Interspecific Relations between Marine Rotifer Brachionus rotundiformis and Zooplankton Species Contaminating in the Rotifer Mass Culture Tank

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1 Fisheries Science 61(4), (1995) Interspecific Relations between Marine Rotifer Brachionus rotundiformis and Zooplankton Species Contaminating in the Rotifer Mass Culture Tank Atsushi Hagiwara, Min-Min Jung, Takumi Sato, and Kazutsugu Hirayama Faculty of Fisheries, Nagasaki University, Bunkyo, Nagasaki 852, Japan (Received December 7, 1994) We investigated the interaction between marine rotifer Brachionus rotundiformis (formerly called S-type B. plicatilis) and zooplankton species which contaminate in the rotifer mass culture tanks. These species include Brachionus plicatilis (formerly called L-type B. plicatilis), Diaphanosoma celebensis (cladoceran), Tigriopus japonicus (copepod) and Euplotes sp. (protozoan). We compared the popula tion growth of 40 ml single-species and two-species of mixed cultures fed on phytoplankton Nan nochloropsis oculata at 8 ~ 105 cells/ml (food limitation level). We observed three manners of interspecific relations that include competition, commensalism and amensalism. Predations or mechanical damages were not observed between tested species. The popula tion growth of B. rotundiformis, B. plicatilis, and D. celebensis in mixed-species cultures were sig nificantly suppressed when compared with those in single-species cultures. This indicates that the com petition for the limited amount of food suppressed mutual population growth. The population growth of B. rotundiformis was not affected by the co-existence of T. japonicus, however. T. japonicus grew better when cultured with B. rotundiformis. Contrary, the existence of B. rotundiformis did not affect the growth of Euplotes sp, but Euplotes sp. population interferes the growth of B. rotundiformis. These suggest the possibility of bacterial intervention in the interspecific relation of T. japonicus and Euplotes sp. with B. rotundiformis. Key words: interspecific relation, rotifer, rotifer microcosm, copepod, cladoceran, protozoan, competition, mass culture In marine fish larval rearing facilities, the marine rotifer Brachionus rotundiformis (formerly called S-type B. plicatilis) and B. plicatilis (formerly called L-type B. plicatilis)1) occasionally co-exist in mass culture tanks, although it is preferable to culture one or the other species depending on the needs of the fish species and larval stages. In recent years, mass culture of B. rotundiformis is more common in larval rearing practices, because of its higher productivity. Contamination of harpacticoid copepods and protozoans are also commonly found in the open rotifer mass culture tanks. Information has been scarce, however, with regard to the interaction among rotifers and these contaminants. In rotifer mass cultures where B. rotundiformis and B. plicatilis coexisted, the domination of B. rotundiformis oc curs at higher temperatures (>27 Ž) and B. plicatilis at lower temperatures (<20 Ž).2) The recent findings on differences in temperature-dependent population growth patterns between these two species3) supports this interpre tation. No research has been conducted, however, to evalu ate the interaction between these two marine rotifer spe cies quantitatively. It was reported that mass production trials of the harpac ticoid copepod Tigriopus japonicus were successful when mixed-cultured with rotifers.4.5) But experiments to exa mine this method have not been conducted. In freshwater environments, pelagic copepods generally do not have sig nificant effects on rotifer abundance.6) Cladocerans, on the contrary, often suppress rotifer populations.6) Results from laboratory experiments using cladocerans (Daphnia and Scapholeberis) and freshwater rotifer species indicate that the mechanical interference and predation by cladoce rans cause high mortality in rotifer populations.7-12) Roles of protozoans appearing in rotifer mass culture tanks were summarized by Maeda and Hino.13) Among those protozoan species, Euplotes and Uronema cannot eat Nannochloropsis, but can eat yeast which is usually added as a supplemented food in rotifer mass cultures. The population growth of B. rotundiformis and B. plicatilis have been thoroughly investigated.3) The marine cladoceran Diaphanosoma celebensis was lately in troduced as a live feed, and its biological characteristics have been investigated in detail.14-16) Studies on euryhaline harpacticoid copepod Tigriopus japonicus have been con ducted to clarify its population dynamics in tide pools as well as to establish intermediate sized live feed for marine fish larval rearing (reviewed by Hagiwara et al. 17)). Despite that specific biological information has accumulated on those organisms, none of the studies evaluated inter specific relations. Owing to the cladoceran-rotifer competi tion papers6-12) reported during the last decade, we expect that competition could be intense in mass cultures. In this paper, we quantitatively examined whether con taminating zooplankton species (B. plicatilis, D. celeben sis, T. japonicus, and Euplotes sp.) could affect the popula tion growth of B. rotundiformis.

2 624 Hagiwara et al. Materials and Methods The marine rotifer B. rotundiformis (Koshiki strain18) and four other zooplankton species (B. plicatilis, D. cele bensis, T. japonicus, and Euplotes sp.) were employed. The B. plicatilis was a Nagasaki strain.18) The marine cladoceran D. celebensis was delivered by W.-T. Yang.14-16) The harpacticoid copepod T. japonicus was collected at Lake Hamana.17) These organisms have been cultured for more than five years in our laboratory. Cultures were main tained in the GF/C filtered and autoclaved natural sea water diluted with distilled water to 22 ppt salinity at 25 Ž and fed on centrifuged Nannochloropsis oculata grown in modified Erd-Schreiber medium.19) Feeding density of N. oculata was 8 ~ 105 cells/ml. Prior to this experiment, we collected Euplotes sp. from a rotifer mass culture tank at the Japan Sea Farming Center, Kamiura Station (Oita Prefecture). After the collection, the protozoan was cul tured in the above conditions for two months.the ex perimental populations, except Tigriopus were initiated from a single individual. Tigriopus populations were in itiated from a single brood. The size of experimental animals were compared in Fig. 1. The experimental design followed the method by Gil bert.12) Culture conditions were the same as those for precultures described above. Culture containers were 50 ml glass beakers with 40 ml culture volume. Beakers were covered with plastic film (Asahi-Kasei Industrial Co.) and kept in total darkness except when observed, less than I h a day. Number of organisms for starting cultures was 20 newly born amictic females for B. rotundiformis and B. plicatilis, 3 young gravid females for D. celebensis, 3 young females with red colored egg sacs20) for T. japonicus and 5 cells for Euplotes sp. Each triplicate culture was pre pared for four single-species cultures and four two-species mixed cultures. Mixed cultures were conducted between B. rotundiformis and other four. The culture period was 16 days. Every two days, the number of organisms was count ed by removing all individuals in a vessel. After the count, the animals were transferred to a fresh N. oculata suspen sion. For T. japonicus, the number of individuals in nauplius stages, copepodites and adults were counted separately. A two-way analysis of variance was conducted to deter mine the combined effect of treatment and time. In order to evaluate the size of zooplankton population over time, we introduced the population growth index (obtained from the calculation of area surrounded by population growth curve, x-axis and y-axis; see Fig. 2-5 in results sec tion). The difference of population growth index between control (single-species) and mixed-species cultures was compared by Student's t-test. Results After Day 4, in all cultures except single-species cultures for T. japonicus and Euplotes sp., all Nannochloropsis cells were consumed before the renewal of culture medium (Figs. 2-6). Predation or mechanical damage between test ed species were not observed during the experiments. Through 16 days of the culture period, no F2 generation ap peared in D, celebensis and T. japonicus. The co-existence with B. plicatilis and D. celebensis in terfered with the population growth of B. rotundiformis. (Fig. 2, 3, 7, Table 1). The suppression of B. rotundifor mis population growth started on Day 4 with B. plicatilis and Day 8 with D. celebensis. The population sizes of BA plicatilis and D. celebensis were similarly suppressed by B. rotundiformis (Fig. 2, 3, 7, Table 1). The population growth of B. rotundiformis cultured with T. japonicus did not differ from that of single-species cultures (Fig. 4, 7, Table 1). T. japonicus grew better when cultured with rotifers than when singly cultured (Fig. 4, 7, Table 1). Such trends appeared after Day 4. Fig. 6 indi cates that the population size of T. japonicus mix-cultured Fig. 2. Number of Brachionus rotundiformis (above) and Brachionus Fig. 1. Morphologies of zooplankton species used for experiments, plicatilis (bottom) in 40 ml single-species (, solid line) and two-spe cies mixed ( œ, dotted line) cultures. Each plot and vertical bar represent mean } SD of three replicates.

3 Interspecific Relations in Rotifer Microcosm 625 Fig. 3. Number of Brachionus rotundiformis (above) and Diaphanoso ma celebensis (bottom) in 40 ml single-species (, solid line) and two-species mixed ( œ, dotted line) cultures. Each plot and vertical bar represent mean f SD of three replicates. Fig. 5. Number of Brachionus rotundiformis (above) and Euplotes sp. (bottom) in 40 ml single-species (, solid line) and two-species mix ed ( œ, dotted line) cultures. Each plot and vertical bar represent mean }SD of three replicates. Fig. 4. Number of Brachionus rotundiformis (above) and Tigriopus japonicus (bottom) in 40 ml single-species (, solid line) and two species mixed ( œ, dotted line) cultures. Each plot and vertical bar represent mean } SD of three replicates. with B. rotundiformis increased both in nauplius and after copepodite stages. T. japonicus nauplii did not grow in the single-species cultures fed on N. oculata. On the contrary, the presence of Euplotes sp. significant ly suppressed B. rotundiformis population growth, while Fig. 6. Number of Tigriopus japonicus in nauplius (above) and copepo dite (bottom) stages (including adult) in 40 ml single-species cultures (, solid line) and mixed ( œ, dotted line) cultures with Brachionus rotundiformis. Each plot and vertical bar represent mean }SD of three replicates. Euplotes sp. was not affected by B. rotundiformis (Fig. 5, 7, Table 1).

4 626 Hagiwara et al. Table 1. Results of two way ANOVA of single-species and mixed species culture experiments to see the effect of coexistence of Brachionus plicatilis (Bp), Diaphanosoma celebensis (Dc), Tigriopus japonicus (Tj) and Euplotes sp. (E) on the population growth of Brachionus rotundiformis (Br), and vice versa Significant levels of p>0.05 (NS), p<o.05 (*), p<o.o1 (**) and p<0.001 (***) were indicated. *1 Population size of each species with and without the other was analyzeḍ *2 Analysis was conducted on population size data from Day 0 to Day 16, Fig. 7. Relative population growth of Brachionus rotundiformis (above, Br) and four zooplankton species (bottom, Bp-Brachionus plicatilis, Dc-Diaphanosoma celebensis, Tj-Tigriopus japonicus and E-Euplotes sp.) in two-species mixed cultures, comparing with the population growth in single species culture (control, dotted line). Relative population growth is a relative value of population growth index (see text) against control. Significant levels of differ ences between single-species and mixed-species cultures were indicat ed. Discussion We observed three different types of interspecific interac tions in mixed zooplankton cultures. These patterns are competition (observed between B. rotundiformis and B. plicatilis, and between B. rotundiformis and D. celebensis), commensalism (observed for T. japonicus when cultured with B. rotundiformis) and ammensalism (observed for B. rotundiformis when cultured with Euplotes sp.). The com petition (Fig. 2, 3) indicated the interaction which results from the limited amount of food. The domination of either B. rotundiformis and B. plicatilis did not occur probably because the experimental temperature (25 Ž) was moderate for both species.3) Large-sized freshwater cladocerans can mechanically interfere with small-sized rotifers. l2) Such effects were not observed for D. celebensis and B. rotundiformis in this study, despite the remarkable size difference between these species (Fig. 1). In the single species cultures, nauplii of T. japonicus did not grow on N. oculata diet (Fig. 6). But Euplotes grew (Fig. 5), despite the fact that this species does not feed N. oculata cells.13) A remarkable increase of population size for T. japonicus was observed in mixed cultures with B. rotun diformis, while Euplotes population was not affected by the presence of rotifers. We did not observe the predator prey relationship between T. japonicus and B. rotundifor mis. These results indicate that T. japonicus and Euplotes sp. can utilize bacteria on rotifer feces and/or in the water column, but they require different bacterial flora for their growth. The B. rotundiformis population growth was sup pressed by the presence of Euplotes, but unaffected by T. japonicus (Fig. 4, 5, 7). It is possible that the population of T. japonicus and Euplotes sp. modified the bacterial flora in the rotifer culture differently. A case is reported that a bacterial strain from Euplotes cultures strongly in hibited the rotifer growth.13) It has been reported that rotifer population growth and mixis induction are regulated by coexisting bacteria flora. 13,19,21,22) Maeda and Hino13) described that Flavobacteri um strains often grown in Nannochloropsis cultures strongly suppress rotifer population growth. The vitamin B12 producing Pseudomonas strains promote B. plicatilis population growth 21) and presence of a Vibrio alginolyti cus strain caused acute mortality in rotifer populations22) Hagiwara et al.19) reported a bacterial induction of B. plicatilis sexual reproduction. Although information is scarce, it is also possible that bacterial flora in the environ ment have a significant effect on the life history of other zooplankton groups, The commensalim and ammensalism observed for T. japonicus and Euplotes sp. may be altered depending on the co-existing bacterial strains. Hagiwara et al.17) reported the maximal r (intrinsic rate of increase) value of singly cultured T. japonicus was 0.28 on feeding Tetraselmis tetrathele. This r value may be increased by feeding an appropriate bacterial diet. We will further inves tigate bacterial interference or promotion of zooplankton population growth to determine if bacteria have a regulato

5 Interspecific Relations in Rotifer Microcosm 627 ry role in the outcome of competition between zooplankton species. In these experiments, we started B. rotundiformis cul tures in the same manner among four combinations of treatments (but different period), by trying to make the conditions of precultures as well as the condition of Nan nochloropsis cultures identical. The population growth of B. rotundiformis was, however, different aamong trials (Fig. 2-5). For example, in the B. rotundiformis vs. B. plicatilis and B. rotundiformis vs. Euplotes sp. experi ments, the single-species cultures of B. rotundiformis reached to 1,000 individuals on Day 4, while it was Day 6 and 10 for B. rotundiformis vs. D. celebensis and B. rotun diformis vs. T. japonicus experiments, respectively. The in consistency of population growth observed between trials may also be ascribed to differences of bacterial flora among precultures or experimental cultures. In addition to the Euplotes employed in this study, there are other protozoan groups which are commonly found in rotifer mass culture tanks, such as Uronema and Vorticel la. Rotifer mass cultures were unstable when Noctiluca scintillans contaminated (K. Hamada, person. comm.). In the larval rearing practices of Ayu Plecoglossus altivelis in freshwater, marine Brachionus are cultured in low salinity usually less than 8 ppt, where coexistence of freshwater cladocerans, such as Moina, is commonly observed.2) The interspecific relation between these species and marine Brachionus will be examined later using the methods em ployed in this research. Acknowledgments We wish to thank T. W. Snell for review and com ments. References 1) H. Segers: Nomenclatural consequences of some recent studies on Brachionus plicatilis (Rotifera, Brachionidae). Hydrobiol., (in press). 2) A. Oka: Biological characteristics of Brachionus plicatilis-breeding environment, in "The first live feed-brachionus plicatilis" (ed. by K. Fukusho and K. Hirayama), Koseisha Koseikaku, Tokyo, 1991, pp (in Japanese). 3) K. Hirayama and I. M. F. Rumengan: Fecundity pattern of S and L type rotifers Brachionus plicatilis. Hydrobiol., 255/256, (1993). 4) K. Fukusho: Mass production of a copepod, Tigriopus japonicus in combination culture with a rotifer Brachionus plicatilis, fed co-yeast as a food source. Nippon Suisan Gakkaishi, 46, (1980) (in Japanese). 5) K. Fukusho and C. Kitajima: Mass production of the copepod, Tigriopus japonicus, in combination culture with rotifer, Brachio nus plicatilis, feeding baking yeast and using large scale outdoor tank (September-December). Suisan Zoshoku, 25, (1977)(in Japanese). 6) J. J. Gilbert: Suppression of rotifer populations by Daphnia: A rev iew of the evidence, the mechanisms, and the effects on zooplankton community structure. Limnol. Oceanogr., 33, (1988). 7) J. J. Gilbert and R. S. Stemberger: Control of Keratella popula tions by interference competition from Daphnia. Limnol. Oceanogr., 30, (1985). 8) C. W. Burns and J. J. Gilbert: Effects of daphnid size and density on interference between Daphnia and Keratella cochlearis. Limnol. Oceanogr., 31, (1986). 9) C. W. Burns and J. J. Gilbert: Direct observations of the mechan isms of interference between Daphnia and Keratella cochlearis. Lim nol. Oceanogr., 31, (1986). 10) R. S. Stemberger and J. J. Gilbert: Rotifer threshold food concen tration and the size-efficiency hypothesis. Ecol., 68, (1987). 11) J. J. Gilbert: The Polyarthra escape from response: Defense against interference from Daphnia. Hydrobiol., 147, (1987). 12) J. J. Gilbert: Competitive interactions between the rotifer Synchae ta oblonga and the cladoceran Scapholeberis kingi Sars. Hydrobiol., 186/187, (1989). 13) M. Maeda and A. Hino: Environmental management for mass cul ture of rotifer, Brachionus plicatilis, in "Rotifer and microalgae cul ture systems" (ed. by W. Fulks and K. L. Main), Proceedings of a U.S.-Asia Workshop, The Oceanic Institute, Honolulu, 1991, pp ) S. Segawa and W. T. Yang: Reproduction of an estuarine Di aphanosoma aspinosum (Branchiopoda: Cladocera) under different salinities. Bull. Plankton Soc. Japan, 34, (1987)(in Japanese). 15) S. Segawa and W. T. Yang: Population growth and density of an es tuarine cladoceran Diaphanosoma aspinosum in laboratory cul ture. Bull. Plankton Soc. Japan, 35, (1988) (in Japanese). 16) S. Segawa and W. T. Yang: Growth, moult, reproduction and filter ing rate of an estuarine cladoceran, Diaphanosoma celebensis, in laboratory culture. Bull. Plankton Soc. Japan, 37, (1990) (in Japanese). 17) A. Hagiwara, C.-S. Lee, and D. J. Shiraishi: Some reproductive characteristics of the broods of the harpacticoid copepod Tigriopus japonicus cultured in different salinities. Fisheries Sci., (submitted). 18) Y. Fu, Y. Natsukari, and K. Hirayama: Morphological differences between two types of the rotifer Brachionus plicatilis O. F. Muller. J. Exp. Mar. Biol. Ecol., 151, (1991). 19) A. Hagiwara, K. Hamada, S. Hori, and K. Hirayama: Induction of monogonont rotifer Brachionus plicatilis O. F. Muller sexual reproduction with bacterial coexistence and addition of rotifer ex tracts. J. Exp. Mar. Biol. Ecol., (in press). 20) R. S. Burton: Mating system of the intertidal copepod, Tigriopus californicus. Mar. Biol., 86, (1985). 21) J.-P. Yu, A. Hino, M. Ushiro, and M. Maeda: Function of bacteria as vitamin B12 producers during mass culture of the rotifer Brachio nus plicatilis. Nippon Suisan Gakkaishi, 55, (1989). 22) J.-P. Yu, A. Hino, T. Nognchi, and H. Wakabayashi: Toxicity of Vibrio alginolyticus on the survival of the rotifer Brachionus plicati lis. Nippon Suisan Gakkaishi, 56, (1990).