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Aquarium.Net March 97

Rob Toonen writes on rotifers and there role in captive breeding of fish, Aquarium Net has numerous articles written by the leading authors for the advanced aquarist

Reefkeeper's Guide to Invertebrate Zoology:

Part 5

Phylum Rotifera

By Rob Toonen

Last month I covered some relatively obscure animals (flat & ribbon worms) that are encountered quite frequently, but about which little general information is available to reef-keepers. This month, I will again cover a relatively "obscure" group, but one for which most reef-keepers will immediately recognize the name: the phylum Rotifera. I had intended to continue on and also cover our first coelomate group (the sipunculans), but have ended up covering more information than I intended in this article, and am forced to save that group until next month along with an introduction to the annelids (better known as bristleworms).

Although rotifers are a small and generally uninteresting group to almost anyone, they are quite well known among reef-keepers as the food of choice for early-stage larval fishes and many late-stage larval Crustaceans (e.g., crabs & shrimps). The topic of larval culture is far too complicated to cover in a single article, but I will try to give some basic information regarding the culture of rotifers in this article for people who have no introduction to the topic. I do not have the space in this article to cover the topic in any detail, but my goal in writing this article is to introduce a naive reader to the techniques and requirements for setting up a culture. Both the culture of rotifers, and a general guide to breeding and raising larvae of marine invertebrates have recently been, or are currently the focus of much more in-depth articles in the Breeder's Registry publication, The Journal of Maquaculture (Rosenquist, 1993; Wilkerson, 1995; Toonen, 1996; Toonen, 1997a,b,c). Moe (1982) covers the topic of rearing larval fishes with particular emphasis on clown-anemone fishes in the genus Amphiprion , and there is a short section on the culture of rotifers in reference to raising these larvae. If rearing larvae is the reason for which you are interested in learning more about culturing rotifers, and you are successful in breeding any inhabitants of your reef tank, I strongly encourage you to contact the Breeder's Registry to share your success and techniques with others.

The phylum Rotifera includes about 1,800 described species, of which approximately 50 are marine. The remaining species are primarily freshwater, although some live in damp soils, or in the water film on some mosses (Brusca & Brusca, 1990). Rotifers are tiny: a few species reach 2-3 mm, but most are less than 1 mm long. The body is typically divided into a head, which bears a ciliary organ called the corona , a trunk, and a foot. The coronal cilia, when the animal is actively swimming/feeding, resemble two spinning wheels -- hence the name "wheel animals" from which the phylum name is derived. The pharynx is modified as a mastax , which contains a pair of internal jaws ( trophi ). These jaws may be of several types depending on the feeding mode of the animal ( ramate , & malleate for crushing/grinding; forcipate & incudate for piercing/grasping). Ciliary suspension feeders use the cilia of the corona to generate feeding currents (this is technically incorrect, but the mechanism of ciliary feeding is quite complex, and I will not go into detail here) which bring particulates (typically tiny organic detritus or small plankton) to the mouth. Such forms have crushing/grinding jaws to macerate the food items prior to passage into the gut. Raptorial feeding is also common among rotifers, and in such animals ciliation on the corona is often reduced or used entirely for locomotion. These animals feed by grasping prey with protrusible jaws; they ingest the prey whole and grind it to smaller particles within the pharynx or they may pierce the body of the prey and feed by sucking out the fluid contents of their prey. Predatory rotifers typically feed on small animals (such as ciliates, protozoans or other rotifers), but are often seen to ingest plant material as well.

The body is 'covered' in a cuticle (actually the cuticle is produced by, and lies within a syncitial epidermis , but for our purposes we can say it covers the body) which may be thin or thick, and ends in a telescoping foot which generally bears spines or a pair of "toes" through which the ducts from the pedal glands pass. These ducts deliver sticky secretions produced by the pedal glands which allow the animals to attach temporarily to the substratum. The foot may be modified for permanent attachment in some sessile forms (e.g., Floscularia ) or completely absent from some swimming forms (e.g., Asplanchna ). This cuticular covering allows the animals to withstand some extreme environmental conditions by entering a metabolically dormant state. For example, the resting zygotes (explained below) of some rotifers have survived freezing in liquid helium (272C!!), while others have been dried out and kept on a shelf for years (some reports claim storage viability as long as 20 years), only to spring back to life when water was added.

Rotifers are unusual in that they always have separate sexes, but with the exception of the genus Seison , males are either reduced (in size, complexity and abundance) or completely absent (Brusca & Brusca, 1990). If you're wondering how these animals can reproduce if they have separate sexes but no males, you are not alone. For many years the absence of males puzzled scientists, but it is now generally accepted that males are indeed absent from the Class Bdelloidea, and that these animals reproduce entirely via parthenogenesis . Parthenogenesis is a mode of 'asexual' reproduction (for lack of a better term), in which females produce eggs ( amictic ova ), but the eggs are produced by mitosis (simple cell division) rather than meiosis (the halving of the genetic material during cell division which forms gametes in all non-parthenogenic species), so the eggs can proceed to develop into offspring without fertilization. Even in the species which have males, the males are often very short-lived, perhaps because they possess a reduced gut. The males are typically produced in response to various environmental stresses (such as high temperature, food limitation or high population density) or seasonal cycles (such as changes in day length or temperature). Developing eggs exposed to these conditions develop into sexual females which produce mictic ova via meiosis. These females can either reproduce via parthenogenesis to form haploid (a single copy of the genetic material rather than duplicate copies being provided by each the mother and the father) males asexually, or after copulating with a male, produce thick-walled resting zygotes from the sexually fertilized eggs. These resting zygotes are extremely resistant to adverse environmental conditions (as described above). When favorable conditions return, these resting zygotes hatch into amictic females which reproduce via parthenogenesis again until stressed. The reproductive rate of these amictic females is very high; a female typically produce 2-8 offspring per day under ideal conditions, and those offspring reach reproductive maturity within a day themselves (Moe, 1982). If we allow each rotifer to have an average of 6 daughters per day and all of them survive to reproduce at the same rate, a single rotifer would give rise to 134,455 offspring in only 7 days (115,000 would still be immature at that point, but you get the idea).

Well, I imagine that I have just covered far more of the biology of rotifers than you probably ever wanted to know, so I will move on to the information for which you are likely reading this article: how to culture rotifers. As I said at the beginning of this article, there are several in-depth treatments of rotifer culture available elsewhere, so if you are really interested in the topic, I suggest that you find some of these references rather than rely on the sketchy account I am about to provide. My purpose here is not to explain exactly how to successfully culture rotifers at home, but rather to introduce you to exactly what is involved in rotifer culture so that you can make a decision about whether you want to attempt this at home, or whether you want to pass on the whole thing. You can either track down a friend/fellow hobbyist from whom you can get some rotifers to start your culture, or you can buy your rotifers from a supplier such as Florida Aqua Farms (33418 Old St. Joe Rd., Dade City, FL 33525 ph. 904-567-8540). One advantage of going with Florida Aqua Farms is that they can supply you with not only the resting zygotes, but also with Microalgae Disks or liquid Roti-rich food, and The Plankton Culture Manual (Hoff & Snell) to tell you what to do along the way. Florida Aqua Farms is not the only place to get rotifer cultures, of course; Ward's Biological and Carolina Biological Supply companies both sell marine rotifer (typically Branchionus plicatilis ) cultures, although this is undoubtably more expensive. These locations are suitable only for obtaining starter cultures of the rotifers. If you intend to buy all the rotifers to feed your fish/invertebrate larvae from such as source, you would save money by simply buying more of the animals you intend to breed. In some of the more advanced pet shops you may be able to find these live foods, or starter cultures, available for sale locally.

OK, enough of the introduction. The most important factor in successfully raising rotifers is to have a good food supply. There are many commercially available pea flour and/or yeast-based foods (e.g., Liquifry Marine) which could in theory be used to feed your rotifer cultures, but in practice, these foods quickly foul the culture water and more often than not lead to the culture crashing rather than growing (I will discuss culture crashes later). Roti-rich, although yeast based, is successfully used by Aquatic Research Organisms (A.R.O.) for large-scale commercial culture of rotifers (see Rosenquist, 1993). Personally, I prefer a good healthy algae culture for keeping the animals growing well -- I think the rotifers reproduce faster and are of higher nutritional quality when grown on live phytoplankton cultures (Moe, 1982 claims the same thing). Most people will find that getting a "good healthy algae culture" going is more work and harder to maintain than are their rotifer cultures. I don't have the space to go into detail on algal culture techniques here, but there are covered in detail in my article in The Journal of Maquaculture (Toonen, 1996). You can also get a lot of information (and order starter cultures directly) from the University of Texas algal collection planktotrophic (feeding) invertebrate larvae.

The basics of unicellular algal culture are to obtain a reliable culture innoculant, provide those algae with "sterile" seawater supplemented with the nutrients essential to growth, and supply those cultures with a lot of light. The algae genera suggested by Moe (1982) for the culture of Branchionus plicatilis include of Dunaliella , Isochrysis , Monochrysis , and Chlorella . Wilkerson (1995) suggests Chlorella , and Rosenquist (1993) states that A.R.O. uses a mixture of Selenastrum and Isochrysis but recommends Chlorella , Dunaliella , Nannochloris , and Tetraselmis as "better [microalgae] for rotifer culture." In my 1996 Journal of Maquaculture article, I give a list of ten microalgae most commonly used for research applications of marine invertebrate culture, and some of the details about the nutritive value/size of each. In that article, I suggest Dunaliella tertiolecta (but not D. primolecta ), Isochrysis galbana , Tetraselmis ( Paltymonas ) suecica , and Thalassiosira ( Cyclotella ) pseudonana ; although I did not mention Chlorella in that article, I believe that it is probably the microalga which is most widely used for rotifer culture. Your choice of a microalga species likely depends on the availability of the innoculant and the ease of culture. In general, any mixture will be a better choice than a single-species food, and any small (3-15µm cells) algae which do not produce defensive chemicals (e.g., Monochrysis , although suggested by Moe, should probably not be used to feed rotifers or larvae because of the accumulation of chemical byproducts) should suit your purposes.

I said that you need sterile (in quotes) seawater, because although you want seawater without other unicellular organisms (including bacteria) growing, it is virtually impossible to achieve truly sterile cultures at home. You can minimize the contamination of cultures by one of several different methods: 1) autoclaving (autoclaves are something to which few people have access, especially in their homes), 2) ultrafiltration (ideally 0.2, but anything less than 0.45 µm will work), 3) pasteurization, and 4) microwave sterilization. I describe all these methods in greater detail in a previous article (Toonen, 1996), but the easiest and most likely to be used at home is the last one. Microwaves have been found to kill most everything but fungal spores, so if you live in a relatively dry part of the country, and fungal spores are not a source of significant contamination, you need only microwave your culture medium. The famous algal expert R.R.L. Guillard, found that the medium need not even boil for the microwaves to kill most things in seawater. Generally if you place the medium in the microwave in the flask in which you intend to culture the algae for about 2 minutes on high, you'll have pretty sterile culture medium. If, however, you happen to live somewhere like Florida or the Carolinas, where fungal spores will definitely be a major problem, you'll need to get a special microwave pressure cooker. I have heard of them, and know people who use them, but have never tried it myself. The major concern for a pressure cooker is that you add some tapwater to the inside of the cooker to prevent your algal growth medium from boiling over and not only making a mess, but wasting the medium.

You also need something upon which the algae can feed. If you are ambitious and have access to the necessary chemicals, algal medium is simple to mix up yourself (there are many recipes online at the Univ. Texas web site, and I provide the one I use for y research in Toonen, 1996). If, however, you are not able to mix your own, you can easily purchase medium from a source such as Carolina Biological or Florida Aqua Farms. If you cannot afford to purchase sterile algal growth medium nor do you have access to the ingredients to mix your own, you can try to culture your microalgae on a simple liquid plant food (I'm told the "chelated iron" forms wrok best, but I've never tried any of them) from your local plant store. These supplements are not sterile, nor are they ideal, but some people can apparently have reasonable success with them. Once the algal medium and a few cells of the microalgae of choice are added, the culture should be grown under constant illumination from at least 80W of flourescent light (I typically use 160W of full-spectrum lighting for my algal cultures). It turns out that the algae grow perfectly well under the 98¢ cool-white lights from Home Depot (which happen to give more PAR than do equivalent aquarium specialty bulbs), but I have to use my old aquarium bulbs from my reef tank for something, so I "recycle" them for use in culturing algae. Natural sunlight is always going to be better than a flourescent imitation in my opinion, so if you happen to have a well-lit window sill somewhere (and your spouse won't kill you for sticking an ugly greenwater aquarium in your window), that is an excellent alternative to a light-box for growing your algae. I maintain a temperature of 22C (72F) for my cultures, but any temperature between 70 and 80F should be fine. Most people will maintain several (up to a dozen) microalgal cultures at any given time. There is a good reason for this habit. First, you can feed the oldest culture (or cultures if you are maintaining more than one species of microalgae in order to feed your rotifers a mixed diet) while giving younger ones a chance to grow and reach a high cell density before being fed. Second, if a culture crashes (which happens more often than one would like), you always have a number of backups rather than starting from scratch. Finally, it is really very little extra work to keep 8 algal cultures compared to keeping 1 or 2, so if you're going to put in the time and effort, you might as well have a bunch of redundant algal cultures just in case....

When you feed the algae to your rotifer cultures, it is a good idea to have these cultures well-lit also. Again there are a couple of reasons to do so. First, the algae can continue to grow and incorporate nutrients until the moment they are eaten if there is sufficient light, rather than consuming their energy stores and being a non-nutritive food. Second, although the rotifers are voracious and efficient feeders, they may always miss a few cells by chance, and the growth of those cells will provide a continuous source of food for the culture. Both the algal and rotifer cultures are very sensitive entities, and as I have mentioned before, are subject to crashes. Crashes can happen remarkably quickly, and typically occur because of two factors: poor water quality and high population density. Anyone who gives this a bit of thought will realize that these conditions are often intimately linked. If you are diligent and attentive, you will be able to save crashed cultures, but if you miss a crashed culture of algae for any prolonged period of time (i.e., more than a day), it will become an anoxic bacterial soup; these not only smell horribly (opening such a culture is bound to drive your family from the house), but are lifeless and cannot be resurrected. Crashed rotifer cultures, however, are much more resilient, and can survive a fair amount of abuse and neglect. As I mentioned above, the resting zygotes of rotifers are extremely resistant to adverse environmental conditions. Carefully removing the sediment at the bottom of a crashed rotifer culture and transferring it to clean, good-quality water should cause those resting zygotes to hatch into new amictic females which will rapidly populate your cultures once again.

Good water conditions can be maintained by a combination of not overfeeding (this is ESSENTIAL with yeast/pea flour-based foods, but in well-lit cultures fed live phytoplankton, this concern is far less important), and periodic water changes. Cultures should be fed daily (or even twice daily) if you intend to harvest your cultures heavily. You want to feed enough of the algae (or artificial equivalent) to keep the culture water slightly discolored. If you're using phytoplankton, I'd aim for a constant haze due to algae in the culture water; if you're using artificial food, I'd aim for a haze for an hour or two after feeding, but ensure that the water clears up before ever feeding with artificial food a second time. You can keep the foods in suspension and prevent stagnation by gently aerating your culture. Depending on how large a culture you are maintaining (Moe, 1982 & Wilkerson, 1995 recommend a 10 gallon culture to rear a batch of clownfish), you can add more or less aeration to keep the food from settling out. In a 10-gallon tank, a single long airstone with low to moderate air output will keep the culture well suspended. In small (1 gallon) culture vessels, I use a glass pipette releasing 1-2 bubbles per second to keep the culture aerated. The correct amount of aeration and food to add to a culture is a bit of an art, and a little experimentation on your part will likely produce a huge increase in harvest as you gain some experience and discovere what works best for you. You may be wondering how you can do water changes on a rotifer culture. The answer is simple: you harvest the animals frequently and replace the harvested water with fresh "sterile" seawater. Joyce Wilkerson (1995) recommends 25% per day, whereas Mark Rosenquist (1993) suggests 10% per day. Some portion of the culture in this range (10-25% per day) is about right, in my opinion. You can do a quick cleaning of the culture tank with a siphon at the same time as removing the rotifers if you siphon the culture water into a bucket and allow it to settle prior to filtering the rotifers out of the culture. I underline quick cleaning here because production seems most consistent in older, well-established and "unclean" cultures rather than new, virtually sterile cultures. I am not the only person to have noticed this, and other authors go so far as to encourage letting your tank become coated with a thick algal mat (e.g., Rosenquist, 1993). You can either strain rotifers through a fine-mesh cheesecloth, one of those really fine mesh white aquarium nets (I imagine they have a special name, but I am unsure what it is), or a home made Nitex© filter (see Toonen, 1997 for further details). Alternatively, you could add settled culture water with rotifers directly to your rearing tank. Whatever you decide to do, you should never let rotifers removed from the culture sit for extended periods prior to feeding; rotifers kept alive without algae on which to feed for more than an hour or two are useless as food (Clarke as cited in Rosenquist, 1993). If you do not plan on harvesting your rotifer culture regularly, decrease the feeding from daily additions of microalgae to aperiodic or weekly additions to decrease the likelihood of high population densities leading to culture crashes. When shutting down a culture, I have simply placed water siphoned from an old culture in a flask near a window. I cover the flask to reduce evaporation, and add DI water as necessary to keep the salinity constant. These cultures survive without additional food for months at a time, and although the densities become very low, when I begin feeding again the culture quickly blooms to it's former density.

Personally, I maintain my culture water conditions as close to the conditions of the rearing tank in which I intend to feed the rotifers. My rationale is that adding water from the culture will not adversely affect the larvae which I am trying to rear, nor will the rotifers be shocked by be dumped into water with a significantly different temperature, salinity or pH. Although I have not followed this practice myself, Wilkerson (1995) recommends keeping the specific gravity of your rotifer cultures between 1.014 and 1.017. She says that this helps to keep the pH of the culture at 7.9 or below, and that she uses the pH decreasers sold for freshwater aquaria to maintain this pH. As I said, I have never tried this myself, and am unsure exactly why she recommends this practice, but she has had excellent success with her rotifer cultures (in 1995 she reported having kept descendants of an individual culture going without a crash since 1991). If you are having difficulty keeping your cultures going, you may want to try her technique to see if it improves your success rate.

Well, as I said at the outset, my goal in this article was not so much to give you a detailed account of how to culture rotifers at home, but rather a general overview of some of the techniques and commitment involved in setting up and maintaining a rotifer culture at home. If you have plans to try this at home, I strongly suggest that you locate all the references mentioned herein, that you read them al, and then distill the disparate view points into a technique that is likely to work best for you. Good luck, and I hope to see many more reports of success with captive breeding efforts appear on the Breeder's Registry!

Literature Cited:

Brusca, R.C., & G.J. Brusca, 1990. Invertebrates. Sinauer Associates, Inc. Sunderland, Mass. 922 pp.

Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Culture of Marine Invertebrate Animals . W.L. Smith and M.H. Chanley (eds.), Plenum Publishing Corp., New York, NY. pp. 29-60.

Moe, M.A., Jr., 1982. (Revised 1992) The Marine Aquarium Handbook: Beginner to Breeder . Green Turtle Publications, Plantation, FL. 318 pp. Rosenquist, M., 1993. Rotifer Culture. The Breeder's Registry Vol.1(#3), pp. 1,3-4.

Toonen, R.J., 1996. Invertebrate culture, Part 1. Journal of Maquaculture Vol.4(#4), pp.

Toonen, R.J., 1997a. Invertebrate culture, Part 2. Journal of Maquaculture Vol. 5(#1), pp.

Toonen, R.J., 1997b. Invertebrate culture, Part 3. Journal of Maquaculture. In prep.

Toonen, R.J., 1997c. Invertebrate culture, Part 4. Journal of Maquaculture In prep. Wilkerson, J., 1995. Captive Food Chain. The Journal of Maquaculture Vol.3(#4), pp. 1-4.

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