underwater unit suffered electronic damage as a result of the failure. Since these units worked well on the trawl wire, the suspicion was that the CTD cable was causing the problem. This ended our attempt to make a 1/4-m tow at this station.

The night dive that was scheduled for 2130 was also scrubbed because the winds (17 to 21 kts) and sea state were marginal at best. We then decided to do a 1-m MOCNESS early since the time was available. The deployment was scheduled for 2230. Leo Bercial, Chaolun Li, Paola Lona, and Joe Catron got the cod-end buckets on and set the net bar traps. Bruce Cowden, the Bosun, came out onto the deck at 2230 and started the operation by picking up the MOCNESS frame with the port crane to move it aft to the deck area underneath the A-frame. The tow signal cable, however, got caught on the support stanchion just as the frame was being lifted rapidly and the cable was stressed to the breaking point. It took about an hour and a half to re-splice and water-proof the cable. About midnight on the 15^th, the 1-m MOCNESS was ready to go back into the water and indeed it did.

16 April 2006: During the wee hours of the 16^th of April, the 1-m MOCNESS tow was successfully completed. While the samples were being processed and the net system being reset for another tow, 30-liter bottle casts to collect water for tintinnid analyses took place. This was followed by a pair of vertical Reeve net tows.

The second 1-m MOCNESS at station #2 started about 0730 on April 16th and was successfully completed by 1000. Again there was a flurry of activity as the samples came off the deck and went into the cold room. Those processing the samples were working quickly because the nets came on board just as the group was finishing up the preservation of the previous tow’s samples.

Although a blue-water dive was planned for the late morning, wind (around 20 kts out of the west southwest) and sea conditions were still marginal, in spite of the fact that it was warm and sunny, and the dive was delayed until early afternoon. Then with conditions a bit worse, it was cancelled.

In the early afternoon, Erich Horgan and others on the afternoon watch worked on setting up the 10-m MOCNESS. A bracket with a new tab, made by the ship’s engineers, was mounted on net bar 3 and then a cable with the swaged fitting was fixed through the tab. The nets were loaded and laid out ready to be deployed. During this tow, a series of test stops and haul backs were planned to make sure the winch could retrieved the wire and to allow the ship’s engineers to make adjustments to improve the winch performance.

In spite of the preparation, the launch and towing of the MOC-10 proved to be an ordeal. The launch was for 1445. The first snag was with the cable termination rigging. When the cable was hauled up tight in the over-boarding sheave, it was clear that the electrical cable was twisted in a way that had to be fixed. So the termination rigging had to be taken apart and reconfigured so that the electrical wire was out of harm’s way. Then the cod-end buckets and nets, which had been laid out so that the cod-ends could be easily deployed, were lowered into the sea. In the process, a long rent was discovered in the net with 3mm mesh that was deployed open (net zero) for the trip to depth. The launch was stopped while the net was sewn up. Starting the launch again, the tie-down straps were released, the tugger lines were slacked and unhooked, and then the winch wire was hauled in to raise the frame off the deck. Immediately it was noticed that the bar for net 1 had released from the toggle release. The launch was stopped again, the air-tugger lines hooked back onto the frame and the frame brought back down onto the stanchion. Then some agonizing time was spent trying to get the net bars up to the top of the frame so that the swaged fitting from net 1 could be inserted into the toggle and latched. This was made exceedingly difficult because the nets were streaming behind the ship and their drag was very hard to counter. Finally the fitting was secured. The tugger lines were again released and the frame was lifted up and rolled down into the water, and the tow began.

During the down-leg of this tow, the wire went out at a steady 30 m per minute until reaching 4500 meters of wire out (MWO). Then the Chief Engineer had the winch stop and haul in about 250 m to test the winch’s haul-in capability and to make some adjustments. After that it went down again. But the ship was having trouble with the wire tending to starboard of the stern because it was no longer towing into the wind. In order to get the ship pointed into the wind, the bridge decided to make a slow turn to port until the ship was again steaming into the wind. That maneuver was started when the wire was out at 5000 MWO. The change in the course had a negative consequence. Towards the end of the turn, which started at 1906 and ended at 2005, the net began rapidly descending vertically, reaching speeds of 60 to 88 m/minute. Essentially the net was in free-fall. An increase in ship speed was requested, but the time lag for an effect was long (on the order of minutes to ten minutes or more). Later when the net system was brought on board, a kink was found in the wire about 5 meters above the cable termination. The kink may have occurred when the free-fall period stopped and tension was restored on the wire. Once the ship was headed into the wind, the wire streamed straight out off the stern and the net frame stabilized. There was one more test of the wire at 5500 MWO, where the winch was stopped and the wire hauled in a few hundred meters at around 15 m/min. This worked OK. Then the wire was paid out to 6000 MWO (the limit for this tow) and the ship’s speed was reduced to allow the net to fall to the desired maximum sampling depth. But this was done with limited success. The net reached a depth of 4315 m before it started to ride back up. And that is where net zero was closed and net 1 opened. Somewhat later the winch operator saw that the level-wind had malfunctioned and that there was a bad wrap on the winch. He had to stop and pay out wire to fix it. The bad wrap turned out to be around 600 MWO. The rest of the haul went fairly smoothly, although there were additional problems with the level wind.

17 April 2006: The trawl came back on board about 0400 on the 17^th of April. Several of the samples from great depths appeared to be contaminated by animals living closer to the sea surface. This is a problem that often goes unnoticed when towing opening/closing nets systems shallower than 1000 m where life forms are abundant. But when sampling the bathypelagic realm where most species occur in very low abundance, even small amounts of contamination from the nets passing through the shallow waters and catching surface forms can be significant. Still the nets caught some important species of zooplankton and fish that had not been caught in previous tows. In addition, a bathypelagic fish was caught that is possibly a new species (see Pelagic Fish report below)

After the trawl was brought on board, two Reeve net and two surface ring net tows were conducted. Although a second trawl was scheduled for Station #2, it was cancelled because of the number of repairs needed to be made to the trawl and trawl wire, and also because the winds and seas were building. Winds in excess of 30 kts were forecast for the area later in the day. Around 0700 on 17 April, the ship started steaming for station #3 approximately 600 nm to the southeast [25°N; 65°W].

While the over-the-side work at Station #2 was trying, the sample collection has provided the biologists with more material from which to work and a number of species were added to the list of those already identified.

After the sampling at station #2 had been completed in the early hours of 17 April, around 0700 the Ron Brown started for station #3 some 600 nm and approximately two days steam to the southeast. It was a time to catch up on the work-up of the samples, review the performance of the sampling gear, and to repair and enhance the 10- m MOCNESS, which had suffered some damage to the nets and towing wire on the last tow.

The weather during the first day of the transit was warm (air and water temperatures about 23 C) and the winds were around 20 kts. In late afternoon, the winds had dropped to around 13 kts and the skies remained nearly cloud free.

In an examination of the second deep 10-m MOCNESS tow, the contamination issue loomed large. For reasons that are not clear, the opening/closing of the nets on this tow did not show any significant angle change, as is usual when one net is closed and the next one opened. A plot of the angle versus time data for both tow #1 and #2, clearly showed the angle change spike associated with the closure of net 1 and opening of net 2, the closure of net two (which also resulted in the closure of net 3 and opening of net 4 [because of the broken tab]) and the closure of net 4. No such spikes were associated with the commands to step the toggle on tow 2. In discussions with Martin Angel, Francesc Pagès, Tracey Sutton, and Dhugal Lindsay, all taxonomic experts on variety of zooplankton and fish groups, only net 1 on tow #2 seemed to be a true bathypelagic catch, albeit with lots of nearer surface species also present in the sample. The other nets had poor catches and few deep-sea animals. In working up the catch from net 2, Martin made the assessment that most of it was from contamination.

In looking carefully at the bars when the system was cocked and the cables were tight, there was a gap between them about 2 to 3 cm tall by 300 cm wide. Although seemingly small, when the area was computed and then multiplied by the length of a tow, a considerable volume of water could have passed through the gap and into the net, even when closed. For tow #2, the ship traveled nearly 33 nm (~61,000 m) and gap volume could have been around 4800 m³. This is huge and could very easily explain the contamination problem. Discussion about how to reduce or eliminate the gap contamination problem took place during the day and a plan was devised to construct some net bar flaps that would hang from each net bar and cover the gap between it and the bar below. After dinner on the 17^th, concerted effort began to perfect the design and then construct the flaps out of a plasticized cloth material that the Bosun provided. Eight panels were needed 147 cm long and 42 cm wide, with two panels per net bar, one on either side of the middle support for the net retaining rods. The first panel was installed and then some additional discussion took place among those concerned with the contamination about the design and possible problems. All thought the flaps would work while the bars were at the top of the frame, but they were skeptical that the flaps would stay outboard of the bars after they were released. In fact they thought they would be folded inside the net and not close the gap. So an idea to put stays made out of the stiff tie-wraps was put forth to keep the flap fairly stiff and even if they folded in, the material would likely still block the opening enough to reduce the contamination. The rest of the evening was spent making up the flaps with tie-wrap stays.

18 April 2006: During the night, the wind picked up and on the morning of the 18^th velocities between 19 to 26 kts were recorded. A front went through the area around 0800 and there was a wind shift from 240° to about 210°. A rain squall line passed through, although there was no rain on the ship. The wind shift put the wind and seas coming in on the starboard side of the vessel, causing the ship motion to increase significantly. This made it difficult for investigators to work on the samples using the microscopes and the lab was a bit empty during the morning.

During the morning Larry Madin, Leo Bercial, and Peter Wiebe installed the net bar flaps that were intended to close the gaps between the net bars and thereby reduce contamination when the nets were closed.

There was some excitement around 1030 on the 18^th when the bridge sighted a small (~15 feet) overturned boat, a RHIB, drifting out in the middle of nowhere (at 26° 53.37'N; 64° 21.73'W). The ship slowed and maneuvered to bring the hull close enough for inspection. As it passed along the starboard side, some large fish were sighted swimming around it. Shortly after, we again picked up speed and headed for Station #3.

In the afternoon, the wind picked up substantially with sustained speeds between 25 and 31 kts from the southwest. With the sea abeam, the water was coming up onto the starboard deck and flooding out into the aft deck area, making working conditions on the deck more difficult.

The issue of the kink in the 0.68" trawl wire was resolved just after lunch as a result of consultations with Jeff Hill (ET), Jonathan Shannahoff (ST), and Bruce Cowden, the Bosun. All indicated that the cable should be re-terminated. Shortly after, the termination was unbolted from the trawl and carried into the staging bay where the wire was cut and the process of removing the stainless steel fitting from the wire and then re-installing it on the new end of the cable was begun. Erich Horgan did most of the work, which was completed around 2200.

During steams between stations, a seminar series of talks had been planned and the first of the series started at 1300 on the 18^th. The first two lectures were by Martin Angel and Leo Bercial. Martin talked about Ostracods and the web site he has constructed to provide students and experts easy access to the literature, keys, illustrations, data about the distribution, abundance and size of some of the 200+ known species of planktonic ostracods in the world’s oceans. Leo talked about the copepod genus, Clausocalanus, that he has been working on. He is developing a genetic method to distinguish easily between species of this genus, a number of which are exceedingly hard to distinguish morphologically.

The second of the safety drills occurred around 1500 and consisted of a ship collision drill and an abandon ship drill. For the latter, people mustered in their alternate sites inside the ship in order to have dry space to put on their survival suits.

Following the drill, the lecture series continued with Nancy Copley describing the Silhouette technique for measuring the abundance, biomass, and size distribution of zooplankton in a sample by taxa.

After dinner there was an intense period of activity to finish setting up the 10-m MOCNESS. A last addition to the frame was a pair of canvas deflector flaps that Larry Madin and Erich Horgan had made a couple of days ago. Holes were drilled in the side I-beam to attach the flaps to the frame. A check of net 1 revealed more extensive damage to the net than had been suspected and this net was replaced by the spare. The nets were then loaded, the cod-ends attached, and nets arranged for launching with all the cod-ends at the bottom of the frame. With all the nets inside the frame, the side deflector flaps were bolted onto the frame with 3/8" cap screws. Finally, grommets were installed in the corners of the deflector flaps and then bungee cord tied to the grommet on one end and a clip on the other. The top and bottom of the flap were attached to the frame with the clips so that the flap would tow streaming around the outside of the frame, thus covering the open portion of the closed nets.

While this was going on, Erich Horgan was finishing the new termination. About 2200 on the 18^th, the cable was attached to the underwater unit on the trawl and tested it with the MOCNESS software. It was able to run with no problem.

The evening came to an end with the winds having died down some to between 15 and 20 kts from the southwest. Sea and air temperature remained the same at 23° C.

19 April 2006: The morning of 19 April started pleasantly with a mix of sun and clouds, an air temperature of 23.5° C, a sea-surface temperature slightly warmer at 24.2° C, and winds between 10 and 12 kts from the southwest.

The Ron Brown arrived at Station #3 about 0630 and work began with a 200 m vertical Reeve net tow to collect fragile near-surface zooplankton and provide investigators with new animals with which to work. Shortly after, a pull test to around 5000 lbs was done to ensure that the new cable termination on the conducting trawl wire was secure. During this operation, a discussion ensued about the first deep tow. It was to have been a 10-m MOCNESS tow to 5000 m, but there was a desire to have a shorter tow in the upper 1000 m, so that the taxonomic specialists could start their work at this station sooner. So the order of the two tows was reversed without remembering that the way the trawl wire was laid on the drum would dictate a much deeper tow at the start of the station.

The setup of the MOC-1 for launch took about 45 minutes and the net system went into the water about 0900. As the net reached the intended maximum depth and retrieval started, a bad wrap on the winch was reported and wire had to be paid out to fix it. In fact, there were bad wraps on a number of the lays and it took paying out almost 5000 m of wire to get to the place where the wire was correctly laid down on the drum. A broken strand in the outer armor was also found at about the place where the wire started developing bad wraps and the loose strand had to be cut and the ends taped securely to prevent more unraveling of the strand. How the strand broke is not known, but it is suspected that it occurred because of wire, under tension, was snapping into gaps left by the level-wind failing to lay the cable evenly on the drum. Because this tow turned into a very deep one, it was decided to do 500 m intervals from 3500 m to the surface with the nets. After adjustments were made to the level-wind, the bosun reported that the level-wind was now working very well and it has done so since. So rather than coming back on board around noon, the tow was now slated to arrive at the surface around sundown.

This was an ill-fated tow, however, for another reason. When the net system arrived at the surface, only a single net bar should have been left to drop. But in fact all were still locked in the release mechanism, except for bar #1, which dropped because the cable/swaged fitting broke about 200 m below the surface (depth determined by a spurious net response at 200 m). So net zero fished down to 3300 m and back to 200 meters and then net 1 fished from 200 m to the surface. The failure of nets to release was because the cables were wrongly mounted into the toggle, so the release commands failed to release the nets. What a learning experience! The problem was magnified by the fact that nearly 5000 m of cable had to be paid out to get the wire on the drum straight. If it had been a normal tow, the problem would have been discovered a lot earlier.

In the late afternoon while the MOC-1 was still coming up, the fire alarm went off and it was not a drill. Ultimately it proved to be a false alarm in a forward area over a tank, but the response was impressive, and all scientists arrived in the main lab muster area in a timely way. An “all clear” was sounded after it was determined that no fire was present.

The early evening was spent doing near-surface (0-200m) ring net tows and 30-liter water collection for microzooplankton. Colomban de Vargas was amazed to find very few planktonic foraminifera in the net tow samples, a situation he has rarely encountered. Other larger species of interest were caught in these tows including the euphausiid, Sylocheiron suhmii, a lovely small transparent shrimp-like animal with elongated eyes with only 3 facets and distinct photophores designed for counter-shading. Once identified, it, along with others, was prepared for gene sequencing.

A night SCUBA dive took place later in the evening (Figure 2) and although the divers reported relatively low abundances of animals, they nonetheless came on board with a good collection of live radiolarians, siphonophores (one with a leptocephalus[eel] larvae being consumed by the gastrozoids), a pyrosome, jellyfish and associated amphipods, and other fragile species that are destroyed in the nets.

fig_02_dscn5398.night_divers_adusted.jpg

Figure 2. Larry Madin, Erich Horgan, Phil Pokorsky, and Keegan Plaskon ready to begin a night-time blue-water dive [Photo by P.Wiebe].

20 April 2006: The first official deep tow of station #3 started around midnight on the 19^th with the deployment of the 10-m MOCNESS under good sea conditions (winds in 10 to 12 kt range). The launch was a bit difficult at the start, but ultimately the frame rolled down into the water fairly smoothly and soon the net was headed down to depth. During the night there was a wind shift and light winds around 5 to 8 kts began from the north (0°). In the early morning it was cloudy with rain squalls in the area and cooler temperatures (20.73° C). It was raining lightly when the MOC-10 tow #3 came on board very nicely around 0930 on 20 April. This tow was also discovered to have problems. In pulling in the nets, it was found that the cod-end from net three had been lost in spite of the fact that the fasteners had been rubber-banded, which has been the standard way to prevent bucket loss. In addition, the tab on net bar #3 that had been fabricated by the engineers again broke, so that the net bar for net 3 dropped when net 2 was closed and it never fished. This opened net four prematurely. Later in the day, the broken tab fixture was repaired by the engineers, who made it more robust. The catch in the rest of the cod-ends, while sparse, proved to have another set of very interesting deeps sea invertebrates and vertebrates. One interesting species was a mysid in the Gnathophausia group. There are several well-known species, but this did not appear to be any of them. In addition, there appeared to be no contamination by animals living shallower in the water column or very little. The modifications made to the system appeared to have worked.

The 1-m MOCNESS was next up. It went into the water about 1130, but at 70 m depth, the deck unit lost connection with the underwater unit and nothing would bring it back. So the system was brought back on board. After a series of tests that determined that the cable was OK, the underwater unit was switched with the one on the MOC-10. The tow was started again about an hour later. This time the underwater unit worked fine and a complete set of samples was obtained in the upper 1000 m. The net came on deck about 1600. Such were the gremlins out there, that when the unit that failed was bench-tested, it worked.

Larry Madin, Erich Horgan, and the two crew divers left the ship about 1630 for the next in a series of blue-water dives, as the afternoon watch processed the MOC-1 samples. While the divers were away from the ship in the zodiac, some surface ring net tows were taken by hand. All went well with the dive and the divers returned with more wonderful animals around 1730.

The last events at station #3 were night 1-m and 10-m MOCNESS tows to 1000 m and 5000 m respectively, and a ring-net tow to 200 m. All of these tows were accomplished successfully. The 1–m system was towed early in the evening followed by the ring net tow. The 10-m system, which was deployed at midnight, came up at 1130 on the 21^st. This time all the nets fished their intended depths (5000 to 4000, 4000 to 3000, 3000 to 2000, and 2000 to 1000 meters) and the samples showed little or no contamination. This was a perfect ending to a station that started off poorly.

The Ron Brown got underway for station #4 just after noon on the 21^st of April in light winds, calm seas, warm air temperatures, and clear skies. Because diving conditions were extremely good and the animal collections were going very well with this technique, a blue-water dive was scheduled for 1330. The divers returned about 1500 and reported that while animals were sparse, they again collected some interesting radiolarians and siphonophores. The remainder of 21 April was spent steaming under very nice sea conditions. The work on board in the laboratories continued unabated.

22 April 2006: The steam to Station #4 (20° N; 55°W) took about 32 hours under mostly clear skies with only a few clouds. In the early morning of 22 April, winds 10 to 15 kts were from the northeast, the sea surface temperature (SST) was 25.56° C, and the air temperature was slightly cooler (24.1° C).

Throughout the day, the investigators worked in the main lab identifying zooplankton and in the sequencing lab they continued to prepare and sequence identified species. During the afternoon of the 23^th, the second session in the seminar series was held with Larry Madin, Hege Hansen, and Tracey Sutton giving the talks. Larry talked about the Liquid Jungle Laboratory, which is a new tropical laboratory for marine and terrestrial research on the Pacific side of Panama. There is a shore-side wet lab in addition to lab space in the main building up on an island hillside, small boats for access to the coastal waters, and a newly installed cabled underwater observatory just offshore. Hege talked about the deep-sea shrimps that were collected on the Mar-Eco cruise to the mid-Atlantic ridge on the R/V GeoSars in summer 2004. She compared the species caught on that cruise with those collected on this one. So far she has found only one species additional to those collected on the ridge cruise and only a single northern species is currently lacking from our current collections. Tracey gave an overview of the groups of deep-sea fish that exist and talked about those that he was finding in our samples. He has found several rare species and one or two that are probably novel. The talks were split into two sessions because the ship’s personnel had a safety meeting at 1415 that went until about 1515.

After dinner, the 10-m MOCNESS was made ready for the next watch to launch and tow. The cod-end buckets were attached to the nets and the nets arranged so that they could be deployed off the stern easily. In addition, some repairs to the newly fashioned trawl deflector flaps were made.

23 April 2006: We arrived at Station #4 about 0035 on 23 April and started the work with a vertical Reeve net tow to 200 m. This was followed by a 10-m MOCNESS tow. The bottom of the tow was at 4500 m instead of 5000 m because of the very rough topography in the area. The SeaBeam bathymetry data showed that there were substantial ridges and valleys in the area and our tow line cut across them. The broken strand of 0.68" conducting cable at about 4700 m that was taped after the last tow, came loose after the tape had worn off going through the traction winch and had to be re-taped when it came past that spot going out and coming back in, but this did not interfere with the haul. The system came up and on board at 1245. As the nets were being hauled in, the catch in the buckets were initially examined. There was a spontaneous “OOOOH!!” as a large dragonfish was found in the bucket of net 4 (2000-1000 m). After examining it carefully in the laboratory Tracey Sutton concluded it was probably a new species, and might even represent a new genus. The catch also contained some lovely large red prawns. Not only was this an excitingly spectacular haul, but also once again the nets had fished properly.

A daylight blue-water dive took place in the early afternoon under light winds (~8 kts) from the northeast and sunny skies. The divers returned with only a few animals. The dominant organisms were phytoplankton, the nitrogen fixer Trichodesmium and mats of Rhizoselenia. The zooplankton were sparse.

While the divers were out, the 1-m MOCNESS was set up for a tow. The cable termination was changed from the MOC-10 to the MOC-1 and then the underwater unit moved to the MOC-1. About 1520, after a deck check to make sure the electronics and sensors were operational, the net entered the water and the tow began. This tow went fine, except that a collar came off of net 3 and the catch was lost along with the bucket.

Russ Hopcroft and Barbara Costas did a series of net tows and water collection casts, while the 1-m MOCNESS was prepared for a second evening tow. The net system went in smoothly from its position within the back of the MOC-10 frame. The tow, which came on board around 0130 on the 24^th, concluded successfully with more interesting animals in the catch. These included a small tropical squid in the surface sample that Dhugal Lindsay knew existed, but had not seen before.

24 April 2006: In the wee hours of 24 April, the divers went out in the RHIB for a night dive in waters that were now around 26.5° C. Again the take was sparse, reflecting the oligotrophic nature of the station area, but they did collect two very interesting delicate ctenophores, Beroe mitrata, and Thalassocalyce inconstans. Larry Madin and Richard Harbison discovered and named the latter species some time ago. The genus means “cup of the sea” after its shape and inconstans because it is in constant motion. Both individuals were still alive in the evening of the 24th and were the subject of observation and photography, prior to being submitted for sequencing.

A 1/4-m MOCNESS tow was scheduled as the last item to be done at Station #4, but the underwater unit again proved to be unreliable on the deck and so the tow was scrubbed. The ship got underway for the last station (#5) about 0400.

Steaming during the day again gave the investigators an opportunity to catch up on the laboratory investigations of the samples collected at the previous station. There was also a concerted effort to update the CMarZ web site and to add additional photos of people and gear at work on the cruise. It was pleasant steaming weather with sunny skies sprinkled with puffy clouds, winds from the east (90°) at about 15 kts giving rise to some white caps and choppy seas on top of an underlying swell. Both the sea and air temperature was around 26° C.

At 1400 on the 24^th, a subset of the scientific party (Martin Angel, Larry Madin, Rob Jennings, Russ Hopcroft, Tracey Sutton, and Peter Wiebe) met in the Chief Scientist’s cabin to take part in a press conference call organized by Fred Gorell of NOAA. In preparation for the call, two photos were put on the web of two of the first zooplankton (the copepod, Paraeucalanus attenuatus and the pteropod, Clio pyramidata) collected on the cruise that were in the first group sequenced at sea. In addition, a photo of Paola Batta Lona “operating” the sequencer was also posted on the web. Reporters on the call included Christina Reed, a freelance science writer, Peter Spotts of the Christian Science Monitor, Warren Wise of the Charleston (SC) Post & Courier, and Noel Anenberg, who writes a children's / educational series for LA Times. In addition, there were several others taking part in the conference call including Ann Bucklin, the CMarZ lead investigator and a co-PI on this OE project, responsible for the shipboard sequencing activities. The conference call lasted about 75 minutes and covered the overall rationale for CMarZ and the cruise objectives. Questions from the reporters then framed the remarks made by our group of scientists about what we had already learned and how the sequencing information would ultimately be used. The session was tape-recorded by NOAA.gov and would be used for production of a story to be podcast. Coincident with the press conference was the third and last Fire and Emergency Drill, from which the participants were excused.

A schedule of MOCNESS tows, blue-water dives, and other net tows and water collections was prepared in the late afternoon for the first day and a half at Station #5. The early evening was spent setting up the 1-m MOCNESS for a tow after the ship arrived on station the next morning.

Then late in the evening around 2300, the third in the seminar series took place. This late hour was chosen because this was when most of the scientists from both watches, i.e. the midnight to noon and noon to midnight, were awake. With the last station quickly approaching and the end of the cruise in sight, there was an increased impetus to make the most of the time remaining. Barbara Costas provided an introduction to the ciliates and in particular the groups that include tintinnids, oligotrichs, and other choreotrichs. She described her work on tintinnids (small ciliate microzooplankton) and the difficulties involved in using the classical methods to preserve and identify them . She has been developing molecular methods to determine species identities, and has found that a number of forms previously described as distinct species appear to have very similar genetics and may not be separate species. A library of ciliate genetics is being built and is at the point in some areas where bulk DNA analysis can be used to track the presence and perhaps the abundance of the different species. Rob Jennings described the steps carried out by Team DNA in the processing of the animals in the sequencing lab. This involved extracting the DNA, amplifying it, running the sequencing reactions, and then running the product on the sequencer. He described how to resolve the data from noise in the reaction by sequencing a forward strand and a reverse strand. Once the sequence has been finalized, the next step was to compare it to known sequences in the bank of sequences to see if the sequence matches one for a known species. Since the cruise began, about 500 species have been submitted to the lab for sequencing and there have been 775 specimen extractions. [Note: These numbers have gone up since station #5 was not included in the estimates]. Russ Hopcroft gave a tutorial on larvacean diversity and ecology. Like the tintinnids, the larvaceans are extremely fragile animals and are difficult to collect intact. They make an elaborate external feeding structure that they use and then discard when it gets clogged up. This may happen as often as 14 times per day. Russ described the house structure, which is unique to each species and therefore can be used to identify the species. There are 3 families, 15 genera, and 69 known species. On this cruise, he has collected fewer larvaceans than expected.

25 April 2006: The Ron Brown arrived at Station #5 around 0830 on 25 April with the decks wet from some early morning rain showers. Unlike the previous few days, the skies were cloud covered. Still the winds were light (5 to 8 kts from the east southeast) and the air warm (25.9° C) and humid. The sea surface temperature was the warmest experienced on this cruise (27.147° C).

Almost immediately there was a net in the water. Russ Hopcroft deployed his Reeve net for a tow to collect larvaceans. In this tow were several larger species of larvaceans that he was expecting to encounter, but had not done so until now. The 1-m MOCNESS went in about 0915.

In the area of station 5, there are physical structures in the water column between 200 and 500 m known as “thermohaline staircases”. When a plot of temperature or salinity versus depth is made, distinct steps in the profile are visible wherein there are zones of ten meters or more of isothermal and isohaline water and then narrow transition zones where both temperature and salinity change abruptly until there is another step. The zones of constant temperature and salinity are active mixing zones as demonstrated by work that Ray Schmitt and colleagues had done in this area some years ago. The staircase structure might also be an area of unique biology, but this has not been previously studied. Knowing that there was the possibility that the staircase structure might be present, we looked carefully at the data from the first tow as it was in progress. The tow went smoothly and during the downcast a plot of the temperature and salinity structure showed that indeed there was a staircase structure in the same depth zone that Schmitt had observed in 1985.

At the end of the tow, while the net was approaching 25 m depth, the winch overheated and shut down. It took a relatively short time for the engineers to investigate the problem and to start the winch operating again. The last net then sampled the upper 25 meters and the net came on board about 1238.

The divers went in around 1300 with winds in the 12 to 15 kts range. They came back with a real beauty of a collection of jellyfish, siphonophores, and salps. This station was very different from the previous two because of the rich surface life they encountered.

In the late afternoon, ring net towing primarily for larvaceans and foraminifera, and water collection for microzooplankton (tintinnids) were carried out. At dusk, the second 1-m MOCNESS was started. This tow was very successful. All the nets opened and closed where intended and the catches were quite good. There were no problems with the winch this time. Once the 1-m net was secured, the cable termination and underwater unit from the 1-m system were moved to the MOC-10, the buckets installed, and the nets arranged for deployment. This took about an hour. By 2200 the MOC-10 was heading down to depth.

26 April 2006: Some twelve hours later at 1000 on 26 April, the trawl re-appeared at the surface. It had caught a wonderful assortment of animals, especially fish. Tracey Sutton was particularly pleased. Martin Angel was busy trying to increase his inventory of ostracod species and exceed his previous inventory compiled for the Eastern Atlantic. Already at the start of this station he had identified nearly a third of the known species of planktonic ostracods. Net one, the first to be fished as the system returned from 5000 m to the surface, was ripped up a bit on the starboard side and a support rope was ripped off the seam to which it had been attached. It is a mystery how this happened.

Shortly after, the 1/4- m MOCNESS was returned to service by using an old 16-bit electronics unit that was brought on the cruise in case other units failed to operate properly. This tow to 500 m went OK, except that the flowmeter stopped working during the net system’s return to the surface. Water filtered for some nets will thus have to be calculated by time the net was open and distance traveled These samples caught with very fine mesh nets (64 um) were primarily used by those investigators interested in microzooplankton.

The second 10-m MOCNESS tow of station #5 started in the early afternoon under fair skies with winds a steady 12 to 15 kts from the east northeast (82°) and tropical air (26.22° C) and water (27.19° C) temperatures. Around 1900, the MOC-10 reached within 200 m of the bottom (~5200 m) with more than 7000 meters of wire out. The rest of the evening was spent flying the MOC-10 up from 5000 m. The tow took longer than expected. The net came up too fast on its own accord to haul wire in quickly, so a good portion of the tow was spent coming in at 10 or 15 m/min.. The first net fished from 5000 to 4000 m. On this last MOC-10 tow, it was decided to use the last net to fish the upper 1000 m and use the other three to cover the 5000 to 1000 m range. The group was really interested to see what large-ish animals might be captured in the near-surface zone with the big fine-meshed nets.

27 April 2006: At midnight on the 26^th, when the science watch changed, Larry Madin came in to take over the “flying” of the MOC-10. There was some discussion about whether the nets had opened and closed where intended because no significant angle change had been observed when the net system was sent the command to close one net and open the next. When the net system finally came on board about 0330 on the 27^th, the concern disappeared. All evidence suggested that the nets opened and closed where intended and the catches were pretty spectacular. Contamination was minimal. Francesc Pagès was excited because previously he had been catching a particular transparent jellyfish about 5 cm in diameter in the 1-m MOCNESS collections that had very few distinguishing characteristics and was an undescribed species as far as he could tell. In the 1000 to 0 net, a much larger individual was caught and it had characteristics that he could now use to make a description. Also Dhugal Lindsay was happy with the squid collection. Several species that had not been caught earlier were in the sample including a Vampyroteuthys. Martin Angel also found specimens of a species that he had been looking for. In addition, Russ Hopcroft gave Martin another ostracod not yet seen on the cruise from a Reeve net collection made soon after the trawl was on board (from 0415 to 0445). So the last station was ending with a flourish.

The 1/4-m MOCNESS tow began around 0600, under cloudy conditions and winds 13 to 18 kts from the east. A few rain squalls moved through the area. This tow was used to sample a special series of depths for Colomban de Vargas and Yurika Ujiie to look at how the vertical salinity and temperature structure in the upper 300 m might be affecting the foraminifera. This targeted depth sampling was based on the information from the down trace and from earlier tows. The 1/4-m tow came back on board about 0900 with samples that were a bit disappointing to Colomban because the catches were fairly sparse and there were few foraminifera.

The last tow of the cruise was made with the 1-m system. It was targeted at the staircase structure mentioned above, which showed prominently on every tow at this station that went below 300 m (Figure 3). Nets were opened and closed so that one fished in an isothermal/isohaline (mixed) zone and then the next in the interface between it and the mixed zone just above. We successfully fished 4 mixed zones and 4 transition regions starting about 550 m below the surface and ending about 400 m.

Over the last three days, Team DNA received more identified specimens for sequencing and also a few more unidentified forms that might be undescribed. The total number of identified specimens in the bank topped 1000. More than 400 sequences had been run and it was anticipated that there would be more than 100 good species sequences by the end of the cruise. Many of the other sequences may turn out to be usable or may need more work to make them good. The nature of the sequence would determine what additional steps might need to be taken, like running an additional PCR under different conditions to optimize the sequence reaction. There is more work to sequencing than simply extracting the DNA, amplifying it, and then running it through the sequencer to get the sequence. Having to repeat steps with different conditions is typical.

With the last over-the-side sampling completed around 1500 on the 27^th of April, the R/V Ron Brown set sail for San Juan, Puerto Rico.

28/29 April 2006: During the two day trip to San Juan, the activity of the science party focused on packing up all the gear and getting it ready for off-loading and shipping on the day the ship reached port. In addition, the investigators wrote up sections of the cruise report.

At 1030 on the 28th, there was a conference call with CoML media relations lead, Terry Collins. Included in the call were Martin Angel, Larry Madin, Rob Jennings, Tracey Sutton, Russ Hopcroft, and Peter Wiebe. Also Ann Bucklin and Fred Gorell were present from a distance. Discussion centered on what was going to be the focus of the CoML press release about the cruise and its findings.

During the morning of the 29^th (around 0900), there was a final 45 minute blue-water dive involving all of the authorized divers on board to inspect the hull of the Ron Brown. Later in the evening after we arrived back in the US EEZ a net tow was taken to obtain live zooplankton for video imaging by Russ Hopcroft to be used as part of the press material package on the CoML web site.

The 20' shipping container van on the ship was loaded by mid-afternoon on the 29^th of April and the remaining gear was assembled in totes and boxes for off-loading and packing in a second van that was to be on the Coast Guard dock in San Juan.

30 April 2006: The R/V Ron Brown entered San Juan, Puerto Rico harbor about 0800 on 30 April. With the tying of the lines on the Coast Guard dock in the Old Town area of San Juan, this CMarZ cruise came to an end. Off-loading of the science gear began shortly after the ship was tied up. By noon, the second van was loaded and ready for shipment back to Woods Hole, Ma.

This CMarZ cruise was a remarkable expedition that brought together classical taxonomists and gene sequencing experts to collaborate at sea and produce impressive results in just three weeks. Zooplankton and fish samples were obtained from 5000 m to the surface at five stations distributed from 33.5 N to 14 N in the western North Atlantic Ocean. From these samples, the investigators identified between 500 and a 1000 species, and they provided more than a 1000 specimens to the DNA lab on board the ship for sequencing. For several taxonomic groups, a significant fraction of the known species were collected and identified. For example, 65 species of ostracod were identified by Martin Angel, representing nearly half of all 140 known ostracod species in the North Atlantic Ocean. Six of the ostracod species are not yet described in scientific literature. Nearly all of them were submitted for sequencing and the first DNA barcode for a planktonic ostracod was obtained on this cruise. More than 40 species of molluscs (pteropods, heteropods, etc.) were identified and more than 100 species of jellyfish, several of which may be undescribed. Several hundred species of copepods were identified and more than 100 species of fish, many rarely caught, and two of which may be undescribed. In addition, several groups brought photographic equipment on the cruise that enabled hundreds of high resolution digital photographs to be made of many of the zooplankton species identified and submitted for sequencing. Russ Hopcroft in particular made many photos that were put up on the web site during the cruise.

Having a gene sequencer and associated DNA laboratory equipment and personnel on board made it possible for a level of interaction between taxonomic specialists and molecular biologists that is seldom achieved in any other setting. The high productivity in terms of the identification and sequencing of known and unidentified specimens was the result of the very positive interactions that occurred while at sea. In spite of the difficulties encountered in MOCNESS sampling, the goals of the cruise were met and overall results were successful.

Zooplankton and micronekton were quantitatively sampled throughout the water column using a 1/4-m, a 1-m, and a 10-m MOCNESS (Multiple Opening/Closing Net and Environmental Sensing System; Wiebe et al., 1985; Figure 4). The MOCNESS telemetered data continuously to the ship, including depth, temperature, salinity, horizontal speed, and volume filtered. This allowed on-the-fly adjustment of sampling depths or times, and completion of a continuous series of stratified hauls in a relatively short time. All data were recorded electronically for subsequent analysis.

The MOC-10 carried 5 separate nets; the mesh size of the nets was a combination of 3 mm and 335 µm mesh. Net 0 had 3 mm mesh and nets 1 to 4 had 335 µm mesh nets of special design that were fabricated for this cruise. In addition, during the cruise, deflector side flaps and net bar flaps were constructed to prevent contamination of the deep samples from plankton in other strata, especially those closer to the surface (Figure 4). The MOC-10 was launched, towed, and recovered through a stern A-frame with the ship maintaining a speed of 1.5 to 2.5 kts. The trawl was deployed with the first net open (3mm mesh) down to the deepest depth desired, normally 5000 m. It was closed at that point, and subsequent nets (335 um) were opened at desired depths as the trawl was hauled obliquely toward the surface. Thus, one MOC-10 net sampled from the surface to the bottom and the other nets normally sampled ~1000 m intervals from the bottom up to a depth of 1000 m (Figure 5).

Above 1000 m, vertically-stratified sampling was done using a 1-m MOCNESS equipped with 9 nets with 335 µm mesh. In addition, a ¼-m MOCNESS with 0.64 µm mesh was used to collect foraminifera and other micro-zooplankton in the upper 500 m (Figure 5). The use of the large trawl below 1000 m enabled large volumes of water to be sampled (tens of thousands of cubic meters) to compensate for the very low abundance of species that occur at bathy- and abyssopelagic depths. The smaller 1-m and 1/4- m MOCNESS’s provided adequate sample sizes in the upper 1000 m.

Several other nets were used for surface or near surface zooplankton collections. A Reeve Net consisting of a ½-m ring net attached to a large-volume cod-end was used to collect fragile gelatinous animals and microzooplankton in the upper few hundred meters. These tows were made opportunistically. Other very fine mesh (5µ and 10µ) ring nets deployed by hand were used to collect microzooplankton during periods when the blue-water dives were taking place.

Collection of living or intact specimens of gelatinous zooplankton is difficult with nets or trawls because the organisms are usually damaged and sometimes destroyed. During the last 30 years, the technique of blue-water diving to make observations and collections of these fragile animals by SCUBA has been developed and this technique was used on this cruise. A group of (usually) 4 divers worked from a small inflatable boat launched from the ship. They were connected by 10 m long tether lines to a central line hanging down from the inflatable and manned by a safety-diver who watched over the others. Each diver moved about within a 10 m radius to locate, observe, and collect free-swimming gelatinous animals. The technique was only semi-quantitative, but allowed collection of live and undamaged specimens, as well as in-situ photos of behavior. Organisms were collected in simple wide-mouth jars and returned to the ship for further study. The same technique was used at night, with the addition of underwater flashlights or headlamps. During this cruise a day and a night dive was planned for each station.

A thirty-liter Niskin bottle was used to collect water for tintinnid analysis and for use with other work with microplankton. The depths selected for sampling were generally based on the water column temperature and salinity structure.

An idealized scheme for sampling at each station was approximated that enabled replicate tows with each MOCNESS to be made during an approximately 48 hr period (Figure 6). MOC-10 tows generally took 10 to 12 hours to complete, MOC-1 tows took about 3.5 hours, and MOC-1/4 tows took about 2 hours. In addition, a 2-hour time block was allocated for two blue-water dives. Not shown on this scheme was time for opportunistic sampling with ring nets or water collection with the Niskin bottle. In reality, neither replicate samples with all net systems nor blue-water dives were obtained at all the stations, because of time and weather limitations, and gear malfunctions.

Samples collected with the MOCNESS’s were processed using a standard protocol (Figure 7).

On Deck: With completion of the tow, the nets were immediately washed with seawater as they were pulled on deck and the plankton still in the nets carefully moved into the cod-end. The cod-ends were placed in buckets with ice packs to cool the samples and moved expeditiously into the walk-in cold room to await analysis.

Specimen removal: One by one the cod-ends were taken into the wet lab for digital photographing, and the picking and removal of large individuals of 1) gelatinous forms, 2) fish, and 3) macrozooplankton/nekton. Pickers described what was being removed and a recorder logged the information. The specimens removed were placed in numbered jars, shell vials, or dishes and the recorder wrote down all specimen information on the data sheets provided, linking the container number to specimen and collection data. This was done so that the actual taxonomic composition and species count for each sample can be reconstructed. The removed specimens were subject to a variety of procedures including further identification, dissection, preservation (in alcohol, frozen nitrogen, or formalin as appropriate), or taken for photographic imaging prior to preservation.

Sample splitting and preservation: Within a few minutes of arrival, the stratified samples (with most large gelatinous forms, fish, and macrozooplankton/nekton removed) were passed to the individuals responsible for splitting the samples (Figure 7). Generally ½ (split A) was preserved in formalin for future studies, including biomass estimates (e.g., displacement volume), species counts, and other quantitative analyses. The other half was split again with ¼ (split B) for live picking in the main lab and subsequent preservaton in alcohol for later taxonomic analysis. The other ¼ (split C) was immediately preserved in alcohol. After picking, the integrated sample (net 0) was generally split into two halves with one preserved in alcohol and the other in formalin. Picking of foraminifera from the live split took a long time, so this fraction was kept in the the cold room and often not preserved in alcohol for several hours. Condition of these samples is questionable.

Figure 7. Schematic drawing of the protocol for processing zooplankton
samples on the CMarZ cruise.

Sample analyses: Species were identifiedby the taxonomic experts on board. Several individuals of each identified species were placed in a labeled vial and submitted to the DNA lab for at-sea DNA extraction, PCR amplification of target genes, and sequencing (with a few specimens retained as vouchers). Samples and specimen numbers were entered into the CMarZ Specimen Log, an ACCESS database. Representative specimens of many species were digitally photographed before preservation and some were photographed after preservation.

Specimens of the skeletonized protists were recovered from epi- and mesopelagic samples using simple decantation processes. The taxa of interest were manually sorted under a dissecting microscope immediately after collection. The isolated cells were cleaned in filtered sea-water using micro-brushes, and put into individual tubes containing 100 μl of GITC buffer. This buffer has been developed in the de Vargas laboratory and allows extracting the nucleic acids from the organisms while preserving their micro-shell. The material was stored at -20°C before further analyses. Total DNA was extracted according to protocols developed by de Vargas et al.

The Station locations (Figure 1), which ranged from the Northern Sargasso Sea to the tropical waters east of the Windward Islands, provided contrasting physical settings for the zooplankton collections. In the Northern Sargasso Sea, the “eighteen degree water” was present from near the surface (~40 m) to more than 400 m deep with only a shallow mixed layer of slightly warmer water at the surface (Figure 8). The main thermocline and halocline occurred between 500 and 1000 m below which temperatures gradually decreased from around 5° C to below 3° C at 5000 m. At station 2, a similar structure was present, although the surface layer was warmer (~20° C) and there was a distinct gradient in temperature and salinity in the “eighteen degree water” zone. The upper water column T/S properties at station 3 were distinctly different with lower salinity water at the surface increasing to a maximum around 100 m, and a much warmer surface temperature (~24° C). In addition, the zone of nearly isothermal and isohaline water seen at the northern stations was no longer present. Instead, below 100 m there was a steady decrease in temperature and salinity to about 900 to 1000 m and then a more gradual decrease in temperature. Salinity reached a minimum at about 900 m and then increased down to about 1200 m before gradually decreasing to 5000 m.

The pattern of low salinity at the surface, a peak at ~100 to 140 m, a minimum at 800 to 1000 m, a secondary maximum around 1400 m, and then a gradual decrease to the sea floor was observed at stations 4 and 5 in increasingly exaggerated form (Figure 8). Sea surface temperature also increased to the south with Station 5 having surface temperatures around 27° C. In addition at station 5, the staircase formations between 250 and 550 m were present as described in the narrative (Figure 3). An explanation for the origin of the water seen in the area sampled at stations 3 to 5 was provided by Ray Schmitt (WHOI) while we were at sea.

“The low salinity water at the surface originates in near equatorial latitudes, where rainfall exceeds evaporation under the Intertropical Convergence Zone (ITCZ). Basically it’s the water that evaporated under the trades coming back down. The Amazon outflow is also important in this freshwater supply. ... The salinity maximum at ~150 m depth is coming from the Northeast. It is formed from the surface waters that have experienced high evaporation under the trades and been transported to the north in the surface Ekman layer. There is a region at ~25 N in the eastern Atlantic where the salinity maximum is at the surface, this water is subducted and carried southwest in the gyre circulation to [this area]. Georg Wust called it the Subtropical Under-Water (SUW). Farther down at about 800 m depth, [there is] the salinity minimum associated with the Antarctic Intermediate Water (AAIW). It’s coming from the south, as part of the thermohaline circulation. Freshness originates from precipitation in the Southern Ocean.

So it’s a layer cake with surface freshness from the South, SUW from the North and AAIW from the South. The salinity gradient in the main thermocline between the SUW at ~37.2 and the AAIW at ~34.8 may be the strongest large scale unstable salt gradient in the world. And thus its propensity for forming these salt finger staircases.”

Blue-water, or tethered open-water, diving is a simple technique that allows observation, photography, and collection of undamaged live zooplankton, particularly larger gelatinous forms that are commonly damaged or destroyed in nets. During the CMARZ cruise, 8 dives were made by Larry Madin and Erich Horgan, assisted by RV Brown crew members, Lt. Liz Jones, Ens. James Brinkley and 1st Asst. Engineer Keegan Plaskon. Dives were supported by the RV Brown’s RHIB workboat, driven by Phil Pokorsky. We made at least one dive at each station, with 5 during daylight and 3 at night (Table 1).

On this cruise the main dive objective was to collect gelatinous animals for identification and DNA bar-code sequencing, as these forms are less likely to be represented in net collections. During the dives we collected approximately 260 individuals of 42 species (Appendix 2). This included 4 species of medusae, 13 of siphonophores, 6 of ctenophores, 7 of molluscs, 10 of thaliaceans, and a few others. In the oligotrophic waters of Stations 4 and 5, we encountered numerous colonial radiolarians and Trichodesmium. In general the abundance of large macrozooplankton on these dives was fairly low, consistent with the low abundances of other zooplankton sampled with the nets.

Some notable findings included two specimens of the epi- and mesopelagic ctenophore Thalassocalyce inconstans, a specimen of the siphonophore Halistemma cupulifera that had caught a leptocephalus (eel) larva, and a surprisingly high diversity of both siphonophores and Thaliaceans. Collected specimens were examined for taxonomic identification, morphological study and behavior, and then added to the group to be sequenced.

Collections of Thaliacea (salps, doliolids, pyrosomes) were rather sparse on this track, although 17 species were obtained either in the net tows, or more often, from the dives.

Most of these were in the shallower nets, and were only found in small numbers (1 to 4 of each).

All salps were identified and measured, and good specimens (mainly from dives) examined and photographed for anatomical details. These descriptions will become part of the detailed morphological information which will accompany the genetic data. A parallel project to develop a morphological and molecular phylogeny of the Thaliacea will be carried out by Madin and Bucklin over the next two years.

Station 1	Station 2	Station 3	Station 4	Station 5
B. rostrata	C. polae	C. polae	B. rostrata	C. polae
C. polae	I. zonaria	P. bicaudata	D. denticulatum	H. virgula
I. zonaria	S. aspera	P. confoederata		I. zonaria
S. aspera	S. fusiformis	S. aspera		P. confoederata
S. cylindrica	D. gegenbauri	S. fusiformis		S. fusiformis
S. fusiformis	D. denticulatum	S. maxima		S. maxima
T. democratica	Doliopsis sp.	D. gegenbauri		T. democratica
D. gegenbauri		Doliolina sp.		D. denticulatum
		D. denticulatum
		P. atlanticum
		Pyrosomella sp.

All species found at the 5 stations can be considered warm-water cosmopolitan forms. Although Station 3 appears to have the greatest diversity and Station 4 the least, the sample sizes are probably much too small to draw any real conclusions.

Our main goal was to increase knowledge of the species composition and vertical distribution of planktonic cnidarians, mostly below 1000 metres depth. About 110 species have been collected, namely siphonophores (70 species), hydroidomedusae (30 species) and scyphomedusae (10 species). About 5 medusae have not been properly identified (nicknamed as Red Ball, Pegantha 24, Pegantha M, White Nausithoe, and Transparent Nausithoe) and some could be new species. One of the nicest and more interesting specimens is an epipelagic narcomedusan of the genus Pegantha (P. 24), whose 2 otoporpae (lines of cnidocysts) per marginal lappet bifurcate ending in 4 statocysts (sensorial organs), a splitting never before reported in any other narcomedusan.

The blue-water dives led by Larry Madin and Erich Horgan collected valuable specimens of rare and little-known species like the recently described siphonophore Lilyopsis fluoracantha (Haddock, Dunn and Pugh, 2005), but most of the specimens belong to species described a long time ago, such as Athorybia rosacea (Forskal, 1775), Rosacea cymbiformis (Chiaje, 1822), and Lilyopsis rosea (Chun, 1885).

The Ring net and the Reeve net proved to be excellent devices for collecting small-sized planktonic cnidarians. In particular, the examination of some early stages of hydromedusae that were collected will shed further light on the growth, development, and morphology of some multispecific genera like Podocoryna and Bougainvillia.

The MOCNESS-1, towed in the top 1000m, collected the greater portion of the specimens examined. Many species widely distributed in epipelagic warm waters were obtained, as well as some specimens of little-known mesopelagic siphonophores like Frillagalma vityazi Daniel and Halistemma sp.

Below 1000 m depth, the MOCNESS-10 collected several little-known species such as the siphonophores Nectadamas richardi (Pugh, 1992) and Lensia quadriculata (Pagès, Flood and Youngbluth, 2006). Likewise some specimens of the genera Apolemia, Erenna, and Bargmannia were also captured, but their specific identification require additional microscopic analyses.

It is too soon to estimate the amount of information obtained in comparison with the sampling effort spent. However, in our opinion, the number of species found is lower than was expected, especially below 1000 m depth where the number of specimens caught was very low, suggesting a rather sparse macro- and megaplanktonic community of gelatinous organisms. This is only partly due to the use of nets for the sampling, since results from other oceans (e.g. Southern Ocean) using similar devices have collected higher gelatinous biomasses. It is interesting to note that very few specimens of the scyphomedusan genera Atolla and Periphylla were collected. Both are common dwellers in the meso- and bathypelagic waters of the oceans.

Preliminary results from the so-called “Staircase Tow” (MOCNESS-1 Tow 13) carried out on 27 April at station 5 suggest some kind of species stratification along the hydrographic staircase. Twenty species of gelatinous zooplankton were collected, namely 13 siphonophores, 3 hydroidomedusae, 3 ctenophores and 1 salp. Only the siphonophore Abylopsis tetragona -a fast swimming species- occurred in the eight ranges sampled between 547 and 461 m depth. Two siphonophores (Lensia ajax and L. exeter) occurred frequently, but irregularly between net 1 (547-527 m depth) and net 8 (464-461 m depth). An increase in the number of species was observed in even-numbered nets (2, 4 and 8) where several species occurred only at single depth ranges. Net 8 collected the three dominant species plus 3 new ones, indicating the beginning of a shift towards more epipelagic species composition.

Ctenophore forms that were identified from net samples included several cydippids belonging to the Haeckeliidae (Aulacoctena acuminata), the Bathyctenidae (Bathyctena chuni), the Pleurobrachiidae (Hormiphora palmata, Pleurobrachia sp.1), the Mertensiidae (Charistephane fugiens), and others. Lobates such as Kiyohimea usagi and Ocyropsis maculata maculata, the Cestoid Cestum veneris, and several Beroe species were also caught in net tows. Blue water diving allowed the collection of several individuals of the lobate Eurhamphaena vexilligera and the Thalassocalycid Thalassocalyce inconstans, in addition to some of the forms listed above.

Twelve species of amphipods were sorted from the live samples, most of them large Physocephalata that were easy to spot. Many more species will undoubtedly be found upon examination of the formalin and ethanol-preserved samples. More than half of the gelatinous organisms captured during the blue-water SCUBA dives were host to hyperiid amphipods at varying stages of development. In many cases it was impossible to identify the hyperiid embryos to species or indeed genus level due to their early developmental stages. Individuals were extracted from the canals or gelatinous matrix of their hosts and placed in ethanol for sequencing. This should allow determination of any species specificity in host/parasite relationships as a factor contributing to species diversity maintenance mechanisms in the pelagic zone. The paucity of species belonging to the Physosomata at meso- and bathypelagic depths may have been a consequence of the dominance of these ecosystems by small siphonophores rather than the larger cnidarians that usually host these animals. Submersible dives should be conducted in this area to directly assess the types and numbers of large ctenophore and cnidarian forms to compare with the data gained on amphipods by net systems such as the MOCNESS.