BULLETIN OF MARINE SCIENCE, 28(2): 363-370, 1978 CORAL REEF PAPER

CIRCULATION AND DIGESTION OF FOOD IN THE GASTROV ASCULAR SYSTEM OF GORGONIAN OCTOCORALS (; )

Gordon R. Murdock

ABSTRACT Colonies of porosa were fed radioactive food while in situ on the reef, then collected after various elapsed times. The distribution of radioactivity in each colony was determined and chemical fractionation of the coral tissues with TCA and ethanol was used to provide an indication of the progress of digestion and assimilation of the food. Much of the food is assimilated near the point of ingestion. Digestion proceeds rapidly and resembles that reported for the sea anemone Aiptasia. Currents measured in the stem canals of nine of gorgonians gave estimates of maximum speeds ranging from 0.55 mm sec'l to 0.97 mOl sec-1 with most values in the upper part of the range. Some observations are presented on the behavior of currents in the gastro- vascular canals.

In the colonial cnidarians, the ectoderm, colony by means of the ciliated canal system gastroderm, gastrovascular cavity, and me- and be taken up by cells in the colony at sogloea are continuous throughout. Al- points remote from the site of ingestion. though the specific morphology differs from She had found in 1905 that fish meat group to group, in all cases gastrodermally stained red with borax carmine and fed to lined canals or spaces interconnect the a was broken down to small pieces coelenterons of all the zooids so that it is in its coelenteron and that these pieces were reasonable to think of such colonies as then phagocytized by cells in the six asulcal organisms with many mouths rather than as mesenterial filaments (those not associated assemblages of individuals. with the siphonoglyph or sulcus). Once in- In the gorgonian octocorals with which corporated into food vacuoles, the particles this study deals, the gastrodermally lined were observed to disintegrate and lose their links between the coelenterons of the zooids color, at which point she assumed digestion consist of ciliated canals. The relatively to be complete. Her description fits the large canals arranged around the axial skele- pattern which has emerged from the more ton and running parallel to it are called detailed and rigorous modern studies (Mus- stem canals, all other canals being termed catine, 1973). solenia (Fig. 1). These canals probably Pratt also maintained colonies of Alcyoni- convey nutrients to areas on colonies lack- um in suspensions of carmine particles for ing feeding polyps (e.g. the holdfasts of a week. Carmine particles were seen in the some colonies), but do they transport nu- gastrodermal cells of the sulcal mesenterial trients elsewhere and are there other means filaments (those nearest the siphonoglyph) of transport which function as well? after 2 days and in the gastrodermal cells Pratt (1905) suggested that food-derived lining the soIenia and in amoebocytes in the materials may be carried through the tissues mesogloea after four. She argues from this of Alcyanium digitatum by migrating amoe- that amoebocytes must be important in dis- boid cells or by being passed from cell to tributing nutrients within the colony. cell along chains of such cells. Later she Pratt's observations are valuable but some suggested (Musgrave nee Pratt, 1909) of her conclusions should be accepted with that materials may circulate through the caution. Borax-carmine stained fish meat

363 364 BULLETIN OF MARINE SCIENCE, VOL. 28, NO.2, 1978

11 7

,, ,, ,, / I I

5 Figure 1. A generalized diagram of part of a gorgonian colony. The arrangement of the stem canals is shown as is that of the solenia in simplified form. Redrawn and modified from Bayer (1956) with permission. (I, mesenteries; 2, mesenterial filament; 3, gonads; 4, axial skeleton; 5, stem canals; 6, solenia; 7, polyp; 8, tentacle; 9, coelenteron; 10, mouth; 11, pharynx.)

was apparently taken up by gastrodermal place within a few hours as did digestion of cells only in the asulcal mesenterial fila- zooplankton which she fed to the , ments while carmine particles appeared in whereas distribution of carmine particles ap- gastrodermal cells throughout the colony. parently required several days. Lastly, she The reason for the difference is not clear. notes in one place that phagocytosis of Also, phagocytosis of stained fish meat took carmine particles "is identical in every rc- MURDOCK: GORGONIAN CIRCULATION AND DIGESTION 365 spect with the ingestion of food material" Rees et al. (1970) have provided infor- while elsewhere she notes that "the amoe- mation on the movement of radioactively boid endoderm cells were observed to eject labelled food through colonies of the hydroid foreign bodies in the form of carmine Pennaria liarella, showing that while various particles." factors could influence the result, radio- Pratt's observations do show that material activity was found throughout the colony can enter the gastrovascular system, prob- within thirty minutes. ably through the polyps' mouths, and sub- The contents of the gastrovascular canals sequently become distributed throughout the of the hydromedusan Phialidium are circu- colony in gastrodermal and amoeboid cells. lated by flagella (Roosen-Runge, 1967) Indeed, while discussing the canal system and perhaps by local contractions. Collars in pcnnatulids in a later paper (Musgrave of flagella and local constrictions in the nee Pratt, 1909), she appears to be speak- canals may influence the pattern of circula- ing of alcyonarians generally when she says tion, but no overall pattern was obvious. "we have strong evidence for believing the It is clear that more information is needed fluid contents of the canal system to be on the functioning of complex gastrovas- composed mainly of seawater, in which nu- cular systems in cnidarians. This study uses trient matter may be dissolved or suspended isotopic tracers and modern field techniques in minute particles ... " In the absence of to provide quantitative information on the stronger evidence this must remain a sugges- normal pattern of events in situ against tion. which laboratory work may be compared. Parker (1920) has said that the flow of water through the canal system of Renilla MATERIALS AND METHODS amethyslina follows a specific route. Brafield The corals studied here came from reefs ( [969) could find no specific route for the along the Florida Keys. Pseudaplexaura flow in Pteroides griseum though there is parosa was studied on and collected from no doubt that seawater marked with Hens and Chickens Reef off Upper Mate- methylene blue injected into the colonies cumbe Key. The other species studied came was transported to other areas. variously from this reef, from Alligator Reef Circulation in hydrozoan colonies has just to the south, and from various small been studied as well. With these colonies, patch reefs in the vicinity of Big Pine Key. however, some quantitative data are avail- The S-35 labelled radioactive mouse liver able. was prepared by C. B. Cook at Duke Uni- Willem (1894) used fish meat marked versity by injecting a mouse intraperitoneally with india ink to follow digestion qualita- with a neutralized acid hydrolysate of com- tively in the siphonophore Apalemia uvaria. mercially grown S-35 labelled yeast (T. He showed that ingested food which had ulilis). A day later, the mouse was killed been partly digested extracellularly in the and the liver was found to have a specific gastrozooid was forced by contractions of activity of 0.15 p.Cijmg. Before being fed that polyp into the whole gastrovascular to corals on the reef, the radioactive liver system of the colony. Crowell (1957) has was minced into pieces about % mm3 in pointed out that in Campanularia, colonial volume, a few of which were then soaked growth rate and the longevity of polyps overnight in seawater in a refrigerator to seem not to differ on one colony between wash out any readily displaceable label. areas which are given food and areas which On the reef, individual polyps on colonies are not. He attributes this to the "vigor" of P. porosa were fed pieces of the radio- of the movement of fluids through the active liver with watchmaker's forceps and gastrovascular canals of hydroids and to the the position of the recipient polyp noted as "effectiveness" of that circulation. carefully as possible with respect to identifi- 366 BULLETIN OF MARINE SCIENCE, VOL. 28, NO.2, 1978

able landmarks such as branch tips or points tides, and large polypeptides or proteins of branching. As SCUBA equipment was respectively (Roberts et al., 1955; Lenhoff, used, it was possible to watch the polyp 1961; and Murdock and Lenhoff, 1968). which had received the food to make sure Observations and measurements of the that the food had been swallowed and to flow of water within the solenia and stem record the precise time at which the food canals were made on animals which either was ingested. It was found that soaking the had been just coIlected or had been main- bits of liver in a fresh homogenate of tained a few days in natural seawater with Artemia salina nauplii or cysts in seawater vigorous aeration. Only colonies which were for about an hour made them more accept- well expanded when undisturbed and which able to the coral polyps. contracted rapidly when touched were used. After a suitable period, the colonies To expose canals for observations, part of a which had been fed were cut just above the branch from a healthy was cut in holdfast, placed in plastic bags and put on half parallel to the branch axis and the axial ice. About 0.5 hr later they were put in a skeleton lifted out. The coenenchyme home freezer on shore. They were trans- around the axial skeleton usuaIly stayed ported to the laboratory on dry ice and kept stuck to the skeletal rod as it was pulled frozen until assayed. away, thus tearing open the stem canals. To In order to determine the distribution of minimize mechanical and physiological arti- radioactivity within colonies which had in- facts due to removal of a part of the canal gested labelled food, successive 5-mm long wall, observations and current measurements pieces were cut from branches bearing the were made only where a canal was torn open polyps fed. Each piece was homogenized just enough for the contents to be seen. and the radioactivity in an aliquot of this Carmine particles suspended in seawater homogenate was determined on a gas flow were dropped into the canals being observed radiation counter. Fractionation of the coral and the motion of those weIl below the tissue with its included radioactive material openings in the walls of the canals was ob- derived from the food was carried out using served with a dissecting microscope fitted the technique described by Roberts et a1. with a measuring eyepiece reticle. Flow (1955) which has been previously used to velocities were measured by timing the follow digestion in other cnidarians (Len- particles as they moved between two points hoff, 1961; and Murdock and Lenhoff, on the measuring reticle. Flow rates were 1968). The tissue, previously homogenized, measured only where they were judged was extracted successively with 5% (wtjvol) maximal on the basis of brief preliminary trichloroacetic acid (TCA) at room tem- observations and only where flow was un- perature, then at 45°C with 80% (vol/vol) impeded over a distance of at least 0.1 mm. ethanol acidified to pH 3.3. Samples of the Several replicate measurements were made two extracts were dried onto planchets for on most preparations. Measurements were counting. A known volume of the alcohol made in more than one canal in each piece extraction mixture was Millipore filtered and cut from a colony, and usually in more than the filter was fixed to a planchet to provide one piece of the colony. a measure of the material insoluble in both extractions. The three resulting fractions are RESULTS termed the TCA soluble fraction, the alcohol Figure 2 contains 24 histograms, showing soluble fraction, and the alcohol insoluble the distribution of recovered S-35 activity fraction. As just noted, this last fraction is along branches on which a polyp had been insoluble in the TCA solution as well. These fed labeIled mouse liver. The time associated three fractions are generaIly interpreted as with each histogram indicates the amount of containing free amino acids, small polypep- time elapsed between feeding labelled liver MURDOCK: GORGONIAN CIRCULATION AND DIGESTION 367

minutes •. 4 10:0-, 60 10°eL :! 60 · 10 o • 0 • 10 10°eL 65 40 o .5 10 70 80 90 1 ':~ '0 20 90 I~Lh c '~Ch 10 o , ·.. 20 30 '~~ 130 I~J~ 10 20 30 • 10 100 200 300 400 .00 37 'O:CL 135 lOOoLD time (minute.) • •• Figure 3. Radioactivity in the TCA soluble frac- · 10 tion expressed as a percentage of the total radio- 40 '~Ch 140 lO:Ck activity in the sample of coral. The results are ~ o I plotted as a function of the time elapsed between · , ingestion of the radioactive food and freezing of .~ 40 155 lOOOeL the colony. The arrow to the left of the ordinate li l~li:b~ 10 20 indicates the mean proportion of radioactivity in ~ 'GO • '0 the TCA soluble fractions from six samples of the m 40 165 lOO~ e> oeL food given the animals. ~ • 10 o .0 '0 40 390 C 'OOoCh '~Jl from a branch is expressed as a percentage ~ .0 • '0 of all the activity recovered from that branch !. 45 lOlL 400 I~Jl so that while different amounts of radio- 10 activity were recovered in different speci- • .0 mens, the area beneath each histogram in 55 415 lOlL 'OOO~ Figure 2 is the same. '0 '0 Notes taken when each polyp was fed 60 I~JL 427 'OOO~ indicate that in all cases but one (155 min), • 10 • 10 the region which gave the most recovered dislance from branch tip (mm) radioactivity contained the polyp which swallowed the labelled food. The one excep- Figure 2. The distribution of radioactivity in 24 tion is probably due to an error of 1-2 mm specimens given radioactive food ill situ on the reef, then collected after some time and frozen. in measuring the location of the fed polyp on The time in minutes elapsed between swallowing an expanded colony in the field. of the food and collection of the colony is indio Figures 3, 4, and 5 show the results of cated to the left of each histogram. Each distri- the chemical fractionation of those pieces of bution is given in terms of proportions of the total radioactivity detected in the samples assayed. P. porosa which showed the highest propor- tion of recovered activity in each histogram in Figure 2. The arrow to the left of the to a polyp and placing the cut colony on ordinate in each case represents the value ice. As mentioned above, the colonies were obtained from fractionation of the food fed frozen about 0.5 hr after they were put on to the animals. Each arrow is the mean of Ice. six values obtained from the fractionation The abscissa of each histogram indicates of as many food samples. the location of the pieces assayed with re- Little if any consistent change is seen in spect to the tip of the branch in question. the level of activity recovered in the TeA The activity recovered from each piece cut soluble fraction. During the first 165 min 368 BULLETIN OF MARINE SCIENCE, VOL. 28, NO.2, 1978

80 100 >- .." •.•. ~ . 60 . 1 80 . '.. ~ >- . ~ :l 40 <: • 11 60 ~ ~.. 20 ~

40 •0 ..~e 100 200 300 400 soo 20

Figure. 4. Radioactivity in the alcohol soluble fraction expressed as a percentage of the total o o 100 200 300 400 radioactivity in the sample of coral. The results are expressed and plotted as in Figure 3. The lime (minutes) arrow to the left of the ordinate indicates the Figure 5. Radioactivity in the alcohol insoluble mean proportion of radioactivity in the alcohol fraction expressed as a percentage of the total soluble fractions from six samples of the food radioactivity in the sample of coral. The results given the animals. are expressed and plotted as in Figure 3. The arrow to the left of the ordinate indicates the mean proportion of radioactivity in the alcohol insoluble fractions from six samples of the food after the food was ingested, however, the given the animals. proportion of activity found in the alcohol soluble fraction shows a consistent increase while that in the alcohol insoluble fraction reverse, the former decreasing and the latter decreases. The values obtained at about 400 increasing again. min for the activity in the alcohol soluble Two lines of information have been ob- and alcohol insoluble fractions suggest that tained on the water currents in the stem at some time between 165 min and 400 min canals of nine species of gorgonians. Table the patterns of change in these two fractions I lists values of the mean maximum veloci- ties observed in the various species. It should be remembered that these are not Table 1. Mean maximum flow rates observed in time averages of the flow velocities in the stem canals stem canals, but estimates of the maximum

Mean measured velocities. The preparations used did not velocity permit reliable measurement of the pattern Species (mm/sec.) S.E. of variation of flow velocity with time ex- Ellnieea knighti 0.97 0.11 cept as noted in the qualitative observations Ellnieea Sp. 0.93 0.04 below. Ellnieea ealyelliata 0.73 0.13 Carmine particles dropped into the lumens ElIllieea lacineata 0.59 0.01 of stem canals which had been torn open Plexallrella nlltans 0.94 0.03 generally moved along parallel to the long Plexallrella diehotoma 0.80 0.04 axis of the canal. In some cases, however, Plexallrella pillmilla 0.74 0.03 particles followed zig zag or roughly helical Plexallra flexuosa 0.84 0.06 paths through a canal, and in one extreme PselldoplexOllra porosa 0.55 0.03 case, a particle was seen moving in circles MURDOCK: GORGONIAN CIRCULATION AND DIGESTION 369 in a plane perpendicular to the long axis of transported beyond the region assayed. A the canal. No means was found for dis- laboratory based study parallel to this one tinguishing between normal behavior and (Murdock, 1978) suggests that most of the artifacts due to the opening of the canals. ingested food material is taken up in the The currents in any two stem canals may vicinity of the polyp which ingested the or may not run in the same direction, but food. generally the currents do not run in the same The fractionation results presented in direction in all the stem canals. Indeed, in Figures 3, 4 and 5 do not allow a detailed one preparation of , time course to be described for digestion of a current which was initially running toward protein by P. porosa, but insofar as the the base of a branch in a stem canal changed characteristics of such a time course are so that two currents diverged from a single evident, they are in good agreement with region. The current basal to that region those obtained for the gorgonian Plexaurella continued running basally while that distal nutans (Murdock, 1978) and for the anem- to the region began running toward the one Aiptasia sp. (Murdock and Lenhoff, branch tip. Such a change was seen only 1968). Thus, the proportion of label in the once. alcohol soluble fraction increases within the Solenia open into (or out of) the stem first two to three hours that food is inside the canals at intervals along the length of the animal while the proportion of label in the branch. Water and suspended particles alcohol insoluble fraction decreases corre- move into or out of the stem canals through spondingly. These represent a temporary these openings. The flow in a stem canal shift of labeled amino acids away from the may pass a solenial opening for some high molecular weight protein fraction into minutes, then suddenly begin to receive the fraction containing polypeptides of inter- water from the solenium. With these mediate weight formed both by degradation changes as with those noted above, nothing of food protein and by incomplete synthesis was seen to suggest that the changes seen of gorgonian proteins from food material. in the flow patterns in the stem canals were Little increase in the amount of label in the correlated with any other gross behavior or TeA soluble fraction is expected as the free change. amino acids represented by this fraction are Because they are generally smaller than apparently incorporated into higher molec- the stem canals and are tortuous and highly ular weight fractions at about the same rate ramifying, the solenia could not be observed that they are generated. as well as the stem canals nor could the The current velocities listed in Table 1 current velocities in them be measured as are adequate to transport ingested food or they were in the stem canals. It is my im- the products of its digestion for considerable pression, however, that the flow velocities distances. Each could transport material in the solenia are similar to those in the more than two meters in an hour, and sev- stem canals. eral would go nearly twice that distance. Many questions remain unanswered, how- DISCUSSION ever. The details of the flow pattern in the As noted in the previous section, the solenia and stem canals and the factors results presented in Figure 2 show that controlling them remain obscure. The nearly all of the radioactivity which was temporal pattern of flow in the canals is recovered was very near the location of the still unknown as well. We do not know polyp which was fed the radioactive food. whether the velocities in Table 1 are As it was not possible to search the whole typical or merely maximal. Last and most colony for transported radioactive matter, critically, it is not clear what determines some of the food material may have been whether material derived from ingested food 370 BULLETIN OF MARINE SCIENCE, VOL. 28, NO.2, 1978 does move through the canal system or is --, and H. M. Lenhoff. 1968. Alcohol taken up by phagocytic cells near the site soluble proteins: their formation and assim- ilation during intracellular digestion in Hydra of ingestion. littoralis and Aipfasia sp. Compo Biochem. Physio!. 26: 963-970. ACKNOWLEDGMENTS Muscatine, L. 1973. Nutrition of corals. Pages 77-115 in O. A. Jones and R. Endean, eds. I thank Dr. S. A. Wainwright for making the Biology and Geology of Coral Reefs, vol. 2. field work possible and helping in it and B. S. Academic Press, New York. Murdock for criticism and help with the figures. Musgrave, Edith M. (nee Pratt). 1909. Ex- Dr. C. B. Cook kindly prepared the radioactive perimental observations on the organs of mouse meat. Drs. E. C. Horn, D. J. Fluke and circulation and powers of locomotion in N. W. Gillham generously provided laboratory pennatulids. Quart. J. Micr. Sci. 54: 443-482. space and equipment and Dr. J. R. Gregg pro- Parker, G. H. 1920. Activities of colonial ani- vided helpful comments and criticism. Dr. F. M. mals. 1. Circulation of water in Renilla. J. Bayer, the University of Kansas Press, and The Exp. Zool. 31: 342-367. Geological Society of America all gave permission Pratt, Edith M. 1905. The digestive organs of for the use of Figure 1. This work was supported the alcyonaria and their relation to the in part by an NDEA Title IV fellowship, and mesogloeal cell plexus. Quart. J. Micr. Sci. predoctoral fellowships from the Cocos Founda- 49: 327-362. tion and NSF. Rees, J., L. V. Davis, and H. M. Lenhoff. 1970. Paths and rates of food distribution in the colonial hydroid Pennaria. Compo LITERATURE CITED Biochem. Physio!. 34: 309-316. Roberts, R. B., P. H. Ableson, D. B. Cowie, Bayer, F. M. 1956. . Pages 166- E. T. Bolton, and R. J. Britton. 1955. 231 in R. C. Moore, ed. Treatise on In- Studies of biosynthesis in Escherichia coli. vertebrate Paleontology, Pt. F. Coelenterata. Carnegie Inst. Wash. Pub!. No. 607, Wash- Geological Society of America and Univer- ington, D.C. 521 pp. sity of Kansas Press, Lawrence, Kansas. Roosen-Runge, E. C. 1967. Gastrovascular sys- Brafield, A. E. 1969. Water movements in the tem of small hydromedusae: mechanisms of pennatulid coelenterate, Pteroides griseum. circulation. Science 156: 74-76. J. Zoo!. Lond. 158: 317-325. Willem, V. 1894. La structure des palpons de Crowell, S. 1957. Differential responses of Apolemia IIvaria Esch., et les phenomenes growth zones to nutrient level, age, and de l'absorbtion dans ces organes. Bull. Acad. temperature in the colonial hydroid Campan- Roy. Sci. Lett. Beaux-Artes Belg. 3rd ser. It/aria. J. Exp. Zool. 13: 63-90. 27: 354-363. Lenhoff, H. M. 1961. Digestion of protein in Hydra as studied using radioautography and fractionation by differential solubilities. Exp. DATEACCEPTED: April 26, 1977. Cell Res. 23: 335-353. Murdock, G. R. 1978. Digestion, assimilation, ADDRESS: Department of Zoology, Duke Univer- and transport of food in the gastrovascular sity, Durham, North Carolina 27706. PRESENT cavity of a gorgonian octocoral (Cnidaria; ADDRESS:Department of Zoology, Clemson Uni- Anthozoa). Bul!. Mar. Sci. 28: 354-362. versity, Clemson, South Carolina 29631.