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Fisheries Science 61(4), 623-627 (1995)

Interspecific Relations between Marine rotundiformis and 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 (formerly called L-type B. plicatilis), Diaphanosoma celebensis (cladoceran), Tigriopus japonicus () and Euplotes sp. (protozoan). We compared the popula tion growth of 40 ml single-species and two-species of mixed cultures fed on Nan nochloropsis oculata at 8 •~ 105 cells/ml (food limitation level). We observed three manners of interspecific relations that include competition, commensalism and amensalism. 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 contrary, often suppress rotifer populations.6) Results Brachionus rotundiformis (formerly called S-type B. from laboratory experiments using cladocerans ( plicatilis) and B. plicatilis (formerly called L-type B. and Scapholeberis) and freshwater rotifer species indicate plicatilis)1) occasionally co-exist in mass culture tanks, that the mechanical interference and by cladoce although it is preferable to culture one or the other species rans cause high mortality in rotifer populations.7-12) depending on the needs of the fish species and larval stages. Roles of protozoans appearing in rotifer mass culture In recent years, mass culture of B. rotundiformis is more tanks were summarized by Maeda and Hino.13) Among common in larval rearing practices, because of its higher those protozoan species, Euplotes and Uronema cannot productivity. Contamination of harpacticoid eat , but can eat yeast which is usually and protozoans are also commonly found in the open added as a supplemented food in rotifer mass cultures. rotifer mass culture tanks. Information has been scarce, The population growth of B. rotundiformis and B. however, with regard to the interaction among and plicatilis have been thoroughly investigated.3) The marine these contaminants. cladoceran Diaphanosoma celebensis was lately in In rotifer mass cultures where B. rotundiformis and B. troduced as a live feed, and its biological characteristics plicatilis coexisted, the domination of B. rotundiformis oc have been investigated in detail.14-16)Studies on euryhaline curs at higher temperatures (>27•Ž) and B. plicatilis at harpacticoid copepod Tigriopus japonicus have been con lower temperatures (<20•Ž).2) The recent findings on ducted to clarify its population dynamics in tide pools as differences in temperature-dependent population growth well as to establish intermediate sized live feed for marine patterns between these two species3) supports this interpre fish larval rearing (reviewed by Hagiwara et al. 17)).Despite tation. No research has been conducted, however, to evalu that specific biological information has accumulated on ate the interaction between these two marine rotifer spe those organisms, none of the studies evaluated inter cies quantitatively. specific relations. Owing to the cladoceran-rotifer competi It was reported that mass production trials of the harpac tion papers6-12)reported during the last decade, we expect ticoid copepod Tigriopus japonicus were successful when that competition could be intense in mass cultures. mixed-cultured with rotifers.4.5) But experiments to exa In this paper, we quantitatively examined whether con mine this method have not been conducted. In freshwater taminating zooplankton species (B. plicatilis, D. celeben environments sis, T. japonicus, and Euplotes sp.) could affect the popula , pelagic copepods generally do not have sig nificant effects on rotifer abundance.6) Cladocerans, on the tion growth of B. rotundiformis. 624 Hagiwara et al.

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

Fig. 2. Number of Brachionus rotundiformis (above) and Brachionus

plicatilis (bottom) in 40 ml single-species (•¡, solid line) and two-spe cies mixed (•œ, dotted line) cultures. Fig. 1. Morphologies of zooplankton species used for experiments, Each plot and vertical bar represent mean •} SD of three replicates. Interspecific Relations in Rotifer Microcosm 625

Fig. 3. Number of Brachionus rotundiformis (above) and Diaphanoso Fig. 5. Number of Brachionus rotundiformis (above) and Euplotes sp. ma celebensis (bottom) in 40 ml single-species (•¡, solid line) and (bottom) in 40 ml single-species (•¡, solid line) and two-species mix two-species mixed (•œ, dotted line) cultures. ed (•œ, dotted line) cultures. Each plot and vertical bar represent mean f SD of three replicates. 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 Fig. 6. Number of Tigriopus japonicus in nauplius (above) and copepo species mixed (•œ, dotted line) cultures. dite (bottom) stages (including adult) in 40 ml single-species cultures Each plot and vertical bar represent mean •} SD of three replicates. (•¡, solid line) and mixed (•œ, dotted line) cultures with Brachionus rotundiformis. Each plot and vertical bar represent mean •}SD of three replicates. withB. 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 Euplotes sp. was not affected by B. rotundiformis (Fig. 5, ly suppressed B. rotundiformis population growth, while 7, Table 1). 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

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 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 Fig. 7. Relative population growth of Brachionus rotundiformis pressed by the presence of Euplotes, but unaffected by T. (above, Br) and four zooplankton species (bottom, Bp-Brachionus japonicus (Fig. 4, 5, 7). It is possible that the population plicatilis, Dc-Diaphanosoma celebensis, Tj-Tigriopus japonicus and of T. japonicus and Euplotes sp. modified the bacterial E-Euplotes sp.) in two-species mixed cultures, comparing with the flora in the rotifer culture differently. A case is reported population growth in single species culture (control, dotted line). that a bacterial strain from Euplotes cultures strongly in Relative population growth is a relative value of population hibited the rotifer growth.13) growth index (see text) against control. Significant levels of differ It has been reported that rotifer population growth and ences between single-species and mixed-species cultures were indicat ed. 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 Discussion B12producing Pseudomonas strains promote B. plicatilis population growth 21)and presence of a Vibrio alginolyti We observed three different types of interspecific interac cus strain caused acute mortality in rotifer populations22) tions in mixed zooplankton cultures. These patterns are Hagiwara et al.19) reported a bacterial induction of B. competition (observed between B. rotundiformis and B. plicatilis . Although information is plicatilis, and between B. rotundiformis and D. celebensis), scarce, it is also possible that bacterial flora in the environ commensalism (observed for T. japonicus when cultured ment have a significant effect on the history of other with B. rotundiformis) and ammensalism (observed for B. zooplankton groups, The commensalim and ammensalism rotundiformis when cultured with Euplotes sp.). The com observed for T. japonicus and Euplotes sp. may be altered petition (Fig. 2, 3) indicated the interaction which results depending on the co-existing bacterial strains. Hagiwara et from the limited amount of food. The domination of al.17) reported the maximal r (intrinsic rate of increase) either B. rotundiformis and B. plicatilis did not occur value of singly cultured T. japonicus was 0.28 on feeding probably because the experimental temperature (25•Ž) Tetraselmis tetrathele. This r value may be increased by was moderate for both species.3) Large-sized freshwater feeding an appropriate bacterial diet. We will further inves cladocerans can mechanically interfere with small-sized tigate bacterial interference or promotion of zooplankton rotifers. l2) Such effects were not observed for D. celebensis population growth to determine if bacteria have a regulato Interspecific Relations in Rotifer Microcosm 627 ry role in the outcome of competition between nus plicatilis, feeding baking yeast and using large scale outdoor zooplankton species. tank (September-December). Suisan Zoshoku, 25, 63-67 (1977) (in In these experiments, we started B. rotundiformis cul Japanese). tures in the same manner among four combinations of 6) J. J. Gilbert: Suppression of rotifer populations by Daphnia: A rev iew of the evidence, the mechanisms, and the effects on treatments (but different period), by trying to make the zooplankton community structure. Limnol. Oceanogr., 33, 1286 conditions of precultures as well as the condition of Nan 1303 (1988). nochloropsis cultures identical. The population growth of 7) J. J. Gilbert and R. S. Stemberger: Control of popula B. rotundiformis was, however, different aamong trials tions by interference competition from Daphnia. Limnol. (Fig. 2-5). For example, in the B. rotundiformis vs. B. Oceanogr., 30, 180-198 (1985). 8) C. W. Burns and J. J. Gilbert: Effects of daphnid size and density plicatilis and B. rotundiformis vs. Euplotes sp. experi ments, the single-species cultures of B. rotundiformis on interference between Daphnia and . Limnol. Oceanogr., 31, 848-858 (1986). reached to 1,000 individuals on Day 4, while it was Day 6 9) C. W. Burns and J. J. Gilbert: Direct observations of the mechan and 10 for B. rotundiformis vs. D. celebensis and B. rotun isms of interference between Daphnia and Keratella cochlearis. Lim diformis vs. T. japonicus experiments, respectively. The in nol. Oceanogr., 31, 859-866 (1986). consistency of population growth observed between trials 10) R. S. Stemberger and J. J. Gilbert: Rotifer threshold food concen may also be ascribed to differences of bacterial flora tration and the size-efficiencyhypothesis. Ecol., 68, 181-187 (1987). among precultures or experimental cultures. 11) J. J. Gilbert: The escape from response: Defense against interference from Daphnia. Hydrobiol., 147, 235-238 (1987). In addition to the Euplotes employed in this study, there 12) J. J. Gilbert: Competitive interactions between the rotifer Synchae are other protozoan groups which are commonly found in ta oblonga and the cladoceran Scapholeberis kingi Sars. rotifer mass culture tanks, such as Uronema and Vorticel Hydrobiol., 186/187, 75-80 (1989). la. Rotifer mass cultures were unstable when Noctiluca 13) M. Maeda and A. Hino: Environmental management for mass cul scintillans contaminated (K. Hamada, person. comm.). In ture of rotifer, Brachionus plicatilis, in "Rotifer and microalgae cul the larval rearing practices of Ayu Plecoglossus altivelis in ture systems" (ed. by W. Fulks and K. L. Main), Proceedings of a U.S.-Asia Workshop, The Oceanic Institute, Honolulu, 1991, pp. freshwater, marine Brachionus are cultured in low salinity 125-133. usually less than 8 ppt, where coexistence of freshwater 14) S. Segawa and W. T. Yang: Reproduction of an estuarine Di cladocerans, such as Moina, is commonly observed.2) The aphanosoma aspinosum (Branchiopoda: ) under different interspecific relation between these species and marine salinities. Bull. Soc. Japan, 34, 43-51 (1987)(in Japanese). Brachionus will be examined later using the methods em 15) S. Segawa and W. T. Yang: Population growth and density of an es ployed in this research. tuarine cladoceran Diaphanosoma aspinosum in laboratory cul ture. Bull. Plankton Soc. Japan, 35, 67-73 (1988) (in Japanese). 16) S. Segawa and W. T. Yang: Growth, moult, reproduction and filter Acknowledgments We wish to thank T. W. Snell for review and com ing rate of an estuarine cladoceran, Diaphanosoma celebensis, in ments. laboratory culture. Bull. Plankton Soc. Japan, 37, 145-155 (1990) (in Japanese). References 17) A. Hagiwara, C.-S. Lee, and D. J. Shiraishi: Some reproductive characteristics of the broods of the harpacticoid copepod Tigriopus 1) H. Segers: Nomenclatural consequences of some recent studies on japonicus cultured in different salinities. Fisheries Sci., (submitted). Brachionus plicatilis (Rotifera, ). Hydrobiol., (in 18) Y. Fu, Y. Natsukari, and K. Hirayama: Morphological differences press). between two types of the rotifer Brachionus plicatilis O. F. Muller. 2) A. Oka: Biological characteristics of Brachionus plicatilis-Breeding J. Exp. Mar. Biol. Ecol., 151, 29-41 (1991). environment, in "The first live feed-Brachionus plicatilis" (ed. by 19) A. Hagiwara, K. Hamada, S. Hori, and K. Hirayama: Induction of K. Fukusho and K. Hirayama), Koseisha Koseikaku, Tokyo, 1991, monogonont rotifer Brachionus plicatilis O. F. Muller sexual pp. 28-38 (in Japanese). reproduction with bacterial coexistence and addition of rotifer ex 3) K. Hirayama and I. M. F. Rumengan: Fecundity pattern of S and L tracts. J. Exp. Mar. Biol. Ecol., (in press). type rotifers Brachionus plicatilis. Hydrobiol., 255/256, 153-157 20) R. S. Burton: Mating system of the intertidal copepod, Tigriopus (1993). californicus. Mar. Biol., 86, 247-252 (1985). 4) K. Fukusho: Mass production of a copepod, Tigriopus japonicus in 21) J.-P. Yu, A. Hino, M. Ushiro, and M. Maeda: Function of bacteria combination culture with a rotifer Brachionus plicatilis, fed co-yeast as vitamin B12producers during mass culture of the rotifer Brachio as a food source. Nippon Suisan Gakkaishi, 46, 625-629 (1980) (in nus plicatilis. Nippon Suisan Gakkaishi, 55, 1799-1806 (1989). Japanese). 22) J.-P. Yu, A. Hino, T. Nognchi, and H. Wakabayashi: Toxicity of 5) K. Fukusho and C. Kitajima: Mass production of the copepod, Vibrio alginolyticus on the survival of the rotifer Brachionus plicati Tigriopus japonicus, in combination culture with rotifer, Brachio lis. Nippon Suisan Gakkaishi, 56, 1455-1460 (1990).