Aquaculture 416–417 (2013) 374–379Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online Short communication Culture of the cladoceran Moina macrocopa: Mortality associated with flagellate infection Sarah L. Poynton a,⁎, Philipp Dachsel b, Maik J. Lehmann c, Christian E.W. Steinberg b a Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Room 855 Edward D. Miller Research Building, 733 North Broadway, Baltimore, MD 21218, USA b Freshwater and Stress Ecology, Institute of Biology, Humboldt University, Berlin 12437, Germany c Molecular Parasitology, Institute of Biology, Humboldt University, Berlin 10115, Germany a r t i c l e i n f o Article history: Received 9 July 2013 Received in revised form 16 September 2013 Accepted 17 September 2013 Available online 25 September 2013 Keywords: Adhesive flagellum Bodoid flagellate Cladoceran Kinetoplastid Moina macrocopa a b s t r a c t Cladocerans are important food animals in aquaculture, key grazers in freshwater ecosystems, and model animals for ecotoxicological investigations. Their epibiont community, extensively studied in Daphnia, includes filamentous bacteria, fungi, algae, peritrich ciliates, and rotifers; although epibionts are usually benign, heavy infections can be detrimental. During our laboratory culture of female Moina macrocopa Straus, we observed a novel flagellate infection associated with mortality. At day 10, all M. macrocopa were alive in uninfected cultures, whereas in untreated infected cultures, the survival was significantly lower: only 26% of cladocerans were alive. In infected cultures treated with humic substances (as 25 mg L−1 dissolved organic carbon), mortalities were comparable to those in the untreated infected cultures; in contrast, in the infected cultures treated with 4 g L−1 sea salt, mortalities were arrested, and 76% of the M. macrocopa were alive at day 10. Moribund cladocerans were transparent, had empty digestive tracts, and greatly reduced motor activity. Free-swimming flagellates moved forward with a wobbling motion, rotating around their long axis; they also attached to cladoceran tissue, the Petri dish, and the glass slide, by the tip of their posterior flagellum. Flagellates preserved for scanning electron microscopy were 6.9 ± 0.7 μm long and 2.1 ± 0.3 μm wide, with a short anterior flagellum (6.8 ± 1.1 μm) and long posterior flagellum (14.1 ± 1.5 μm). Multi-functionality of a flagellum, for locomotion and adhesion, is relatively rare, and previously reported from genera within the Kinetoplastea, suggesting that the flagellate on M. macrocopa may belong to this group. To combat flagellate mass occurrence in Moina cultures, we recommend a treatment with 4 g L−1 sea salt. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Cladocerans of the genus Moina, and Moina macrocopa Straus in particular, are progressively important in aquaculture and ecotoxicology. Moina spp. are increasingly used as food for larval and post-larval rearing of crustaceans (Alam et al., 1993) and teleost fish in culture (He et al., 2001; Ingram, 2009; Peña-Aguado et al., 2009). Due to a relatively high protein and nutrient content, Moina spp. is a superior live food compared to Artemia (Alam et al., 1993; Loh et al., 2012). Furthermore, the use of freshwater zooplankton, such as M. macrocopa, may be more convenient for feeding freshwater species than is use of saltwater Artemia (Alam et al., 1993; Loh et al., 2012). Although Moina is widely distributed, from temperate to tropical regions, commercial scale quantities of this cladoceran are not easily obtained from natural habitats (Loh et al., 2013). Mass cultivation for live feed has been successful, and Moina tolerates low oxygen and ⁎ Corresponding author. Tel.: +1 410 502 5065. E-mail addresses:
[email protected] (S.L. Poynton),
[email protected] (P. Dachsel),
[email protected] (M.J. Lehmann),
[email protected] (C.E.W. Steinberg). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.09.029 high ammonia, reproduces rapidly, and grows rapidly on a range of food sources (Loh et al., 2013). There continues to be considerable focus on investigating different foods for mass culture of M. macrocopa (Kang et al., 2006; Loh et al., 2009, 2013). In the laboratory, Moina spp., and Daphnia magna Straus are widely used model animals in ecotoxicity testing of synthetic and natural xenobiotics. Of particular note is that Moina sp. may be used as the replacement for Daphnia in regions where the latter does not occur naturally (Ferrão-Filho et al., 2010; Mano et al., 2010; Sarma and Nandini, 2006). The successful and reliable culture of cladocerans as food for aquaculture species is dependent on many factors, including maintenance of healthy stocks, and effective diagnosis of disease-causing organisms such as parasites. Cladocerans are hosts to a diversity of epibiont taxa, including filamentous bacteria, fungi, algae, peritrich ciliates, and rotifers (Ebert, 2005; Green, 1974). Heavy coatings of epibionts can be a weight burden, increase drag (Gilbert and Schröder, 2003), reduce population growth (Green, 1974; Stirnadel and Ebert, 1997), and those on the thoracic limbs can lower the resistance of their host to oxygen deficiency (Pacuad, 1939). Among the parasitic taxa infecting cladocerans are bacteria, fungi, microsporidia, cestodes, and nematodes, which may cause macrocopa. Since the classical study by Green (1974). macrocopa was fed daily. ad libitum. another group of small freshwater crustaceans (Hitchen.. in M. they do infect copepods. The flasks were kept in a temperaturecontrolled room at 20 ± 1 °C. Each xenobiotic experiment was initiated with 10 replicates. usually ephemeral. in which we aimed to determine whether the heritage of cross tolerance was epigenetically controlled and based on DNA methylation in the presence of humic substances. (b) posterior part of the second antenna of the cladoceran.. and their response to DOC and salt. To pursue this. although some replicates were lost. 1997). M.. During these xenobiotic experiments. macrocopa cultures. one flagellum attached to a communally-secreted stalk. While the epibiont and parasite fauna of cladocerans is well known for Daphnia (Ebert. / Aquaculture 416–417 (2013) 374–379 behavioral changes (Decaestecker et al. 2010) and reduced egg production (Green. the number of live and b a c d Fig.S. Makrushin. allowing us the opportunity to study them. Skulberg. M. Our clone was originally isolated from a puddle in Rio de Janeiro. Pansporella perplexa Chatton.. Stress ecology studies and source of Moina The background for the present investigation was our maintenance of cultures of M. Poynton et al. d). 2005)..L. Suhett et al. and in a recent DNA methylation study (Menzel et al. with the coccal green algae Raphidocelis subcapitata (Korshikov) Nygaard. which are often rich in dissolved organic carbon (Petrusek. Chloranigiella epizooticum Korschikoff. 1974). 2012. 90 or 100.. and illuminated by cool white light in a 14:10 h light:dark rhythm. macrocopa in a 200 ml Erlenmeyer flask. such as Megachytrium sp. Scale bars = 100 μm (a.. in each of which were 10 M. there appears to have been only one report of a parasite in Moina. During the experiments. and then tested their cross tolerance against sea salt (following Suhett et al. Stirnadel and Ebert. Epistylis helenae Green. 2010). Light micrographs of Moina macrocopa and live bodonid flagellates from the M. we 375 pre-exposed Moina to humic substances. Cephalothamnium cyclopum Stein (incertae sedis Kinetoplastea) forms stalked colonies on the copepod Cyclops sp. . macrocopa reproduces partheogenetically under stable laboratory conditions (contrasting with sexual reproduction in times of stress).M. macrocopa for stress ecology studies.. (thus the number of live cladocerans expected at each time point per experiment was 80. 2. d) two flagellates showing the short whiplash anterior flagellum and the long posterior/recurrent flagellum. (c. Every second day. 2011). macrocopa (Makrushin. Kristiansen & O. water bodies from temperate to tropical regions. Komárek. (2011)). b) and 10 μm (c. 2010). 2011). 1974. there was always a minimum of 8 replicates for each experiment. (a) Healthy parthenogenetic Moina macrocopa female. the fauna of the increasingly important genus Moina is little known. and thus they might be found on cladocerans. in xenobiotic experiments. by reporting our light microscopy and scanning electron microscopy observations on the dense infections of flagellates associated with mortality of cultured M.1. the other is used in food gathering (Hitchen. 1974). Although flagellates have not been reported from cladocerans. We now extend knowledge of pathogenic infections in Moina spp. Only neonates of the 3rd generation under identical laboratory conditions were used for the experiments. arrows indicate the flagellates. note also the yellow-brown refractile inclusions. namely the microsporidia Gurleya sp. 1994). and Brachionus rubens Ehrenberg. and all offspring from this asexual reproduction were female. 1a) is a characteristic inhabitant of small. and has been successfully used since then in life table and cross tolerance studies in stress ecology (Hofmann et al. assuming no mortalities). J. macrocopa used in xenobiotic exposure experiments. identifying a variety of epibionts and parasites on M. M. 2. 1. macrocopa (Fig. Maintenance of cladoceran cultures and cross tolerance experiments The stock culture was maintained in artificial Daphnia medium (Klüttgen et al. Materials and methods 2.2. 2002). Brazil (ElmoorLoureiro et al. some cultures became infected with flagellates. 2005. in the infected and untreated cultures.05. a few drops of a 1% methyl cellulose solution were added.3 7. and reattach. and their motor activity was greatly reduced.9 11. and flagellates were also seen outside the body (Fig. Differences were considered statistically significant when p b 0. which was developed specifically for lifespan curves (Bioinformatics at the Walter and Eliza Hall Institute of Medical Research (http://bioinf.4–8. Microscopy and video recordings Light microscopy and video recordings were used to document shape and motility of the flagellates. Video clips showing the flagellates inside the cladoceran tissue (low magnification)..3 100 98. The anterior flagellum was 5. macrocopa (Bouchnak and Steinberg. they could also swim freely and then reattach to the Moina tissues.4.au/software/russell/logrank/)). because salt is commonly used to treat ectoparasitic infections in fish. Morphometrics The morphometrics of the flagellates. Infection and mortality 2. However.1 28.4 In the uninfected cultures.5 70. and in the culture medium (high magnification).5. resulted in significantly reduced host mortality. the flagellate-infected controls suffered mortalities.2 16. all of the M.7 ± ± ± ± ± 0. were tested for statistical significance by the log-rank test.8 ± 1. macrocopa alive at day 10 (Table 1). 2008). Although most of the flagellates were attached to the host tissues by their long posterior flagellum. untreated cultures was significantly lower than in the uninfected cultures (p b 0. their digestive tracts were empty. Results and discussion 3. flagellates were coated with gold and analyzed on a LEO 1430 scanning electron microscope. Morphometrics were determined from examination of SEM specimens. 3.1 26. n = 8).L. the addition of 4 g L−1 sea salt. 2007.7 25. During the 10 day treatment period. Day Uninfected untreated Infected untreated Infected +25 mg L−1 DOC Infected +4 g L−1 sea salt 2 4 6 8 10 100 100 100 100 100 100 97. infected cultures were treated with either: (i) humic substances (as 25 mg L−1 DOC) because they can reduce growth and survival of aquatic parasites and pathogens (Meinelt et al.5–15.5.edu. in the infected cultures treated with 4 g L− 1 sea salt. macrocopa had died after one week. mortalities were arrested. Data are percent of day 0 individuals that were alive at each time point (means ± SD). standard deviation): 5.5 μm wide (mean = 2.0 ± ± ± ± ± 0. flagellates were usually attached by their posterior flagellum to rigid structures. 1a. b). when viewed under the scanning electron microscope were (minimum.8 77. or (ii) sea salt (as 4 g L−1 sea salt).5 14.. and thus their damaging effects to the cladocerans.0 7. Infected cultures and treatments During the experiments. Survival in the infected. the healthy individuals were slightly opalescent. However. 2.6 7.8 76.0 0.. can be viewed online as supplementary data (see Appendix A). The addition of humic substances was ineffective against the flagellates affecting the Moina. 2a–d). it was significantly lower than in the uninfected controls (p b 0. approximately half of the M. and the longer posterior/ recurrent flagella trailed (Fig. such as glass slides or a Petri-dish. Poynton et al. Dead individuals were removed immediately after counting.05).5 100 97.5% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 100 mM cacodylate buffer (pH 7. Surface ultrastructure of the flagellates was observed by scanning electron microscopy study of individuals retained on the tissues of the cladocerans. Association with cladoceran tissue There were numerous flagellates inside the body cavity of the live M.6 38. After fixation. In the culture media. The shorter anterior flagella had a whip-like action. detach. the concentration was well within the previously documented tolerance range of the M.0 0.4.2. In contrast. macrocopa clone we studied (Suhett et al.6 18. 3. Moribund cladocerans were transparent.9 ± 0. macrocopa. and dehydrated through a graded ethanol series. Suhett et al. and the short anterior flagellum was very active. each with 10 individual cladocerans. and the data at each time point [% of day 0 individuals still alive] were means for the replicates in each group.7. Data analysis The entire lifespan of an exposure group was derived from mortality data. and the posterior/recurrent flagellum was 13. 2014.1 μm long (mean 6. frequently rotating around their long axis. When attached by the tip of their long posterior/recurrent flagellum.5–2. Tissues were fixed with 2.3. Table 1 Temporal changes in mortalities in four Moina macrocopa cultures.5 90.3. approximately .9 ± ± ± ± ± 0. which in turn reduced their viability.4 μm long (14. to the cladoceran or the culture vessel. We presume that the flagellates were sensitive to the change in osmolarity. their digestive tracts were full of green algae.376 S. n = 15) and 1. the two flagella emerged together. After washing three times with hexamethyldisilazane (Electron Microscopy Sciences). 1c–d). and only 26% remained alive at day 10. Although survival in this treatment was significantly higher than in the infected cultures treated with humic substances [25 mg L− 1 DOC] (p b 0. maximum.1.3 ± ± ± ± ± 0.5 μm long (mean = 6. 2011). 2008). and were not significantly different from the infected cultures that were not treated. and increase lifespan of hosts. To slow the flagellates. To try to reduce subsequent mortalities.1.4 17.. mean.wehi. / Aquaculture 416–417 (2013) 374–379 dead cladocerans was recorded.1 ± 1.8–8. the flagellates whirled around. Movement When free-swimming. and the exposure medium was exchanged. 3.4 43. In the infected cultures treated with 25 mg L−1 DOC. the flagellates moved forward with a wobbling motion. Differences between groups over the entire 10 days.0 0. mortalities were high.6 11. n = 12) (Fig. although humic substances can reduce growth and survival of some aquatic parasites and pathogens (Meinelt et al. 2011). The log-rank test has previously been applied to lifespan data for M. live and dead cladocerans were counted every second day.. samples were rinsed three times for 10 min with 100 mM cacodylate buffer. The flagellates could quickly attach. 3. Each xenobiotic exposure experiment was initiated with 10 replicates. and they moved very actively. with 76% of the M. we observed that while the uninfected controls remained healthy (these were the cultures with no addition of humic substances or salt). as described above. a concentration well within the tolerance range of the clone (Suhett et al. 2007. 2011). 2.0 3.1 ± 0.01).01).0 88.4) for 30 min at room temperature. free-swimming flagellates were rarely observed. and dead individuals removed. In contrast.0 0. n = 13).0 4. macrocopa were alive at day 10 (Table 1).3. note also the two pores (visible in d). d) longitudinal view showing the anterior of the flagellate.25–0. no hairs are visible [enlargement of part of panel b].15–0. (e) flagellate undergoing longitudinal binary fission. there were two pores each approximately 0. / Aquaculture 416–417 (2013) 374–379 a c 377 b d f e g Fig. 1c–d). 2d. The surface of the body was smooth. However.0 μm from the anterior end of the cell (Fig. Scale bars in the micrographs are 1 μm. 2. and the attachment of the flagellate by the flagellar tip. the short whiplash anterior flagellum and the long trailing posterior/recurrent flagellum. b) Whole flagellates showing their heterodynamic flagella. recently made by Adl et al. we observed multiple distinct yellowish-brown refractile inclusions. as it is a conventional practice. we are aware of the proposal. while in others there were longitudinal ridges (Fig. Note also the numerous rod-shaped bacteria. (f) surface of flagella.50 μm in diameter (Fig. and the two emergent flagella. . which has begun at the anterior. (2012). the flagella surface was smooth (Fig. In some cells. and (g) surface of posterior/recurrent flagellum. we have chosen to continue to use the term “flagellum” for each of the locomotory organelles.S. 2c. 2g). d).20 μm in diameter. In live flagellates. It is not yet clear whether the new terminology proposed by Adl et al. longitudinal ridges appear present. Poynton et al. no hairs are visible [enlargement of part of panel a]. Individuals divided by longitudinal binary fission. and thus such organisms as we now describe. approximately 0. would be considered biciliated. situated 1. which commenced at the anterior end of the cell (Fig. In some individuals. (2012) will be widely adopted. (a. to refer to a eukaryotic flagellum as a cilium. 2e). note the two pores at the right. In our descriptions of the flagellate. 2f). which is smooth. (c.L. 1.5 μm posterior to the emergence of the flagella (Fig. Scanning electron micrographs of flagellates from laboratory culture of the cladoceran Moina macrocopa. e). .. Res. Khoo. A. 70. Aquaculture 287.. Cladocera) and perspective as live food for marine fish larvae: review. in culture (as cited in the Introduction to this paper). may have contributed to the morbidity and mortality we now report. as shown in the scanning electron micrographs (Fig. Khoo.T.. Smirnov. Green.F.J.Bamidgeh 64.. H. Z. and the seasonal dynamics of M. V. of the poor condition of the cladocerans. National Library of Medicine (US).J. Ratte. The present report appears to be the first documentation of flagellates being associated with morbidity and mortality in a population of Moina spp. Based on our observations of live organisms. J.W. Declerck. Alan Ong. S. Recommendations To confirm the identity of flagellates from infected Moina. J. Y... H.. L.D.E... macrocopa in natural ponds in Iran (Khalaf and Shihab. Qin. or Eubodonida) (Adl et al... However.L.M. S.. as described in the section on “Recommendations” below.B. Lara. 3. Hampl.K. 61. 2012. reproduction and effect on two rotifers. and flagellates preserved for light and electron microscopy). Moina macrocopa.W.. P. T. macrocopa. E. and retortamonads. 2009. C. Epidemiology.. 2012.. 221–231. 743–746. Acknowledgment We are pleased to thank Dr. E. Oecologia 144. 382–390. The revised classification of eukaryotes. Braz.S.E. and the work was conducted ethically and conforms to the uniform requirements for manuscripts submitted to biomedical journals. Supplementary data Supplementary data to this article can be found online at http://dx. Y. Lopes. Hoppenrath. M. R. Azevedo.L. Pollut. Biol. body size. II. 2012). Aquaculture 109. 21. Use of Moina micrura (Kurz) as an Artemia substitute in the production of Macrobrachium rosenbergii (de Man) post-larvae. Gall. S. Bouchnak. Bass. C. specific quantitative real-time PCR targeting SSU rDNA. C. Ingram. To facilitate Adl.6. Zool. they may be combatted by increasing the salinity of the culture medium up to approximately 4. J. F.5 g L−1 that is lethal to M.M. D.2013. G. trout cod and Macquarie perch (Percichthyidae) in fertilised earthen ponds.A. Klüttgen. which is below the salinity of 5. Loh. Decaestecker. D. macrocopa.O. V. 2012)... 2g). J. Anomopoda) in South America.M. Molecular tools for the detection and identification of Ichthyobodo spp.. enriched with lipid emulsions. and Evolution of Parasitism in Daphnia.. D. W.A. 48. Moina macrocopa and M. All experiments were carried out in compliance with the corresponding laws in Germany. age) and flagellate infection.. macrocopa (Suhett et al. A. M.H. 675–683. J. Pathogenicity The evidence suggests that the flagellate was a cause.e. Ferrão-Filho. 417–515. doi. the flagellate we now report appears most akin to Bodo.5. H. 2a. Water Res. 1974. the flagellum of kinetoplastids has a unique paraflagellar rod.. 225–226.. 2012. Hii.. Parasitol..L. Parabodonida. References 3. Khalaf. Rhynchobodo. Simpson. Aquacult. and novel primer sets for identification using PCR and sequencing (Isaksen et al. 2005... and the location of the kinetoplast DNA (thereby allowing assignment to the Order Neobodonida.. 5. distinguishes it from the cryptomonds and euglenids which have hairs on the flagella. 1974. including the cryptomonads. Menzel. ADaM. J. including bacteria and viruses. Lynn. 1820) (Cladocera.W. / Aquaculture 416–417 (2013) 374–379 3.. E. Heiss. 23–31. Ong. Rueckert. A. Ecology. 1227–1237. Fish faeces as a potential food source for cultivating the water flea. S.. Light microscopy should include staining of the cells by DAPI.. The fine structure of the colonial kinetoplastid flagellate Cephalothamnium cyclopum Stein..K.. Trop. this quality is unique to this group of flagellates (Vickerman. The unadorned surface of the flagella of organism we have described.S. 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