o2^ EXPERIMENTAL CULTURE OF THE ESTUARINE ECTOPROCT CONOPEUM TENUISSIMUM FROM CHESAPEAKE BAY JUDITH E. WINSTON Reference: ?iol. Bul!., 150: 318-335. (April, 1976) EXPERIMENTAL CULTURE OF THE ESTUARINE ECTOPROCT CONOPEUM TENUISSIMUM FROM CHESAPEAKE BAY ^ JUDITH E. WINSTON 2 Department of the Geophysical Sciences, University of Cliicago, Chicago, Illinois 60637 Virtually all of the 3500 extant species of ectoprocts feed b\' filtering plankton. But in spite of the fact that they are common components of both marine and estuarine suspension-feeding- communities, little is known about the kinds of par- ticles which are utilized by ectoprocts as food. Gut contents of ectoprocts were described by Flentschel (1922) for Sargassttm- encrusting Membrampora tiiberailata, and by LIunt (1925) for two species from the Plymouth fishing grounds. The gut of Meiiibranipora tiiberailata held dia- toms, coccolithophores, peridinian dinoflagellates, and Physalia nematocysts. The two Plymouth area ectoprocts contained small diatoms, silicoflagellates, peridinians, coccolithophores, algal cysts and detritus. Hunt also observed the capture of small flagellates by ectoprocts and speculated that they might serve as food sources. Literature on the culture of marine ectoprocts has been scarce until recentl)'. Hasper (1912) fed young colonies of Bozverbankia pustulosa on the diatoms Nit2schia closterium jornia fiiiniita {=^ Phaedactyluin tricornutuni?) and the cyanophyte Pleurococcus mucosas. Schneider (1959, 1963) cultivated Biigula azncularia on the colorless dinoflagellate Oxyrrhis marina. Bullivant (1967, 1968) found that colonies of the denost?me Zoobotryon verlicillatum grew well on the chrysophyte flagellate Monochrysis lutheri and the aberrant diatom Cyclotella nana and the coccolithophore Criocosphaera carterae, but could not grow on the dinoflagellate Amphidiniuni carterae, the green flagel- late Dunaliella tcrtiolecla, or the diatom Thalassiosira fliiviatilis. Further experi- ments showed that colonies of the cheilostome Bugiila neritina grew well on Monochrysis, but not on Phaeodactylum-. Recentl}' Jebram (1968) has used Oxyrrhis marina (fed upon Dunaliella) and Cryptoinonas sp. to culture several ectoproct species including Alcyonidiuni sp., Boiverbankia gracilis, Farella repens. Plectra crustulenta, E. monostachys, Cono- peum seurati, C. reticulurn and Biigula sfolonijera. Menon (1972) has also used Cryptomonas sp. to maintain colonies of Elcctra pilosa, Conopeiim reticuhiin and Membranipora ?neuibranacea for experimental work. The existing literature seems to suggest then, that each ectoproct .species may react differently to each of a variety of food species. The factors responsible for such difl:'erential growth responses are unknown and recluir? examination. ^^ Contribution no. 671 from tlie Virginia Institute of Marine Science and no. SI from the Harbor Branch Foundation, Inc., Ft. Pierce, Florida. Based on a portion of a Ph.D. disserta- tion submitted to tlie University of Chicago, August, 1974. This research was supported by grants from the Hinds Fund, University of Chicago. 2 Present address: Smithsonian Institution, Fort Pierce Bureau, Rte. 1, Box 196, Fort Pierce, Florida 33450. 318 THE CULTURE OF CONOPEUM TENUISSIMUM 319 This report examines the growth reactions of one species of ectoproct fed a variety of foods. The purpose of this experimentation is to identify some of the factors which may be responsible for differential growth responses and to examine the role of nutrition as it affects colony morphology. The estuarine cheilostome Conopeum tenuissimnm was chosen as the ecto- proct to be used for the series of culture experiments. This species was common in the research area and is an important component of estuarine fouling com- munities along the east coast of the United .States from Maine to Florida. METHODS Conopeum colonies used in the culture experiments were collected in the York River at the Virginia Institute of Marine Science (VIMS), Gloucester Point, Virginia. Artificial substrates, i.e., bryozoan traps, were suspended from the VIMS pier for several days. The glass slide sub.strates were then removed and examined for Conopeum colonies. When one was located near the center of a slide it was isolated by careful cleaning and removal of all other organisms on the slide. The size of the experimental colonies at the start of the culturing period ranged from 2-28 zooids. Six to eight colonies were grown on each experimental food medium. The slides bearing the colonies were placed in rectangular pyrex dishes capable of holding one liter of water. Slides rested on the bottom or against the sides of the dishes .so that expanded polypides were in either an upright or a horizontal position. The colonies were cleaned and rotated in position in the dish each time the feeding medium was changed. It was assumed that the position of colonies had no eft'ect on growth as long as there was no build up of debris. This is indi- cated in nature by the occurrence of colonies on all surfaces of eelgrass and other substrates. The experiments had l)een planned to begin in late June, 1972. At this time the Virginia coast and Chesapeake Bay were struck by the rains and flooding associated with tropical storm Agnes. This flooding caused salinities over the entire Bay to drop drastically. At Gloucester Point, Virginia where salinities in late June are usually around 16-17%c, the salinity dropped from l5.8^o on the 23rd of June to 8.8%.. on the 28th. It appeared that it might be several months before the York River salinity reached its pre-Agnes level, and it was feared that this low .salinity might affect growth. Thus it was decided to raise the colonies on York River water mixed with enough water obtained from the Eastern Shore of Virginia (marine) to reach a salinity of about 16%^. This mixture was filtered through the laboratory filter system. Water was changed three times weekly, and tanks were cleaned and colonies fed at the same time. Temperatures for the 1972 experimental period ranged from 23.0-26.5? C and salinities from 15.8-17.5%^. Each of the media used to culture the Conopeum colonies is described below. The chrysophyte flagellate Monochrysis lutheri (5.4 ?/.m) was chosen as the first experimental food because it had been found to promote growth in numerous other invertebrate species including oyster larvae. Dunaliella tertiolecta (6x9 ?im), a chlorophyte flagellate, has also been utilized in culturing experiments, but reports of its food value have varied. 320 JUDITH F.. WIXSTON Va-12 is an unidentified ehrysopliate flagellate isolated from a "red tide'' in the York Ri\'er. Cells of this species ax'crage 4.5 X 4.2 /j.m in size: the shape is slightly longer than broad, the apical end somewhat smaller than the posterior end; some cells appear slightly concave on one side. I'his species was tried be- cause oyster larvae being cultured at VT^^S had shown good growth when \'ii-]2 was used in combination with other loods. Nannocldorix occtilala is a nonmotile chlorophyte only 2-3 /j,m size. This small species has also been used successfully in oyster larviculture at VIMS. Cyclutella nana (6.4 /iUi). a small centric diatom with a siliceous cell wall, was chosen because of its size and because it had been utilized in the culture ot other invertebrates. Gymnodiniimi simplex is an unarmored dinoflagellate 6 X S /tm in size. l?e- cause dinoflagellates are often an im\)ortant component of nearshore and cstuarine ])h\-toplankton, I decided to try to culture Conopeum on si)ecies that would be found in its natural habitat, (iyninodiniiim simplex, while never a dominant in the York Ri'.-L-r waters, occurs with some frequency (,MacKiernan, 1968) throughout the year. jlnaeyslis iiiariviis is a small (1-2 /jni) rounded species of cyanophyte. In the inner parts of the bay, blue-green algae are often of considerable importance. Therefore, it seemed flcsirable to test the value of a member of this groii]:) as a food for Conopeum colonies. While various worlcers ha\-e attempted to cidture freshwater organisms on blue-green algae, there is no information on their use as a I'ood for marine or estuarine invertebrates. liecause other invertebrates had been sliown to grow better on a combination of foods than on a single algal food, two mixtures of foods were tried also: Monoelirysis-Diiiialiella, and Monochrysis-Thinaliclla-Cyclolella. In preliminary exj)eriments during the jn-evions year, oyster tank detritus had failed to support growth of Conopeum colonies, so it was decided to try leeding colonies dircctU' with a culture of bacteria which had been isolated from tlie sides of the owster tanks at VI.MS. This bacterium h;is not been positively identi- fied but is probably a pseudomonad or Vibrio species (S. Rivkin, VLMS, personal communication). In atldition to the culture of Conopeum on the different diets listed above, colonies were also cultiu'ed on five different concentrations of Monoehrysis and also five concentrations of Dunaliella. ^ The algal cultures used were obtained from Dr. FranklN-n D. Ott of the VIMS algal culture lab. 'Jlie cultures were grown in large aerated glass vessels with constant illumination |)rovided. Nannochloris was cultured at room temperature; the other species, at 16? C ? 4? C. The medium used in culturing the algae consisted of filtered York River sea water enriched by a stock nutrient mineral medium. The algae were filtered through a 50 ii.m filter to remove any undissolved par- ticles of medium, etc., and added to one liter of York River water wliich had been ])assed through a sand filter and then through two 1.0 ?i.m Cuno cotton filters. At the time the medium was renewed, the colonies were carefully cleaned with a soft brush and the di.shes were .scrubbed clean of any detritus, dead algae, and fecal material that might have settled. THE CULTURE OF CONOPEUM TENUISSIMUM 321 y\t each feeding- the following quantities of algal medium were used: (1) for single foods, 60 ml of algal suspension; (2) for the two-food mixture, 30 ml of each algal suspension; (3) for the three-food mixture, 20 ml of each algal suspen- sion; (4) for the first control, 60 ml of algal medium only; (5) for the bacteria, one ])ipetteful of bacteria; and (6) for colonies being given the various concentra- tions of Monochrysis or DunaUella, 15, 30, 60, 90, or 120 ml/liters of sea water at each feeding. Although the numbers of algae present i)er ml of culture suspension varied both according to species and according to stage in the growth of the algal population cycle, it was desirable to have an estimate of the quantities being fed to the ecto- procts. To facilitate estimation of food concentration each time the algal food suspensions were obtained a few ml were removed, the cells killed with a drop ot formalin and immediately counted under the microscope using a hemocytometer. The experiments ran for about 40 to 42 days for two reasons. P'irst, by this time reproduction would have occurred in nature, and it wa.s desired to see if some foods supported reproductive activity ; and secondly, by this time the colonies could grow to the edges of the slides and thus nullify further quantitative growth records. The growth of colonies was recorded by photography at ?east five times during the experimental period : at the start, at the finish and at three other times during the six week growth period. The few exceptions were due to photographs lost in processing, and the reduced photography of series exhibiting very little growth. The photographs were arranged to prove a setjuential picture of the changes in size and shape of each colony as it grew. Measvu-ements were made of the num- ber of zooids and number of generations (zooids in a direct line out from the ancestrula). These data were used to construct both growth curves for individual colonies and mean growth curves for all colonies receiving a particular diet. RESULTS General patlenis oj eolony groiuih. In the initial four to six days of culture all yoimg colonies of Conopeimi in- creased zooid nutnbers at an exponential rate. This re.s])onse is independent of diet as even starved colonies exhibited such initial growth. After this initial growth period both zooid number and colony growth pattern were found to be diet dependent. Each food treatment produced consistent characteristics of colony growth. The best single species food, DunaUella, produced a healthy roundish colon)' similar in shape to colonies ob.served in nature (Fig. lA, B). In contrast, a poor food such as Cyelotella produced a colony response typified by radiating biserial chains with few zooids (Fig. ID). Most of the zooids [)resent were heavily calcified and lacked functional polypides. Grozvth responses to single species foods Growth of Conopenni colonies was quantified by direct zooid counts. Table I and Figures 2 and 4 summarize and contrast the responses of the colonies to various foods. From Table I and Figure 2A it is evident that Dunaliella and Gyvinodinimii produced the best growth of all single foods tested. DunaUella- FiGURK 1. Examples of variation in growth form among Cono/>ciiiii Iciiuissiinmn colonies raised on different foods. (A) Example of a colony raised on a good food, the chlorophyte flagellate Dnnaliella Icrtiolccta, after 40 days of growtli. Note the large number of zooids and almost circular shape of the colony due to distolateral budding to fill in the space between tlie major branches. (B) Morphology of a colony grown in the natural York River environ- ment, after 20 days of growth. Dark coloration is due to food in the gut of polypides. P0I3'- pides in the outermost rows of zooids are still developing and have not yet begun to feed. (C) Example of a colony raised on a fair food, the chrysophyte flagellate Monochrysis ?ulheri, after 42 days of growth. Colony shows typical biradiate, branching shape, due to budding along major growth axes only. (D) Example of a colony raised on a poor food, the diatom CyclolcUa nana, after 41 days of growth. Colony has small number of zooids, arranged in six branches. Most zooids arc lacking polypides and are heavily calcified. fed colonie.s (7/15-8/26; 42 days) exhibited the healthy circular form (Fig. lA). At the end of the 42-day culture period all colonies appeared healtiiy. Observa- tion of polypides showed some with intertentacular organs for release of eggs ; these zooids contained ovaries with developing eggs. Other zooids had regenerat- ing polypides and developing ovaries. Gymnodimnm was also a good food for Conopeiim (Fig. 2A). The Gym- nodin?mn-i(t? colonies (7/15-8/19; 8/21-10/2; 42 days) always had a pinkish or orangeish tinge due to the dinoflagellates in their guts. This unique colora- tion was the same as that observed in colonies taken from the York River, sub- stantiating the idea that this species might naturally feed upon suitably sized dinoflagellates. It was not possible to examine the total growth response of Conopeinn to Gymnodinimn, as a failure in the VIMS algal culture system eleven THE CULTURE OF CONOPEUM TENUISSIMUM 323 TABLE I Ralio of mean number of zoo ids to mean number of g?n?rations in Conopeiim colonies after six -weeks of culture. A generation is the longest chain of zooids in a direct line outward from the ancestrula, usually measured on primary (distal) chain. Food ilyed Mean niimher of Mean number Ratio of zooid colonies KCncratlons ot zooids number to per colony per colony generation number Mono-Dun-Cyclo 36 1629 45:1 Dun-Mono 2') 1291 45:1 Dunaliella 33 2484 75:1 Gymnodinium 22 1409 64:1 Monochrysis 40 370 9.3:1 \"a-12 31 201 6.5:1 A nacystis 12 48 4:1 Cyclotella 10 35 3.5:1 Nannochloris 15 42 2.8:1 Bacteria 7 16 2.3:1 Control (alffal nicdiuni) 6 11 34 3.1:1 Control (sea water only) .S 8 21 2,6:1 days after commencement of the culture run killed the Gymnodiniiiui culture. The colonies initially responded with rapid growth .similar to that of Dunaliella- fed colonies. As the quality of the Gymnodiniiim. decreased, the more or less even outward growth stopped and colonies begun producing radiating biserial chains. Once the algal culture was dead colony growth gradually ceased. This culture was repeated (8/21 to 10/2) and again the algal culture fluctuated in concentration and vigor. While the food supply was declining, the growth rate of the ectoprocts decreased, with many of the polypides in the central portions of the colonies degenerating. Once the food started to increase in density, the rate of growth of the colonies also increased. At the end of the culture period two of the four cultured colonies contained some zooids full of sperm as well as some with developing eggs and polypides with intertentacular organs. Two other colonies, both smaller than those above, showed no evidence of reproductive activity. Foods producing fair growth of Coiio?yeii?n were Monochrysi.^ (7/15-8/26; 42 days) and Va-12 ( iMg. 2C). Hoth foods produced colonies of intermediate form (Fig. IC). Zooid production was considerably less than for Dnnaliella-ied colonies (Table I) and neither food supported production of polypides with intertentacular organs or other reproductive structures. The foods producing poor Conopeitni growth were Nannochloris, Cyclotella, Anacystis and bacteria. The Ncinnochloris-fed culture (7/15-8/25; 41 days) produced a mean growth curve (Fig. 2D) very different from the growth on the other chlorophyte species Diiualiclla. At the end of the culture period one colony was broken and appeared to lie decaying. Among the other colonies, some had broken zooids while a few appeared to have functional polypides and were pro- ducing occasional new buds. Conopcum cultured on Cyclotella nana (7/15-8/25; 41 days) appeared in poor condition. At the end of the culture period there were functioning polypides only 324 JUDITH E. WINSTON --C Mono -Dun-Cyclo -D Mono - Dun DAYS OF CULTURE o 20 30 40 50 DAYS OF CULTURE FiG?Ri? 2. Mean growtli curves for Conopcum tcnuisshnum colonies cultured on various algal foods. (A) Colonies raised on two good foods: Dunaliella, based on growth of seven colonies; Cymnodimmn. based on growth of four colonies. (B) Colonies raised on algal food mixtures: Monochrysis-Dunaliclla-Cyclolella, based on growth of six colonies; Monochrysis- DunalieUa, based on growth of eight colonies. (C) Colonies raised on two fair foods: Mono- chrysis, based on growth of five colonies, Va-12, based on growth of seven colonies. (D) Col- onies raised on two poor foods : Navnochloris, based on growth of five colonies ; Cyclotella, based on growth of eight colonies. THE CULTURE OF CONOPEUM TENUISSIMUM 325 in the outermost zooids. In these colonies tlie gymnocyst appeared to be more heavily developed than in those fed on any other food and only inner portions of some colonies had any lateral spines (Fig. ID). Conopeiim-ied Anacystis marinus (7/15-8/19; 35 days) grew poorly, but initial growth was greater than exhibited by colonies fed Nannochloris or CycloteUa. Unfortunately the stock culture of Anacystis was among the stock cultures affected by the VIMS culture failure (July 26, ?972), and it could not be regrown in time to start a new experiment. For the first eleven days of the culture period, the mean growth rate exceeded that of other poor-food cultures ; but after the loss of the Anacystis culture, the growth rate was greatly retarded and lagged behind all other algal foods. There were, however, some live zooids present in all colonies at the end of the 35-day period. Bacterial food (7/15-8/25; 41 days) appeared to be the worst of all foods tested for Conopcmn. Growth was less than that exhibited by colonies given no food at all. At the end of the culture period, one colony had decayed completely. All other colonies had at least one functional polypide, but were obviously in a state of decay with many broken zooids. Grozuth responses to food combinations Conopeinn was cultured on a bi-algal mixture of Monochrysis and Diinaliclla (7/15-8/26; 42 days). This mixture proved to be a good food, giving better growth results than any single foods except Diinaliella and Gymnodiniuni (Fig. 2B). Post-culture examination showed one zooid with an ovary and several poly- ])ides with intertentacular organs. The shape of the colonies grown on this mixture was intermediate between that of colonies on a Monochrysis-?\et and that of those on a Diina!ie!la-d\et Conopeinn colonies were also cultured on a tri-algal diet of Monochrysis. Diinaliella, and CycloteUa (7/15-8/26; 42 days). This tri-algal mixture also proved to be a good food. The mean growth rate was similar to the growth rate of colonies fed the Monochrysis-DunaUella mixture, the only difference being that colonies given the three food combination had a slightly higher final mean zooid count (Table I). The tri-algal mixture also supported colony production of re- productive structures and an intermediate colony shape. Groivth of Conopeum in the natural environment Attempts were made to assess growth of Conopeum in nature by placing colonies isolated on slides in a holder suspended from the VIMS pier into the York River. Unfortunately after about 20 days, overgrowth by other organisms was so intense that accurate zooid counts were impossible. A second attempt to assess natural growth was made by placing isolated colonies in an oyster culture tank receiving a continuous flow of York River water. This system eliminated the overgrowth problem. It was felt that this simulated natural conditions, except for lack of predators and a slightly lower water temperature. A comparison of the growth rate in the oyster tanks to cultured growth rates substantiated the belief that laboratory diets and cultures do serve to approximate growth in nature and can be used and interpreted in that light. 326 JUDITH E. WINSTON Control cultures It was also deemed important to examine whether or not the algal medium itself could cause significant growth in Conopeum colonies. Colonies were cul- tured 41 days (7/15-8/25) on filtered sea water, using 60 ml of algal medium per feeding. Some initial colonj' growth occurred, but this was thought to be a result of polypide feeding on tiny diatoms which entered the culture ina the glass slides used to isolate the colonies. At the conclusion of the culture, zooids of most colonies were broken or empty. The growth response was similar to that pro- duced in colonies fed the diatom CycJotella. A second control experiment examined the growth potential of a colony given only its own food reserves and cultured in filtered sea water. Initial growth response produced a growth curve with a slope similar to those of Cyclotclla, Nannochloris, and the algal medium. However, between two and three weeks after the start of the cultures the colonies started to decay. One colony had died by the 22nd day of culture. This colony had only two initial zooids at the start of MEAN ZOOID INCREASE A Dufial'iella . , , , P , 1?1?1 1 1 1 i-| 1 1?1?1 1 1 1 1 1 1 1?1?r B Mnnochrysis ( HIGH SALINiTV) 1 SlOnn cells/ml .... ] 10?flOO cei:s/ml .1 203,000 cells/ml ^^^.r^ _ . j '" ..?^^SSSi- 1 Any.nnn r.Riis/mi c Monochr, sis ( LOW SALINITY) W-m 102.000 cells/ml , ? ;-:1 20a000 cells/ml ?-???-??.:| 305,000 cells/ml FIGURE 3. Growth of Conopeum tcnuhsimmn colonies on various concentrations of algal foods (given as mean zooid increase): (A) Dunaliclla, 15, 30, 60. 90, 120 ml per feeding; (B) Monoclirvsis (\6'U salinity), IS, 30, 60, 90, 120 ml per feeding; (C) Mnnochrysxs {\2%o salinity), IS, 30, 60, 90, 120 ml per feeding. Figures indicate average concentration of the food (in cells/ml/1 sea water) for each of the treatments. THE CULTURE OF CONOPEUM TENUISSIMUM 327 culture and therefore a low nutrient reserve. However, another tvvo-zooid colon}' survived 41 days, produced a total of six zooids and had one zooid with a polypide at the end of the culture. Of the other six colonies, four appeared to have at least one live zooid ; the other two colonies were in a definite state of decay. Growth response to various concentrations of food Since food concentrations varied with both species and time, it was desired to test the effects of food concentration on colony growth. The results of three culture experiments utilizing similar concentrations of foods indicate that Cono- peiini growth was a direct function of food concentration up to about 60 ml. Above this amount there was no significant increase in growth. Figure 3 sum- marizes the results of the concentration of experiments. The various concentrations of Monochrysis used to culture Conopeum generally supported moderate colony growth and a colony shape indicative of foods of moderate value (Fig. IC). Due to a change in laboratory water conditions, colony cultures using Mono- chrysis were carried out at two salinities. For the first series, salinity was 12%o (7/17-8/26; 40 days). Colonies fed various concentrations of Monochrysis at the low salinity sup- ported growth, but there was frequent colony death. There were no colony deaths at the higher salinity, but there was evidence of zooid breakage. Other differences believed related to salinity were: (1) growth rates for the colonies fed small amounts of Monochrysis were lower for the colonies raised at the higher salinity than for those raised at the lower salinity; and (2) those colonies fed at lower salinities had more closely-grouped growth curves than those rai.scd in the higher MEAN ZOOID INCREASE Wm?Ma.,',,., d Monochrysis T;e$^y-1 1 i if/ya JJ Ar?r/st'i. C> clot ella Algal medium Xffl I Filtered seawater Bacteria Dunali^li? Mono -Cyclo. -Dun. Oymnodini'jrn Mono. - Dun. M?A\ GROWTH INCREMENTS ? El ? 0"6 6 -!3 13^21 21 - 28 28-35 35-42 FIGURE 4. Growth of Conopeuyn tenuissimum colonies on all foods investigated (given as mean zooid increase). 328 JUDITH E. WINSTON salinit}'. The reasons for these differences are unknown but suggest further research. Conopentn. cultures on various concentrations of DnnalieUa (8/21-10/2, 42 da}'s) were compared to il'/?;wcir/;r3'?/?-cultured colonies. The JJunaliclla-ied cultvires had to be moved from VIMS to Woods Hole, Massachusetts (9/7/72), and the sub- serjuent growth of these colonies reflected some degree of stress. The colonies did recover however. From Figure 3 it is obvious that those colonies fed the smallest amount (15 ml) of DiinaUella did not grow ap|)reciabl3' more than those fed (he smallest amount of Monochrysii: at a similar salinity. Those fed 30 ml, however, showed slightly more growth than those fed 30 ml of Monochrysis. It is interesting that even in colonies fed the smaller amounts of Diinaliclla, some showed the growth pattern characteristic of those on good diets, with zooids filling in between the main branches, rather than just growing outward in uniserial chains from the ancestrula. The colonies fed 60, 90, and 120 ml of DnnalieUa had very similar and closel}' spaced growth curves. Those fed 120 ml showerl (he least indivirlual variation between individual colonies. Colonies fed 60 90, and 120 ml of Diinaliclla achieved better growth than those fed 60, 90, and 120 ml of Monochrysis. The I)iina!iel!a-ied colonies showed quite circular growth patterns compared with those fed Monochrysis. When the colonies were examined at the end of the experimental period, none of the colonies showed any signs of reproduction except for the colonies fed 120 ml, which had polypides with intertentacular organs and ovaries in the zooids. It was ob- served that many of the polypides in the colonies fed 90 and 120 ml amounts of DnnalieUa had lopho])hores that were shorter than normal and Ixxlies that were like large sacks, possibly an adaptation to bloom conditions. DiscussroN^ To aid in the interpretation of results, bar graphs showing the amount of growth (as mean zooid increase) for Conopcuui colonies on all diets were con- structed (Figs. 3 and 4). For the food concentration experiments. Figure 3 shows only total mean zooid increase. The more detailed graph (Fig. 4) shows the amount of growth that occurred on each diet after 6, 13, 21, 27, 35, and 42 days. Growth at each of these points was measured directly from colony photographs when possible, and in a few cases interpolated from the mean growth curve. Examination of these figures and Figure 1 leads one to conclude that different species of algae affect both pattern and amount of colony growth. The effect of food on colony growth patterns is of interest with respect to life strategy of this estuarine species. Colonies on a good diet (Fig. lA) developed buds not only along the major growth axes, but also distally and laterally to fill in the spaces between the branches, giving the colony a circular rather than double-fan-like form. Colonies fed fair foods {Monochrysis and Va-12) gen- erally grew in a biradiate pattern, in chains only one to two zooids wide (Fig. IC). Though the chains leading out from the ancestrula in the distal and proximal direc- tions were often the longest, in older colonies this was only on the order of a few generations of zooids compared with the length of the secondary chains (which generally developed from distal-lateral buds produced from the young colony, THE CULTURE OF CONOPEUM TENUISSIMUM 329 either before the experiment started or during the first week of growth). The secondarj' chains were usually at about a 45? angle (the angle at which distal- lateral buds are produced) to the primar}' chains: thus, colonies commonly consisted of six main chains or branches (three to cither side of the ancestrula) in a bi- radiate pattern. Shorter tertiary chains developed l?y distal-lateral budding of the zooids of the primary and secondary chains. This development in all directions outward from the young colony suggests that colony energy is being put toward the location of a more nutritionally favorable microenvironment. It is worth noting that colonies fed good foods did not have a larger number of generations (measured as number of zooids in a chain outward from the ancestrula). For colonies fed Dimaliella the mean number of generations was 33 ; for those fed Gymnodiniurn, 22 : for the two and three-food mixtures, 29 and 36 respectively. For colonies given Va-12 it was 31 ; for those fed Monochrysis, it was 40. The poorly nourished colonies had mean generation numbers ranging from seven (bacterial-fed) to 15 {Nannochloris-it?). What this means in terms of colony strategy can perhaps be roughly quantified by a simple ratio between final mean zooid number and final mean generation lunnber (Table I). In well-nourished colonies, the amount of energy put into total colony growth including storage for maintenance and reproduction (as mea- sured by final mean zooid number) versus the amount put into outward expansion (as measured by final mean generation number) ranges from 45 : 1 to 75: 1, while ratios for those on fair foods ranged from 6.5: 1 to 9.3: \. For colonies on poor diets, the range was from 2.3: 1 (bacteria) to 4: 1 (Anacysiis). Thus colonies on a poor or fair diet were putting a proportionateh' much greater amount of energ}' into a "search" for a more favorable microhabitat, while well-nourished colonies were expanding as much as possible within their favorable habitat and were probably accumulating excess food energy (as shown by the onset of reproduc- tive processes in these colonies). What this could mean to colonies in their natural estuarine environment is apparent from a discussion of the life cycle (Dudley, 1973). If the colonies are not able to grow, store some energy and reproduce within about four weeks, colonies on hard substrates will become covered b)' other fouling organisms, such as colonies of the faster-growing, but less numerous ectoproct Menibrani[>ora tennis, as well as by tunicates and barnacles. Their other primar)' substrate, the eel-grass, is short-lived in summer and may come detached and decay ; therefore rapid completion of the life cycle is favored. Moreover, colonies in nature which find themselves in a nutrient-poor microenvironment due to current conditions probably also put all colony reserves into single chains of zooids, favoring expansion into an area where zooids can feed. Examination of colony growth curves (Fig. 2) shows that the response does not differ significantly from the sigmoid growth curves characteristic of many animals (Odum, 1971). Growth curves of young colonies taken from the natural environment had an exponential form (Dudley, 1973). This type of curve also appeared in the early growth of cultured colonies. As the cultured colonies in- creased in size, the growth curves began to approach the sigmoid form. Exponen- tial population growth can continue only as long as environmental factors are not limiting. As the number of zooids increase and factors such as space for colony 330 JUDITH E. WIXSTOX expansion decrease, the growth rate slows until it reaches the carrying capacitj^ of the environment. In the case of the cultured Conopeiiin colonies the chief con- straint on growth was probably food supply. To simplify the experiments, colonies were fed a constant amount of food, and the food was not increased as the colonies grew ; thus food supply might have been adequate for young colonies, hut inade- ([uate for the 1000-2000 zooids present in colonies at the end of the experimental period. Other limiting factors may also be operating. It has been found true for many organisms that .somatic growth diminishes or stops before sexual maturation, as energy is needed for the development of reproductive structures, eggs, brooding of larvae, etc. ITowever, few studies are available, concerning the effects of the reproductive jjrocess upon the growth of colonial organisms. As has been shown to be true for other organisms from i>rotozoans to crusta- ceans, different species of algae were found to be of var3-ing food value to Conopcitm (see Fig. 4). Two single foods, Dunaliella tertiolecta and Gymnodinium simplex, supported good growth of Conopeiiin. The results with Dunaliella are interesting since species of this genus have been shown to be of quite variable value to other organi-sms (Walne, 'l963, 1970; (iibor, 1956; Pilkington and Fretter, 1970). In addition Rullivant (1967) found no growth in Zoobotryon colonies fed Du- naliella except for an elongation of the stolons M'hich seemed to be a reaction to adverse food conditions and Schneider (1959) stated that Dunaliella sp. passed intact through the gut of Bitgula aviciilaria. The results with Gymnodinium are perhajjs even more interesting. A-^ery few dinoflagellates have been used in culturing experiments, and the few species tried (which were generally .s|)ecics known to produce toxic metabolites) have usually given ])00r results, f?uillivant (1967) found no growth and some disintegration in colonies of Zoobotyyon-iarently more efficientl)' utilized. The small centric diatom CycloteUa nana (= ThaUassiosira pseudonana) also appeared to be of little food value to Conopeiim. BuUivant (1967) found that CycloteUa produced some growth of Zoobotryon, a gizzard bearing ctenostome. but not of Biigiila, a species lacking a gizzard. I have also found that CycloteUa does not support growtli of Bugiila stolonijera. The colonies cultured on bacteria showed even less growth than those receiv- ing only filtered sea water. While there has been much speculation in the litera- ture concerning the role of bacteria in the nutrition of suspension-feeding organisms, most attempts to cidture invertebrates on bacteria have ended in failure. Bacteria in combination with phytoplankton may play some role in bivalve nutrition, but it is still unclear whether they are generally useful or harmful (Ukeles, 1971). It appears that certain species of bacteria produce toxic metabolites and others do not (Calabrese and Davis, 1970). The bacteria utilized in the Conopeiim experi- ments could not have produced extremely toxic products as the colonies did not 332 JUDITH E. WIXSTOX die immediately. The reason for poor growth may he one or a combination of the following: the cells were present in too low a concentration to have nutritional value; or, as has heen shown to be the case for many marine bacteria (Provasoli, Conklin and fi'Agostino, 1970), they created an unfavorable environment for the ectoprocts by acidifying the sea water and reducing the oxygen concentration. 'J'he nutritive value of algae appears important in that not all foods that supported growth were adequate for reproductive activity-. The fact that not all the foods tested were able to sui>port reproductive activity of Conoj^euin may be accounted for either by assuming that: (1) not all algae are alike in food value, especially in the amounts of trace elements, vitamins or other factors neccs.sary for reproduction ; or (2) colonies cannot undergo reproduction until they reach a certain size, as measurcfl in the innuber of zooids. Actually both factors may be 0|)craling, but in the absence of any knowledge of a minimum-size requirement (and such a re- quirement would seem to be less likely in an r-strategist like Conopctivi, than in a generally long-lived species which puts its energies first into obtaining as much substrate as ])ossible by a.sexual growth), it is most probable that the food is lacking in some element necessary for the initiation of the reproductive processes. The survival of some colonies for as long as six weeks, during which time they received only filtered sea water, suggests that colony reserves are important in insuring the survival of the species in the event of unfa\'orable conditions. As is apparent from I<'igure 4, growth of the colonies fed good, fair and poor foods is distingui.shable even after only six days of culture. The control colonies (filtered sea water) grew as much as the colonies fed poor foods for the first six days. ISctween six and thirteen days the controls showed more growth than the colonies fed bacteria, but were apparently becoming more and more unhealthy. By the time they were photographed again at 21 days, zooids had degenerated and decayed so that the total mean zooirl number was less than it had been after only 13 days. From 21 days to 42 da\-s the colonies made only a tiny increase in growth (from X = 12 to .V = IS zooids/colony). This very small increase may have been due to remaining food rescr\'cs or to ingestion of benthic diatoms or bacteria that could not be completely removed from the dishes or the surfaces of the colonies. For colonies receiving 60 ml of sterilized algal medium per feeding, growth continued up until the third week (Fig. 4), and even after six weeks was almost equivalent to the growth of colonies receiving Cyclotella. As was mentioned in the results section, continued growth may have been due to the fact that numbers of small benthic diatoms were encouraged by the algal medium and mi,ght provide some nutrition for the Conopeum colonies. Conopeittn growth response to a combination of algal foods was better than to most single foods. Colonies fed a mixture of two foods, Monochrysis and Dnnaliella, showed better growth than colonies fed all hut two single foods (Fig. 4). jNIoreover, the growth curves for all of the colonies grown on the two-food mixture were remarkably similar. The results are not surprising since a mixture of foods has been found to support better growth than a single algal food for many other suspension feeders (Davis and Guillard, 1958; Ukeles, 1971; Provasoli, Conklin and D'Agostino, 1970). Growth of Conopctim on a mixture of three foods {Monochrysis-Dunaliella- Cyclotella) was slightl}' better than on a mixture of two foods and better than on THE CULTURE OF CONOPEUM THNUISSIMUM 333 an}^ single food except Diinaliella. Calabrese and Davis (1970) stated that a mix- ture of several species of algae caused larvae of Crassostrea and Venus to grow more rapidl}'. The}' found that a mixture of chrj'somonads and green algae (no species given) appeared to provide a more balanced diet than any single food. The fact that, in a 2-3 food mixtiu-e, the Conopeum colonies were receiving only one-half or one-third the amount of the "good" food suggests that the important factor lies in some form of nutrient balance rather than in the quantity of nutrient offered. The culture experiments using various concentrations of algae showed that food value increased with increasing amounts of food only up to a certain concentration. Figure 3 shows the effect of increasing food concentrations on colonies fed Dunaliella and Monochrysis (at high and low salinities). In the colonies fed ?unaliella (Fig. 3A), growth increased at food concentrations up to 177,000 cells/ml (average quantity), but seemed to be slightly decreased in colonies receiving an average of 236,000 cells/ml. This decrease could be accounted for b}' two factors. First, as several workers have noted, suspension feeders will ingest food in increasing quantities only up to a certain "satiation concentration" related to the ma.ximum amount of food the individual can process in a certain time period (Eullivant, 1967). If the highest concentration tested was above the maxi- mum that the colonies could consume during the time between feedings, then there could be no increase in growth due to increased concentration. Secondly, it is also possible that Dnnaliella produces a metabolite that at high concentrations has a negative effect on growth (which might explain the varied results noticed by other workers in culturing experiments with members of this genus). Colonies grown on Monochrysis in general showed an increase in size with increasing amounts of nutrient, but there was more irregularit}' in the pattern. Colonies grown at the lower salinity (Fig. 3C) appeared to grow better at the two lower levels of food than did those grown at the higher salinity (Fig. 31)). At the highest concentration (406,800 celLs/ml) offered, both series achieved approxi- mately equal growth. JNI)' sincere appreciation goes to Dr. T. J. j\[. Schopf for his continued guidance throughout the course of this work. I thank the staff" of the Virginia Institute of Marine Science, particular!)' Dr. J. Dupu}', for providing laboratory space, Dr. F. Ott for maintaining algal cultures, and ^ir. .S. Rivkin and Ms. N. AVindsor, for their aid and encouragement in the lab. Finall)', I thank Stephen Dudley, for his assistance with photograph)' of the cultures and his careful and critical review of the manuscript. SUMMARY 1. The pattern and form of colony growth in the ectoproct Coiiopciiin tenuis- simmn was found to be diet-dependent. 2. Poorly nourished colonies were characterized by their straggling shape and low zooid number to generation number ratios. These colonies apparently attempted to maximize substrate covered, facilitating location of a more favorable nutrient regime. 334 JUDITH F.. WINSTON 3. Well fed colonies were rounded in shape and characterized b}- high zooicl number to generation number ratios. These colonies maximized the number of zooids produced in their already favorable area, creating colony reserves, and pre- paring for reproduction. 4. Initial growth in all colonies was exponential, but after a few days of culture growth, rates varied with food used. The chlorophyte flagellate J)iinalieUa tertiolecta and the dinoflagellate Gyiimodiniuiii simplex were good foods, support- ing growth to more than 1,000 zooids and sexual maturation within the 42 day culture period. The chryso])hyte flagellate Munodirysis Inthcri and Va-12 (an un-named chrysophyte flagellate) proved to be fair foods, supporting moderate growth, but no sexual maturation. The blue-green alga Anacyslis marinns, the chlorophyte Nannochloris occulata, and the diatom Cyclotella nana supported little or no growth, while bacterial food did not support growth of Conopettm. cultures. 5. Combinations of algal food produced good colon}' growth, A three-species mixture was better than a two-species mixtin-e, suggesting diflierential yet additive nutritional contributions b)' each algal species. 6. Increased food concentrations supported increased growth only up to a cer- tain concentration. This growth response varied with salinity. LITERATURE CITED BL'I.LIV.\XT, J. 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Dougherty, Ed., The Loiucr Mctasoa, Uni- versity of California Press, Berkeley, California. UKELES, R., 1971. Nutritional requirements in shellfish culture. Pages 43-64 in K. S. Price and D. L. Maurer, Eds., Proceedings of the conference on coviwcrcially valuable shellfish, University of Delaware, Newark, Delaware. WALNE, P. R., 1963. Observations on the food value of seven species of algae to the larvae of Ostrca cdulis. J. Mar. Biol. Ass. U.K., 43: 767-784. WALNE, P. R., 1970. Present problems in the culture of tlie larvae of Ostrea cdulis. Ilel- golaender Wiss. Meeresunters., 20: 514-525. WINSTON, J. E. See Dudley, J.