The American Zoo and Aquarium Association
The American Zoo and Aquarium Association
This article originally appeared in Proceedings of AZAA Regional Conference, Audubon and Zoological Garden in New Orleans, 1996, pp 95-99. All rights reserved by the AZAA. Permission to reproduce is granted by the publisher and the author to The Breeder's Registry and is not transferrable.
Stephen C. Kempf
Associate Professor
Department of Zoology and Wildlife Science
331 Funchess Hall
Auburn University, AL 36849
kempfsc@mail.auburn.edu
Michael Brittsan
Curator of the Shores Department
Columbus Zoo
9990 Riverside Dr.
Powell, OH 43065
Large scale saltwater aquaria are an exciting and increasingly common means of conveying an understanding and appreciation of the marine environment to public audiences. These displays portray habitats of considerable complexity ranging from tide pool to coral reef communities. Along with this complexity comes a burden of maintenance considerably higher than that associated with fresh water aquarium systems. A significant aspect of this maintenance is regular manual cleaning of aquarium walls and glass, and the removal of unwanted organisms that tend to overgrow and obscure sessile species or species replicas that are being displayed.
Species of the small brown anemone Aiptasia, including A. pallida and A. pulchella, are common "weed" organisms in zoo aquaria as well as those of the private hobbiest. This anemone is very well adapted for aquarium life for two reasons. 1) It prefers high illumination because much of its nutrition is derived from millions of photosynthetic algal symbionts, called zooxanthellae, that live in the anemone's tissues (Trench, 1993), and 2) its most common form of reproduction is asexual (Hunter, 1984). This asexual reproduction is accomplished by a process called pedal laceration which simply means that small pieces of regenerative tissue separate from the anemone as it slowly moves over objects in the aquarium. In small numbers, these anemones add interesting highlights to any tank; however, once introduced the regenerates produced by pedal laceration can, if conditions are right, proliferate at a very high rate. As a result, regular and time consuming manual removal is often required if displays are to be kept attractive and informative.
It would be useful if a natural predator of Aiptasia species could be found that would thrive in aquarium displays. This would not only solve the problem of manually removing these anemones, but would also be an instructive example of how predator-prey relationships work to prevent species from overgrowing the capacity of their natural environment.
Recent investigations in one of the author's (SCK) laboratories have described a molluscan predator, the nudibranch Berghia verrucicornis (Figure 1), that feeds on species of Aiptasia (Carroll and Kempf, 1990, 1994; Kempf, 1991). This nudibranch species is found on shallow reef flats in the Caribbean where Aiptasia pallida commonly occurs. Berghia is well suited as a predator of Aiptasia in display aquaria for two reasons. First, as is the case for most nudibranch species, Berghia feeds specifically on one genus of organisms (in this case Aiptasia) and not on species from other genera. Second, this nudibranch species life history includes a non-feeding larval stage that will settle and metamorphose in the aquarium environment a few days after hatching. This means that once a population of Berghia is established in a marine aquarium, it will continue to propagate itself as long as Aiptasia are present to feed on.
Recently the authors have initiated a collaborative effort to develop the potential of Berghia verrucicornis as a natural and cost effective means of controlling Aiptasia populations in display aquaria. At times, simply releasing a pair of adult Berghia into an aquarium continuing Aiptasia will result in the establishment of a population of these nudibranchs after a few months; however, since this is not always the case, it may be prudent to start aquarium populations using juveniles that have been cultured as described below. Once viable aquarium populations of Berghia have been established in this manner, these culture methods may be discontinued.
Culture of Berghia verrucicornis requires supplies and equipment listed in Table 1. A number of these items are readily available from local sources. Preferably, all glassware and brushes should be new. It is important that items that will contact culture water or animals have never contained chemical substances and have never been washed with soap, detergent or solvents. Minute amounts of chemical substances that adsorb to the surfaces of glassware, etc. are often toxic to invertebrate larvae.
We have never had success culturing nudibranch eggs or larvae using freshly mixed artificial sea water. Instead, all sea water for culture is drawn from established aquaria that contain sea water that is either natural or prepared from a commercial formula (e.g. Instant Ocean) with either de-ionized or distilled water. This sea water is filtered using a Nalge filter apparatus, a 0.45 um Millipore filter and vacuum filtration = Millipore filtered seasoned aquarium water (MFSA).
Two adult Berghia verrucicornis are cultured in each 12 cm glass culture bowl in approximately 300 ml of MFSA. The bowl and water are changed daily, at which time egg masses are collected and cultured as described below. At each culture change, a number of large Aiptasia are cut up using forceps and "iris" scissors, and the pieces placed in each Berghia culture bowl. We find it is best if the Aiptasia are cut-up radially such that each piece possesses part of both the oral disk/tentacles and the body column. If the Aiptasia come from the gravel bottom of an aquarium, be sure to remove an pieces of gravel adhering to the pedal disk before trying to cut them up with the iris scissors. A single large Aiptasia (5-8 cm in length when expanded) may be cut into 4-8 pieces, and 2-4 pieces in each culture bowl containing 2 Berghia. As the Berghia feed they will turn brown due to symbiotic algae present in the Aiptasia. Culture bowls can usually be maintained at room temperature (20-25 C).
Egg masses of Berghia verrucicornis consist of a twisted white string that is sometimes laid in a spiral, attached to the substratum. Young, well fed Berghia can be expected to lay one egg mass every 1-2 days. As animals get older, egg mass production will decrease. Egg masses may occur either free floating or attached by one edge to the culture bowl. Attached egg masses are removed from the bowl by carefully scraping them away from the surface with the edge of an old credit card pressed firmly against the bottom of the bowl. Egg masses are picked-up with forceps and transferred to 500 ml glass beakers containing approximately 300 ml of MFSA. An airline is inserted into the large end of a short Pasteur pipette and the culture is aerated such that the egg masses are gently lifted off the bottom and circulated in the beaker. Care should be taken not to over aerate the cultures as excessive agitation of the egg masses may interfere with the embryos' development. Egg mass culture beaker and water should be changed daily.
Embryonic development of Berghia verrucicornis takes about 10 days at 22-24 C. At about 7 days of development, the cultures aeration is decreased such that the egg masses are no longer lifted off the bottom of the beaker. At 9 days of development, the egg masses are removed from the culture beaker and placed in a small glass bowl (5-8 cm diameter) containing about 1 cm depth of MFSA. The egg masses are then artificially hatched by "pumping" them in and out of a short Pasteur pipette until most of the egg capsules are separated from the egg string. Remaining pieces of egg string are then removed from the bowl with forceps and discarded.
Larvae will generally be released from their egg capsules during the artificial hatching process. Most of those that are not will eventually break out of the capsules on their own. The newly hatched larvae are gently swirled in the small bowl such that they collect together in the center. These larvae are then transferred by pipette to a 12 cm diameter culture bowl containing about 300 ml of MFSA and 6-10 very small Aiptasia that were placed in the bowl on the day preceding hatching (=metamorphosis cultures). The metamorphosis cultures are covered with parafilm or saran wrap being careful not to let the cover touch the water, and are left undisturbed for 5 days. The parafilm or saran wrap will reduce water evaporation and an associated increase in specific gravity.
At 5 days the cultures are examined with a dissecting microscope or magnifying glass. Newly metamorphosed juveniles will appear as tiny (~0.5 mm) white slugs, many of which may be clustered at the bases of the small anemones. Other juveniles may be crawling on the bottom of the bowl or attached to the underside of the water surface. If most of the larvae are attached to the surface, they should be disattached by dropping droplets of water on them from a Pasteur pipette. After examining the culture and disattaching juveniles from the surface, the disattached juveniles are given about 5 min to re-attach to the bottom of the culture bowl. The culture water is then changed by simply pouring the old water from the bowl and adding new MFSA to the same level. Culture water is subsequently changed in a similar fashion every 3-4 days for the next few weeks. As the juveniles grow they will develop cerata on their dorsum. As juveniles grow, they should be split between additional bowls such that total biomass does not exceed the "carrying capacity" of a bowl. Once they have reached a length of 4-5 mm juveniles may be released into aquaria that contain Aiptasia. Over the next few months populations of Berghia should establish themselves in the aquaria. Once this occurs, Berghia culture as described above, may be discontinued.
If juvenile Berghia fail to grow and develop cerata, it will probably be the result of trying to feed them anemones that are too large. Tiny pieces of cut-up anemone do not work well for feeding newly metamorphosed juveniles because they tend to decay and foul the water to the extent that the juveniles die. We have found that "bleached" Aiptasia, those that have been kept in the dark long enough to lose most of their symbiotic algae, provide a better source of tiny anemones since they pedal lacerate and produce very tiny regenerates much more frequently than symbiotic anemones. For this reason, it may be wise to set-up an established, appropriately filtered and aerated, 10 or 20 gal aquarium with a good population of Aiptasia, place a tinfoil covered box over it to exclude light and feed the Aiptasia newly-hatched brine shrimp every 2 days. The Aiptasia in the aquarium will "bleach" over a period of 1-2 months and begin to produce tiny, pedal lacerate regenerates that may be used to feed newly metamorphosed, juvenile Berghia.
Our thanks to Dr. John Miller of Baldwin-Wallace College who pointed out the interesting nudibranch that started reproducing in his lab aquaria. This work was supported in part by grants to SCK from the National Science Foundation (DCB-9018698), Office of Naval Research (NAVY-N00014-931-0919), and Alabama Agricultural Experiment Station.
Carroll, D.J. and S.C. Kempf 1990. Laboratory culture of the aeolid nudibranch Berghia verrucicornis (Mollusca, Opisthobranchia): Some aspects of its development and life history. Biol. Bull. 179: 243-253.
Carroll, D.J. and S.C. Kempf. 1994. Changes occur in the central nervous system of the nudibranch Berghia verrucicornis (Mollusca, Opisthobranchia) during metamorphosis. Biol. Bull. 186: 202-212.
Hunter, T. 1984. The energetics of asexual reproduction in the symbiotic sea anemone Aiptasia pulchella (Carlgren 1943). J. Exp. Mar. Biol. Ecol. 83: 127-147.
Kempf, S.C. 1991. A 'primitive' symbiosis between the aeolid nudibranch Berghia verrucicornis (A. Costa, 1867) and a zooxanthella. J. Moll. Stud. 57: 75-85.
Trench, R. K. 1993. Microalgal-invertebrate Symbioses - A Review. Endocytobiosis Cell. Res. 9: 135-175.
Equipment and supplies for Berghia culture
Airline
Air valves
Aquarium air pump
Instant Ocean or similar product
A source of Aiptasia
A source of de-ionized or distilled water
A source of sea water from an established aquarium
One 10 gal aquarium with appropriate filtration
One cardboard box sufficiently large to fit over the 10 gal
aquarium
Filter apparatus (Nalge Co., Cat. #310-4000, 47/50mm)
0.45 um Millipore filters (Millipore Corp., Cat. #HAWP 047 00)
1 liter vacuum filter flask (Fisher Scientific, Cat. #10-182-508)
500 ml beakers or equivalent sized drinking glasses
Glass culture bowls, approximately 12 cm in diameter
Small glass bowls, 5-8 cm in diameter
Cleaning brushes for glassware
One pair of iris scissors (Fisher Scientific, Cat. #08-953-1B)
One pair of forceps (Fisher Scientific, Cat. #08-953G)
Short Pasteur pipettes (Fisher Scientific, Cat. #13-678-20A)
Rubber pipette bulbs (Fisher Scientific, Cat #14-065B)
One old credit card (or similar flat piece of thin flexible
plastic)
Figure 1. An adult Berghia verrucicornis. The anterior end is at the top. The numerous "frilly" projections on the dorsum are the cerata. In animals that have been feeding on symbiotic Aiptasia the cerata will be dark brown.
[NOTE: no physical figure was available at the time of this web translation.]
