Interim Report : Pilot-Scale Phase (PSP) of Diadema Aquaculture Research POR December 23, 2010, by Dave Vaughan

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1 Interim Report : Pilot-Scale Phase (PSP) of Diadema Aquaculture Research POR December 23, 2010, by Dave Vaughan The Diadema Project was developed as a two pronged approach for the restoration of Diadema antillarim, as a keystone herbivore on to the reef. The two pronged approach included a land based culture section and a field based restoration section. The land based culture section was organized at three levels of size and development, consisting of: Small Scale Experimental (SSP); Pilot-Scale Production (PSP); and Large Scale Production (LSP). The scope of work of this project proposal (POR ), was to transition with the transfer of the accomplished procedures and information from the small-scale level experiments into the pilot-scale level of culture production. This Scope of Work is being accomplished through the initiation of the pilot scale culture runs, refining research that was accomplished with the Small Scale Experimetnal runs with broodstock conditioning, spawning, and larval rearing through metamorphosis. This program section has been continuing with focus on critical issues of problem areas that need refinement. The major role also is the transfer of that technology and information through communications and exchanging of information with staff. This has been done through all hatching runs this year. First there has been an inclusion of the field based restoration staff from the FWC (Gabe and Whitney), that have assisted in all of the larval cycles and obtained hands-on experience. Second, Dave Vaughan (Mote), from the PSP section has kept involved with telephone, and website communications as well as in-person visits during that time. A big improvement in communication and technology transfer is the establishment of a web base station specifically for the Diadema project ( This Basecamp website coordinates all of the files, pictures, reports and all communications with the project partners. This web base station allows for all participants in the Diadema user group to access all of the reports, s, pictures and discussions on-line and archived. This has allowed for discussions and exchange of information during all larval runs and by all affiliated with the project. This website included preparation of Diadema Protocols as well as the continued progress of the Small Scale Experimental (SSP), funded through interim funding (POR 2008/2009). The Pilot Scale Production (PSP) accomplishments of goals, tasks and scope of work include: the initiation of transfer of technologies from the SPS section; the scale up of algal production for PSP runs and back-up supply of algae for SPS section; the refinement of larval culture containers; the scale-up of larval culture containers; and the culture run of larvae from small containers to large containers plus alternate setting and metamorphosis containers. Task 1. Martin Moe (SPS) and Dave Vaughan (PSP) spent time in training and learning larval culture container manufacturing and larval tank feeding, draining and cleaning procedures. Two original U shaped larval tanks were transferred to the PSP facilities, plus the new modified U shape tank mold was moved to the PSP facility to manufacture 4 new improved versions of the 50 Liter acrylic larval culture vessels.

2 These 50 Liter larval tanks were operated successfully through the transition from SPS to PSP systems using the protocols set up similar to those used by Martin Moe. Shown are 3 of the 4 new modified U shaped acrylic larval culture tanks at PSP (Mote-TRL). Task 2. The Scale up of the larval tanks was successfully accomplished by using 1,500 Liter U shaped tanks as the scale up from 50 Liter culture vessels. Four, pilot scale larval tanks were transported from Mote Aquaculture Park (MAP) in Sarasota that were used for shrimp larval culture to the PSP facility at Mote Tropical Research Lab (TRL). New stands were constructed and two of these were place inside next to the algal production tanks and two were placed outside to take advantage of natural sunlight. Two runs will approximately 5,000 larvae proved successful larval development with 7 and 10 day increments before larval drain-downs and tank cleaning was necessary. The draining down of these tanks took approximately the same maintenance time as a 50 Liter culture vessel. And more importantly was able to extend the 2-3 day cycle to 7-10 days before requiring drain-down and cleaning. At projected densities at 1-3 per ml. would allow for 1.5 to 3.5 million larvae to be held for each tank. This would also allow for lower densities to be held allowing for increased food availability for each larvae and hopefully increased condition of each larvae prior to settlement. 2

3 Picture of two of the large 1,500 Liter U shaped larval tanks and picture through a microscope of 30 day old larvae after a 7 day duration cycle. Task 3 Algal food production. This task focused on the starting of microalgae at a pilot-scale level and included using 1 liter culture vessels for holding cultures and 5 gallon carboy cultures for production of enough inoculums for the 50 gallon tubes. This system was run using three of the preferred species including: Isochrisis galbana; Chaetocceros gracillis; and Rhodomons lens. It is expected that any of these tanks and the larval tanks can be used to produce macroalgae as well for the broodstock and juveniles. Pictures of 50 gallon algal tubes with all 3 algal species and picture of 5 gallon carboys with each of the 3 algal species being grown for larval cultures. 3

4 Task 4. Pilot scale nursery of juveniles. The larger scale juvenile trays and raceways are now being tested with the 42+ day old larvae that were placed in settlement trays. Four foot by eight foot long trays (6 deep) and raceways (18 deep) are under trials now for successful settlement of larvae ready for metamorphosis. This is a critical area of investigation and will be a major focus of the next half of the year. Picture of a portion of the 8 foot long settlement tray with a coating of diatoms cultured in the algal tanks and natural algae for the development of the juvenile stage of the Diadema. Larval trial will continue with these larger scale equipment and procedures for the second half of the project (1/2011-5/2011), with anticipation of production of enough juveniles to provide for the associated field trials and behavior trials. 4

5 POR Report on POR Special Project: Diadema chronicles: Culture efforts at SSP facility in 2010 December 23, 2010, by Martin Moe During 2010, continuation of Diadema culture efforts at Martin Moe s SSP facility at Islamorada were enabled by Special Consideration funds provided by POR in the fall of These funds allowed this important work to continue in This is a report on the Diadema culture activities enabled by those funds. The work of the SSP facility during 2010 were aimed at consolidating the larval rearing success of 2009 and firmly establishing the basic elements of a large scale, reliable rearing technology for Diadema. Unfortunately this was not accomplished but progress has been made and the reasons for this lack of success have been identified. Diadema larvae rearing runs, 2010 Run # 14, 2/10/10, 53 days Run # 15, 2/24/10, 40 days (partially concurrent with # 14) Run # 16, 4/9/10, 54 days Run # 17, 4/19/10, 44 days (partially concurrent with # 16) Run # 18, 7/20/10, 43 days Run # 19, 9/22/10, 60 + days Total days of Diadema larvae rearing runs in 2010, 294 About 100 healthy, feeding Diadema juveniles were produced in rearing run # 12 in the spring and summer of That rearing demonstrated that the spawning techniques, physical structures of the larvae rearing technology, the basic nutritional requirements of the larvae, and the basic requirements for settlement and survival of the early juvenile were established. Spawning runs #13, #14, #15, #16, all produced many early juveniles (thousands) that did not survive the early juvenile development period of 5 to 7 days post metamorphosis. Various factors, possible effects of inadequate larvae nutrition, inadequate settlement substrates, and other factors were considered and improved during those rearing runs but these corrections, although improving the technology, did not result in juvenile survival. Larvae with pedicellariae and large rudiment with external tube feet from run #16, of April 9, 2010 day 30

6 Juvenile, about five days from metamorphosis, from run #14, of Feb, 10, 2010, day 40 New juveniles, oral pole (underside) above, aboral pole (top side) below from run #14, of Feb. 10, 2010, day 35 Analysis and water quality experimentation strongly indicated after run #16 that the problem with juvenile survival was very likely a great drop in ph, from the normal 8.15 to 8.2 range down to a 7.8 to 8.0 range in the tanks and reservoirs of the lab room. This drop in ph was shown to be caused by the maintenance of a tank system with 45 adult Diadema brood stock urchins. The adult Diadema consume a great deal of macro algae and produce a great deal of CO2. This CO2 accumulates in the close confines of the small laboratory, closed up to maintain proper temperatures in winter and summer, enters all the tanks and containers water in the room, and produces excess carbonic acid which drops the ph of all the water in the room. Installation of an exhaust fan, keeping the lab vented as much as possible with open doors and fans, and addition of alkalinity and calcium to the inside reservoir allows maintenance of ph in the normal range of 8.15 to 8.2 in all water but that of the brood stock system. 2

7 Despite these corrections, the brood stock system, a 220 gallon unit made up of two 110 gallon tanks and a filter system, can only maintain a ph range of 7.76 to 7.85, depending on room venting capability and extent of feeding of the brood stock. When the ph problem was first discovered the ph of the brood stock system was in the 7.6 range with a free CO2 level of 5 to 6 ppm. Although most juvenile and adult sea urchins have a capacity to physiologically maintain an internal normal ph at lower than normal external ph, even these urchins showed a marked reduction in spine length. Their spines grew much longer and sharper when the ph was maintained at 7.8 rather than 7.6. No doubt the brood stock urchins would do even better at a normal ph of 8.2. The most reasonable explanation for the total mortality of all early juveniles produced in the previous four rearing runs was that the low ph of the cultures post settlement, 7.8 to 7.9, prevented the early juveniles from rapidly calcifying the hard body parts, test, spines, and jaw mouth parts that are required for survival within a few days after metamorphosis. Only something as universal and essential as correct ph could be responsible for such total mortality in each run at the same point in juvenile development. I presume that in the partially successful run #12 of 4/6/09 the temperature conditions at that time allowed for open doors in the lab and water exchanges with water only briefly held in the inside reservoir provided a high enough ph at some of the time as to allow some survival of the early juveniles. A normal ph was maintained in the larvae rearing culture vessels during rearing runs #17, #18, and #19, and settlement and juvenile grow out facilities and settlement substrates were prepared in anticipation of resolving the problem of early juvenile mortality. Unfortunately, no late larvae competent for metamorphosis were produced in any of these runs, which was extremely disappointing. The three reasons for these failures were evident. 1. The micro algae, Rhodomonas sp. (either lens or salina) for reasons unknown appears to be essential, or at least extremely important, to the normal and relatively rapid development of a mature rudiment and competency for settlement and metamorphosis. This is an important area for future research, for now, Rhodomonas is necessary. The micro algae species that I used in the rearing runs that produced early juveniles, including run #12 that produce surviving juveniles, were Rhodomonas (Rho), Chaetoceros gracilis (Cha), Pavlova (Monochrysis) and/or Isochrysis gabena (T. Iso), and occasionally some zooxanthellae (Zoo), Clade B. These micro algae are available in quantity from Algagen, a company in Vero Beach, Fl. The proper culture of micro algae is a complex and time consuming endeavor, one beyond my capabilities. I can, for the most part, keep Cha, Pav, T. Iso, and zoo cultures for the two to three month duration of a rearing run, but Rho is difficult to culture, ship, and keep and I have to rely on regular shipments from Algagen to maintain the Rho stocks needed for a rearing run. There were difficulties, especially in runs # 18 and #19 in obtaining Rho. On several occasions, the cultures did not survive shipment (especially in warm weather) and either arrived crashed or crashed soon after arrival. In most instances I was able to resurrect the cultures with continued incubation at low light levels, but this takes one or two weeks. Also the Rhodomonas cultures that I had to feed from were very thin, either at receipt or after recovery and it takes a week to 12 days for these cultures to acquire some density. Feeding has to be carefully controlled so as not to use up the culture before it has time to acquire density. Thus the availability of Rho was occasionally or frequently severely restricted during the last three rearing runs. The larvae survived this and other problems but there may have been effects of nutritional inadequacy that compromised larval development. 3

8 2. A disease or syndrome, beginning with a disintegration of the rapidly growing tissue at the tips of the arms and then over a few days developing into a total regression of arms and body producing a nubbin of a larva that soon wasted into nothingness, continued to affect the rearing runs despite the maintenance of a proper ph. This syndrome seems to affect larvae during the days between 10 to 20 of the rearing run. It may from water quality or a bacterial or fungal infection. It can be reversed and eliminated by total water change and/or a treatment with methylene blue but it does temporarily compromise the growth and development of the larvae. It does not, however, prevent the larvae from attaining competency 15 to 20 days later in the run. This syndrome occurred twice during the last run, #19, day 10 and day 16. The water change regime during this time was Monday, Wednesday, and Friday, skipping a third day to the next Monday. This seemed adequate at the time, an improvement over the change every 3 rd day, but a change to a 50% water change on the day between total water changes was an improvement and seems to have reduced or eliminated the occurrence of the arm tip degeneration syndrome. 3. Vorticella! It is my opinion the ravages of loss of Rho and arm tip degeneration in a culture could be corrected and withstood bringing a significant number of larvae to competency if not for Vorticella. I don t know what specific species of Vorticella is present in the cultures, but it appears typical to the genus. Vorticella in the trophic vorticellid phase of the nonsexual cycle colonize small bits of algae flock and other tiny particulates in the culture, reproduce rapidly and in huge numbers completely consume the micro algae in the culture, starving the Diadema larvae. There may also be a release of a biochemical compound that negatively affects the urchin larvae since after the appearance of Vorticella, Diadema larvae seem to decline to a degree greater then just food competition would indicate, but this is just speculation. The algae balls are coated with hundreds to thousands of individual ciliates and this colony is the same size as the larvae and about the same density. Thus it is almost impossible to separate the Vorticella from the larvae and actually apparently impossible to eradicate the Vorticella from the culture, although I have found a way to control their numbers. Vorticella is a bell shaped ciliate attached to one end of a flexible stalk. The other end of the stalk is attached to a substrate. It reproduces asexually by fission; the bell shaped ciliate divides in two so that there are two heads or bells on the end of the stalk, the stalk then also divides and two individuals are the result. There are two forms in this non sexual reproductive cycle, the attached trophic vorticellid and a smaller free swimming telotroch phase. Interestingly, the telotroch phase is formed in response to high carbon dioxide tensions, an environment that the telotroch can better survive then the vorticllid. It was not until after the high carbon dioxide levels in the lab were reduced by venting that Vorticella, which was occasionally present before, became the extensive problem that it was in the last few rearing runs. The teleotroch ciliate can also secrete a cyst membrane under inhospitable environmental conditions and can then excyst from the cyst membrane when conditions improve, which is an excellent way to persist and be carried from one culture or environment to another. Vorticella also reproduces sexually. Preconjugation division leads to formation of microgamete and a macrogamete. The microgamete has the same form as the telotroch but is quite a bit smaller. The free swimming microgamete and macrogamete fuse and conjugation takes place. After conjugation the macrogamete can resume the nonsexual cycle as can the teleotroch. Thus this ciliate, although apparently not directly injurious to Diadema larvae, have a life cycle and a benthopelagic capacity that creates a perfect storm of food competition for the larvae that seemingly cannot be eliminated once it invades a culture. 4

9 I have been able to keep in under control, somewhat, through use of swirlies at each total water change. A swirly is the use two bowls to manipulate the concentrated mix of larvae and Vorticella coated algae balls. With Wendy s plastic frosty spoon, I swirl a cup or two of water with the Diadema larvae and algae ball mix in one bowl, the very slightly higher density of the larvae sends them into the center of the vortex at the bottom of the bowl with the algae balls more to the periphery of the bowl, and then as the vortex slows down, I pour the contents slowly from one bowl to the other, The concentrated larvae at the center bottom are the last to leave the bowl so I can stop the pour at just the right time and then the relatively isolated larvae can be put back into the culture vessel, sans almost all of the algae flock and Voticella. It takes three or four swirlies to make a functional separation. I lose some of the larvae and can't remove all of the algae/vorticella, but it works well enough for two days giving the larvae the upper hand, er spine, in the competition for food that takes place in the culture. However, over time, 30 days or so, the handling and collateral loss of larvae severely compromise the culture with injury to the larvae and loss of larvae. It does work to the point that the larvae cultures can persist. The current run is now at 61 as of this writing and numbers about 20,000 larvae. Vorticella is still very heavy on the algae balls that carry over from the water change and swirlies but the process reduces the incidence of Vorticella /algae balls to the point that the larvae can persist. However, they are not developing to competency which may or may not be due to food competition with Vorticella. Vorticella on algae strand in larvae culture, run #19, of Sept. 22 on day 40 Bowl at the end of a swirly just before the pour off. Run #19, of Sept. 22, day 50 5

10 Collection of larvae and remaining algae/vorticella balls at end of swirly. Run #19 of Sept. 22 on day 50. The best control is, of course, not to introduce Vorticella in the first place, which is difficult if the source of the introduction is not known. There are several possibilities. 1. The most likely source is the brood stock system. It is possible that Vorticella at some stage of the life cycle enters the culture at the time of spawning along with the fertilized eggs. Care is taken to clean the brood stock urchins before spawning and to clean the eggs by screening before placement in the culture vessels, but this still remains a possibility despite these precautions. Even a very small inoculation with the eggs could, over time, 15 to 25 days, result in a large infestation despite frequent water changes. It may also be possible for the cysts of the telotroch that might be in the brood stock system to become airborne and infect the larvae cultures from a brood stock source. 2. The water, despite the chlorine treatment and the one micron filter, might somehow be the source. UV treatment might help but that would be expensive in time, money, and space to install. 3. The microalgae, especially the microalgae cultured over several generations in the lab, might be the source since various types of ciliates are often found in these cultures. 4. The lab is not perfect, far from it, in the type of procedures designed to eliminate the possibilities of contamination from utensils and containers. Acid washing is not practiced nor are other typical sterilization protocols of biological laboratories. Lack of time, personnel, funding, and space prevent adoption of such protocols. The current run, #19, started on Sept. 22, is now in day 63. There are about 20,000 larvae remaining. They are hanging in there despite the Vorticella and total water changes and swirlies every two days, they look OK but they are not thriving, apparently not developing toward competency despite the presence of large, internal, but undifferentiated rudiments, and I think it is only a matter of time before these larvae eventually decline into nothingness, but maybe not... I don t know why they stopped development at about day 45 despite the same nutritional elements (Rho availability increased when all the larvae were consolidated into one culture vessel), good water quality assured by frequent water change, and relief from intense competition for microalgae with Vorticella. But this lack of development past day 45 to 50 is consistent with larval development in other long runs where competency and metamorphosis was 6

11 not achieved before day 50 or so. Perhaps a length of time over 50 days or so without competency creates changes in larval development that adapt the larvae for long journeys in the plankton and a different sequence in environmental conditions is necessary for the larvae to regain a path to competency. This is a very complex larvae and there are many questions that lack answers. Larvae from run #19 of Sept. 22, on day 62. With Vorticella under some control the larvae persist with internal rudiments but do not develop to competency. The basic spawning and larvae rearing process for Diadema developed at the SSP facility over the last few years, although not perfect and certainly improvable, seems adequate for initial large scale production of juvenile Diadema. All the individual elements of the technology have been successful during various rearing runs, but unfortunately, due to elements beyond my immediate control, I have not yet been able to put all these elements together in one trouble free, completely successful, rearing run that produces large numbers of stable, feeding, surviving juveniles, This is very frustrating because it seems that this should not be too difficult to do at this point. However, continued conduct of Diadema larvae rearing runs is problematic. I have no funding at this point and the expenses of the lab, primarily micro algae and electricity, for each run are significant. Also all the lab equipment has to be kept in the high humidity and salt air environment of the little lab and this has a very detrimental effect on electronics and metals, The high intensity microscope illuminator needs a new bulb and socket assembly, microscopes and pumps need a thorough cleaning and maintenance, the main pump is making a funny noise, many lights are rusty and need replacement, the air conditioner cooling grid is disintegrating and it will no longer turn off, but the circuit breaker works so I can still run it. It will take a month or so to replace, repair, and do maintenance on lab equipment. And the work load, even with occasional help from the FWRI lab (which is greatly appreciated) is staggering. This last run, now at 63 days, basically every day, sometimes 10 hours a day, is typical of the six runs this year and it is getting to be too much for an old retired guy. However, I really want to tie this all up with one successful run that demonstrates the capability of the entire process, and so I will try to conduct another rearing run early next year. Although, if Dave is successful with the large larvae rearing tank approach on his next run (we can spawn the brood stock at any time), or with the small culture vessel technology, then maybe I ll just help him get it going. 7