Asplanchna-induced polymorphism in the rotifer Keratella slacki1

Limnol. Oceanogr., 29(6), 1984, 1309-l 3 16 0 1984, by the American Society of Limnology and Oceanography, Inc. Asplanchna-induced polymorphism in the rotifer Keratella slacki1 John J. Gilbert and Richard S. Stemberger Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 Abstract Asplanchna releases into its environment a filterable factor which induces eggs of Keratella slacki to develop into individuals with slightly larger (x 15%) bodies, considerably longer (a 30%) anterior spines, much longer (= 130%) right posterior spines, and sometimes short, left posterior spines not otherwise present. The Asplanchna-induced morph is much less susceptible to Asplanchna predation than the noninduced morph, its long spines making it about two times less likely to be captured after being attacked and about five times less likely to be ingested after being captured. The pronounced asymmetry of the posterior spines may minimize the cost of spine production without sacrificing protection. The rotifer Keratella slacki (Berzins 1963; Koste and Shiel 1980), an Australian endemic, is very common in billabongs (Shiel and Koste 1983) and displays considerable variation in body and spine lengths (Shiel 198 1). Shiel reported that individuals always had two posterior spines, the right one much longer than the left one. He also found that the combined lengths of these spines increased linearly with body length and that the most exuberant forms co-occurred with the predatory rotifer Asplanchna. We were interested in testing the hypothesis that Asplanchna induces spine development in K. slacki, just as it does in Brachionus calyci- Jlorus (Gilbert 1966, 1967). We describe here laboratory experiments showing that a single clone of K. slacki can exhibit an even greater variation in body and spine lengths than that exhibited by the 25 different populations examined by Shiel and that this variation is due to a developmental polymorphism induced by a filterable factor produced by Asplanchna. We also show that two predatory copepods do not produce such a factor and that K. slacki with large, Asplanchna-induced bodies and spines are greatly protected against Asplanchna predation. The Asplanchna-induced polymorphism we report in K. slacki is very similar to that described in B. calyciforus (Gilbert 1966, 1967) but differs from it in several respects. In K. slacki, the posterior spines do not articulate with the body. Also, in K. slacki, l This study was supported by National Foundation grant DEB 81-21639 to J.J.G. Science posterior spine development is asymmetric, with the right spine often increasing greatly in length and a left one typically being induced only when the right one is very long. The possible significance of this asymmetry is discussed. We are indebted to R. J. Shiel for sending the K. slacki, and we thank M. M. Bean for technical assistance. Materials and general methods Keratella slacki was collected by R. J. Shiel from the Adelaide Botanic Gardens, Adelaide, South Australia, on 24 October 1983. Several clones derived from the specimens sent to our laboratory were cultured on Cryptomonas sp. in a defined medium (Stemberger 198 1). The Cryptomonas was collected by one of us (J.J.G.) from Pleasant Lake, New London, New Hampshire, in summer 198 1 and cultured on the defined medium. Asplanchna brightwelli (clone 4B6 1) and Asplanchna girodi (clone 5Al) were collected (Gilbert 1975; Gilbert and Litton 1978) and cultured on Paramecium aurelia in the defined medium by methods similar to those previously described (Gilbert 1968, 1975). The cyclopoid copepod Tropocyclops prasinus was collected by one of us (R.S.S.) from Post Pond, Lyme, New Hampshire, in spring 1983 and was cultured in the defined medium on a mixture of Cryptomonas erosa and the soft-bodied rotifer Synchaeta pectinata. The Synchaeta 1309 was cultured on Cryptomonas erosa in filtered water from Post Pond. One individual of another, unidentified cyclopoid copepod was collected with the K. slacki and was maintained on this rotifer in the defined me-

1310 Gilbert and Stemberger dium. All algal, rotifer, and copepod cultures were kept at 20 C on a photocycle (14: 10 LD). Filtrates of Asplanchna- and copepodconditioned media were prepared by filtering cultures of the animals through glass microfiber filters (Whatman 934-AH). Population densities in these cultures were several thousand individuals (adults and juveniles) per liter for A. brightwelli, A. girodi, and T. prasinus; the one adult unidentified cyclopoid was kept in a 5-ml culture and thus was at a density equivalent to 200 individuals per liter. The effects of predator-culture filtrates on the body and spine lengths of K. slacki were tested by inoculating one or several K. slacki into I- or S-ml volumes of these filtrates and also into control filtrates. One-milliliter volumes were placed in the concavities of glass depression plates within moist chambers, and 5-ml volumes were placed in plastic Petri dishes (35 x 10 mm). One to five drops of concentrated suspensions of Cryptomonas sp., prepared by centrifugation, were added to the filtrates. The K. slacki in the filtrate cultures were changed every second or third day into freshly prepared filtrates and Cryptomonas. Populations developed rapidly, and after 3-9 days they were preserved for measurement in buffered 10% Formalin with 4% sucrose. Body and spine lengths, as delimited by Gilbert (1967) and Green (1980), were measured with an ocular micrometer to the nearest 5 or 9 pm with a Zeiss Universal compound microscope at 240 magnifications or a Wild M-5 stereomicroscope at 100 magnifications. In one experiment, ra- tios of right posterior spine length to body length were calculated for individuals cultured in different filtrates; these were then arcsin-transformed to permit comparisons with the Student s t-test, since many of the ratios were ~0.3 (Sokal and Rohlf 1981). The effect of an Asplanchna-induced increase in body size and especially spine length in K. slacki on the susceptibility of this rotifer to Asplanchna predation was tested in two types of experiment-prey removal and direct observations. Individuals with long, Asplanchna-induced spines were obtained by culturing K. slacki in filtrates of A. brightwelli cultures, control individ- uals with much shorter spines by culturing populations in filtrates of the defined medium. In one type of experiment, 8-20 nonovigerous individuals of each of these two morphs were placed together with 3-l 0 adult A. brightwelli in 5 ml of defined medium with Cryptomonas sp. in each of several Petri dishes (3 5 x 10 mm) for about 6 h. These experiments were conducted in the dark to eliminate patchy distributions of predator and prey caused by phototactic responses. Then the survivorship of each morph was determined by counting the remaining living K. slacki in each dish. The fate of missing individuals was directly determined by examining the stomachs of the Asplanchna for recently ingested prey and the bottoms of the culture dishes for either dead individuals or the skeletons of regurgitated prey. Frequencies of eaten and surviving individuals of each morph were compared with rowby-column tests of independence and the G-statistic. In the other type of experiment, the abilities of the long- and short-spined morphs of K. slacki to avoid capture and ingestion by Asplanchna were determined by watching encounters between these morphs and A. brightwelli. Nonovigerous individuals of each morph were placed in 5 ml of defined medium in Petri dishes (3 5 x 10 mm) with Asplanchna that had been cultured on K. slacki and starved for 18 h, and interactions were then observed with a Wild M-5 ste- reomicroscope at 12 and 25 magnifications. Encounters were defined as collisions of K. slacki with the central part of the corona of Asplanchna. Captures were defined as retentions of attacked K. slacki completely or partially within the pharynx of Asplanchna. The feeding behavior of Asplanchna is described elsewhere (Gilbert 1980a; Gilbert and Stemberger in press). The frequencies of attacks following encounters, captures following attacks, and ingestions following captures were determined for each morph and compared with row-by-column tests of independence and the G-statistic. Details, results, and discussion of experiments Control of developmental polymorphism-a preliminary experiment showed that K. slacki cultured in filtrates of an A.

Polymorphism in Keratella 1311 Table 1. Means (&SD) of body and spine lengths of 10 subadult to adult Keratefla slacki from populations cultured in filtrates of untreated medium (UM) and Asplanchna girodi-conditioned medium (AM). Student s l-tests were used to determine the probabilities (Z ) that differences between means could be due to chance. Measurement Olm) Body Right posterior spine Left posterior spine Antero-median spines Antero-lateral and -intermediate spines. Filtrate UM AM P 126.5k4.1 136.8k9.0 GO.01 29.1kll.O 72.Ok 19.0 co.00 1 0 l.ok3.2 >O.l 36.5k3.4 46.4k5.8 KO.001 15.0&o 21.6k2.6 KO.001 girodi culture had larger bodies and disproportionately much longer spines than those cultured in filtrates of untreated medium (Table 1). Each of the populations cultured under these two conditions was derived from a single female from a population (clone A) cultured in untreated medium. The populations were cultured in l-ml volumes of the filtrates, and animals of different ages were taken for measurement after 3 and 6 days of culture. Every animal in each population was measured. The spine development induced by the filterable factor from the Asplanchna culture consisted of a lengthening of the right posterior spine, the de novo growth of a left posterior spine (10 pm long) in one individual, and a lengthening of the antero-median, -intermediate, and -lateral spines. Several preliminary experiments showed that K. slacki cultured with the unidentified cyclopoid or in filtrates of cultures of this cyclopoid were similar in body and spine lengths to those cultured in filtrates of untreated medium. These experiments were conducted in 1 - and 5-ml volumes and lasted 3 or more days. A more definitive experiment tested the effects of filtrates of A. girodi and T. prasinus cultures on the body size and spine development of K slacki. Populations of K. slacki, each initiated by three ovigerous adults (clone B), were cultured in 5-ml volumes of these filtrates and in filtrates of a suspension of Paramecium aurelia in the defined medium. These last filtrates served as controb for the Asplanchna-culture filtrates, since the Asplanchna was fed on Paramecium. As the K. slacki populations reproduced more rapidly in the Asplanchna- and Tropocyclops-culture filtrates than in the Paramecium-suspension filtrates, those in the former were preserved for measurements of body and right posterior spine lengths after 6 days and those in the latter after 9 days. All animals of all ages in the populations from the Tropocyclaps-culture and Paramecium-suspension filtrates were measured; most of the animals in the population from the Asplanchna-culture filtrates were selected at random and measured. The results of this experiment are presented in Figs. 1 and 2 and Table 2. The ratios of the right posterior spine length to body length in the populations from the Tropocyclops-culture and Paramecium-suspension filtrates were very similar and much lower than those in the population from the Asplanchna-culture filtrates (Fig. 1, Table 2). Statistical analysis of the arcsin-transformed ratios showed that the mean ratios in the former two populations were not different from one another (Student s t = 0.890, P = 0.376) but each was highly significantly different from the mean ratio in the latter population (Student s t-values > 8, P-values I 1 X 10-9). The variation in the ratios of spine to body length of the K. slacki in this experiment was much greater for the individuals from the Asplanchna-culture filtrates than for those from the other two filtrates (Fig. 1, Table 2). In the individuals from the Asplanchna-culture filtrates, there was a pronounced, positive, linear relationship be- tween right posterior spine length and body length (Fig. 2). A regression analysis of these data showed that the relationship between spine length (y) and body length (x) was Y = 1.91.X - 192.8, with the coefficient of determination (r2) equal to 0.707.

1312 Gilbert and Stemberger o-.09.lo-.i9.20-.29.30-.39.40-.49.50-.59.60-.69.70-.79.80-.89.90-.99 Right posterior spine length to body length ratio Fig. 1. Frequency distributions of right posterior spine length to body length ratios of Keratefla slacki cultured in filtrates of Paramecium aurelia suspensions (0), Asplanchna girodi cultures (O), and Tropocyclops prasinus cultures 0. The results of this experiment also showed that left posterior spines generally appeared only in individuals with long, right posterior spines. Six of the 56 animals from the Asplanchna-culture filtrates had left posterior spines, and all of these had right posterior spine lengths of at least 9 1 pm (Fig. 2). The left posterior spines of these animals had a mean length of 4 1 pm and were, on average, 0.36 times the length of the right ones. Only one of the 107 animals from the other filtrates had a left posterior spine (18 pm) along with a short (45 pm) right one (Fig. 3~). The full range of the polymorphism in body and spine lengths exhibited by clone B of K. slacki under various culture conditions is shown in Fig. 3. These drawings illustrate the several relationships that have already been mentioned: increases in right posterior spine length are correlated with increases in body length; left posterior spines tend to occur in individuals with long, right posterior spines; and increases in anteromedian, -intermediate, and -lateral spine lengths are correlated with increases in right posterior spine length. In addition, these drawings show some variation among individuals in the relative lengths of the antero-intermediate and -lateral spines, the curvature of the antero-median and posterior spines, and the angle at which the posterior spines grow out from the body. Ecological significance of Asplanchna-induced polymorphism-two different types of experiments showed that K. slacki with large, Asplanchna-induced bodies (mean length = x 145 pm) and right posterior spines (mean length = x 87 pm) were less likely to be captured and ingested by Aspkanchna than those from untreated media with smaller bodies (mean length = * 125 pm) and spines (mean length = ~37 pm). In the first type of experiment, populations of these two morphs of K. slacki were placed together with A. brightwelli, and after about 6 h the number of surviving individuals of each morph was determined. The results (Table 3) show that the short-spined morph was eaten to a significantly greater extent than the long-spined morph. All but two of the nonsurviving K. slacki in these experiments were seen in the stomachs of the Asplanchna at the end of the experiments. The two that were not were found as empty loricae packed together on the bottom of a culture dish, obviously having been regurgitated by one of the Asplanchna. In the third Table 2. Ratios of right posterior spine length to body length in Kerateffa siacki cultured in filtrates of a. Paramecium suspension, an Asplanchna girodi culture, and a Tropocyclops prasinus culture. Filtrate Mean SD Range n Paramecium Asplanchna Tropocyclops 0.32 0.06 0.144.43 58 0.52 0.16 0.2 l-o.8 1 56 0.31 0.07 0.14-0.47 49

Polymorphism in Keratella 1313 127 - II8-0 Table 3. Mortality of long-spined (LS) and shortspined (SS) Keratella slacki cultured with Asplanchna 0 CB 0 bright welli. 2 109-2 c 100 - -g 91 - ': 82- '5 73-.E 64-2 55- ifi Z 45. g 36-27 a 0 ua v 0 Not Exp. Morph Eaten eaten G-statistic P a 1 LS 1 9 2.53 0.112 0 0 v SS 4 6 2 LS 0 8 9.29 0.002 v a ss 5 3. A 3 LS 3 15 4.88 0.027 ss 10 10 l-3 LS 4 32 13.93 1.90x 10-4 ss 19 19 I I I I I II8 127 136 145 155 164 Body length (pm) Fig. 2. Relationship between right posterior spine length and body length in a population of Keratella slacki cultured in filtrates of an Asplanchna girodi culture. Open circles indicate individuals which also have a left posterior spine. Measurements are to the nearest 9 pm. experiment, the means of the right posterior spine lengths of the 3 short-spined and 10 long-spined K. slacki ingested by the Asplanchna were 37 and 78 pm. In the second type of experiment, the relative susceptibilities of the short- and longspined morphs of K. slacki to Asplanchna predation were determined by observing encounters between individuals of these two morphs and A. brightwelli. The results (Table 4) show that both morphs were equally likely to be attacked following an encounter but that the long-spined morph was much less likely to be captured after being attacked and then ingested after being captured. The probability of a short-spined individual being ingested after an encounter (0.172) was 10 times greater than that of a long-spined individual (0.0 17). This difference in susceptibility of the two morphs was greater than that observed in the survivorship experiments, where the proportions of short- and long-spined individuals ingested were 0.50 (19/38) and 0.11 (4/36). The probable reason for the less dramatic dif- ference in the survivorship experiments is that the relative availability of short-spined individuals was constantly decreasing due to Asplanchna predation. General discussion We have shown that living Asplanchna, both A. girodi and A. brightwelli, release into their environments a filterable factor which induces slightly greater body growth and much greater spine growth in K. slacki (Table 1, Fig. 3). The extent of this induction varies considerably among individuals in a population cultured with the Asplanchna factor (Figs. l-3, Tables 1 and 2), but generally the body increases about 15% in length, the right posterior spine increases about 130% in length, the three pairs of anterior spines each increase about 30% in length, and a left posterior spine develops de novo in some individuals with an especially long right one. Although no detailed investigation was made of the time when the Asplanchna factor affects K. slacki development, on many occasions we noted that eggs already ex- Table 4. Ability of Asplanchna brightwelli to attack (a), capture (c), and ingest (i) long-spined (IS) and shortspined (SS) Keratella sfacki after an encounter (e). Keratella Feeding responses of Asplanchna morph and statistic a/e c/a i/c LS 53/62(0.85) 23/53(0.43) l/23(0.04) ss 41/51(0.80) 32/41(0.78) 7/32(0.22) G 0.52 12.52 3.77 P 0.47 0.0004 0.052

.. 1314 Gilbert and Stemberger a Fig 3 Polymorphism in Keratella slacki. Individuals are from cultures of clone B without (a-c) and with (d-h) A&anchna girodi factor. Facets on dorsum of lorica shown only for specimens b and f. Scalar is 100 pm.

Polymorphism in Keratella 1315 truded from the maternal pseudocoelom were never influenced by the factor. Thus, the Asplanchna factor appears to operate through the mother during oogenesis, just as in the case of Asplanchna-induced spines in B. calyciflorus (Gilbert 1966, 1967). Experiments with filtrates of media conditioned by the unidentified cyclopoid and by T. prasinus showed that neither of these copepods, nor their prey, produce a factor which increases body size and spine development in K. slacki (Table 2). Thus, the inducing factor must be quite specific, perhaps to the genus Asplanchna, and cannot be a general product of metabolism. It is interesting to compare some of our observations on K. slacki with those of Shiel (198 1). The strong, positive relationship we noted between body length and right posterior spine length (Fig. 2) was also noted by Shiel, although he plotted body length against the cumulative length of both posterior spines and used mean values of these dimensions from each of 25 different populations. The variation we noted in the spine lengths (Fig. 3) was considerably greater than that observed by Shiel, even though we examined a single clone and he examined 25 populations. For example, Shiel stated that the K. slacki in his populations always had two posterior spines, while we found that most of our individuals, including those with long, Asplanchna-induced spines, had only the right posterior spine. Also, under some conditions, we observed individuals with no posterior spines (Fig. 3). Both T. prasinus and Asplancha have recently been shown to produce filterable factors which induce posterior spine development in Keratella cochlearis (Stemberger and Gilbert 1984). This is the first case where a developmental polymorphism in either a rotifer or a crustacean zooplankter is known to be induced by predators from two different phyla. The Asplanchna-induced polymorphism in K. slacki described here is the second example of a predator-induced polymorphism in the genus Keratella and the sixth case of an Asplanchna-induced polymorphism in rotifers. The other rotifers known to respond to Asplanchna in this way, in addition to K. cochlearis and B. calycijlorus, are Brachionus bidentata, Brachionus urceolaris sericus, and Filinia mystacina (Pourriot 1964, 1974). Predator-induced polymorphisms in rotifers and other zooplankters, such as Daphnia (Grant and Bayly 198 1; Krueger and Dodson 198 l), clearly are much more prevalent than has been suspected. The Asplanchna-induced polymorphism in K. slacki is ecologically very significant. Several species of Asplanchna (A. brightwelli, A. girodi, A. sieboldi, A. priodonta) cooccur with K. slacki (Shiel 198 l), and these probably are much less likely to eat K. slacki with large, Asplanchna-induced bodies and spines than those without. In our experiments, A. brightwelli captured and ingested long-spined individuals much less often than short-spined ones, eating the latter 10 times more often than the former (Table 4). The protection afforded by the long spines was also demonstrated by the greater survivorship of the long-spined individuals in cultures with A. brightwelli (Table 3). Long-spined K. slacki are partially protected from Asplanchna because they are effectively much larger and hence more difficult to suck in through the mouth when the pharynx dilates and also more difficult to swallow. The overall length of the Asplanchna-induced morph, including antero- median and right posterior spines, is about 175% that of the noninduced morph (see above). Also, the greater length of all the spines on long-spined individuals probably makes these individuals more likely to catch on the soft integument of the pharynx and hence less likely to be maneuvered completely into the pharynx and then into the esophagus. The long, Asplanchna-induced spines of B. calyczj lorus are similarly effective in limiting predation by Asplanchna (Gilbert 1967, 198Ob). One of the most interesting aspects of the developmental polymorphism in K. slacki is the asymmetry of the posterior spines. Similar asymmetries in this genus have been noted by many investigators in K. valga, K. procurva, and K. tropica (see Edmondson and Hutchinson 1934; Ahlstrom 1943; Green 1980; Shiel 198 1). In our cultures of K. slacki, individuals typically have only a right posterior spine, whether this spine is

1316 Gilbert and Stemberger short or very long, but sometimes develop a left posterior spine, especially when the right one is very long. If posterior spine production is costly, either energetically or in terms of decreased survivorship in the absence of Asplanchna, then production of a single posterior spine could be cost-effective if one spine were as effective as two. It seems possible, for example, that one long posterior spine could limit ingestion by Asplanchna just as much as two. If this were the case, then an explanation is needed for the selective advantage of the short, left posterior spines found on some individuals with long, right posterior spines. It is possible that individuals wifh a single, long spine on the right side are hydrodynamically less stable than individuals which also develop a short left spine. This hypothesis is currently under investigation. References AHLSTROM, E. H. 1943. A revision of the rotatorian genus Keratella with descriptions of three new species and five new varieties. Bull. Am. Mus. Nat. Hist. 80: 41 l-457. BERZINS, B. 1963. Two new Keratella, Rotatoria, from Australia. Hydrobiologia 21: 380-383. EDMONDSON, W. T., AND G. E. HUTCHINSON. 1934. Yale North India Expedition. Report on Rotatoria. Mem. Conn. Acad. Arts Sci. 10: 153-186. GILBERT, J. J. 1966. Rotifer ecology and embryological induction. Science 151: 1234-1237. -. 1967. Asplanchna and postero-lateral spine production in Brachionus calyct@lorus. Arch. Hydrobiol. 64: l-62. --. 1968. Dietary control of sexuality in the rotifer Asplanchna brightwelli Gosse. Physiol. Zool. 41: 14-43. -. 1975. Polymorphism and sexuality in the rotifer Asplanchna, with special reference to the effects of prey-type and clonal variation. Arch. Hydrobiol. 75: 442-483. -. 1980a. Feeding in the rotifer Asplanchna: Behavior, cannibalism, selectivity, prey defenses, and impact on rotifer communities. Am. Sot. Limnol. Oceanogr. Spec. Symp. 3: 158-172. New England. -. 1980b. Further observations on developmental polymorphism and its evolution in the rotifer Brachionus calyc~$orus. Freshwater Biol. 10: 28 l-294. -, AND J. R. LITTON, JR. 1978. Sexual reproduction in the rotifer Asplanchna girodi: Effects of tocopherol and population density. J. Exp. Zool. 204: 113-122. - AND R.S. STEMBERGER. Inpress. Preycapture in the rotifer AspZanchna girodi. Int. Ver. Theor. Angew. Limnol. Verh. 22. GRANT, J. W., AND I. A. BAYLY. 1981. Predator induction of crests in morphs of the Daphnia carinata Ring complex. Limnol. Oceanogr. 26: 201-218. GREEN, J. 1980. Asymmetry and variation in Keratella tropica. Hydrobiologia 73: 24 l-248. KOSTE, W., AND R. J. SHIEL. 1980. Preliminary remarks on the characteristics of the rotifer fauna of Australia (Notogaea). Hydrobiologia 73: 221-227. KRUEGER, D. A., AND S. I. DODSON. 1981. Embryological induction and predation ecology in Daphnia pulex. Limnol. -Oceanogr. 26: 219-223. POURRIOT, R. 1964. Etude experimentale de variations morphologiques chez certaines esptces de rotifires. Bull. Sot. Zool. Fr. 89: 555-56 1. -. 1974. Relations predateur-proie chez les rotiferes: Influence du predateur (Asplanchna brightwezzz) sur la morphologie de la proie (Brachionus bidentata). Ann. Hydrobiol. 5: 43-55. SHIEL, R. J. 1981. Planktonic Rotifera of the Murray- Darling river system, Australia: Endemism and polymorphism. Int. Ver. Theor. Angew. Limnol. Verh. 21: 1523-l 530. -, AND W. KOSTE. 1983. Rotifer communities of billabongs in northern and south-eastern Australia. Hydrobiologia 104: 4 l-47. SOKAL, R. R., AND F. J. ROHLF. 1981. Biometry. Freeman. STEMBERGER, R. S. 1981. A general approach to the culture of planktonic rotifers. Can. J. Fish. Aquat. Sci. 38: 721-724. AND J. J. GILBERT. 1984. Spine development in the rotifer Keratella cochlearis: Induction by cyclopoid copepods and Asplanchna. Freshwater Biol. 14: in press. Submitted: 9 January 1984 Accepted: 29 May 1984