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Culture of the cladoceran Moina macrocopa: Mortality associated withflagellate 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, USAb Freshwater and Stress Ecology, Institute of Biology, Humboldt University, Berlin 12437, Germanyc Molecular Parasitology, Institute of Biology, Humboldt University, Berlin 10115, Germany
Cladocerans are important food animals in aquaculture, key grazers in freshwater ecosystems, andmodel animalsfor ecotoxicological investigations. Their epibiont community, extensively studied in Daphnia, includes filamen-tous bacteria, fungi, algae, peritrich ciliates, and rotifers; although epibionts are usually benign, heavy infectionscan be detrimental. During our laboratory culture of femaleMoinamacrocopa Straus, we observed a novel flagel-late infection associated withmortality. At day 10, allM.macrocopawere alive in uninfected cultures, whereas inuntreated infected cultures, the survival was significantly lower: only 26% of cladocerans were alive. In infectedcultures treatedwith humic substances (as 25 mg L−1 dissolved organic carbon),mortalitieswere comparable tothose in the untreated infected cultures; in contrast, in the infected cultures treated with 4 g L−1 sea salt, mor-talitieswere arrested, and 76% of theM.macrocopawere alive at day 10.Moribund cladoceranswere transparent,had empty digestive tracts, and greatly reducedmotor activity. Free-swimming flagellatesmoved forwardwith awobbling motion, rotating around their long axis; they also attached to cladoceran tissue, the Petri dish, and theglass slide, by the tip of their posterior flagellum. Flagellates preserved for scanning electron microscopy were6.9 ± 0.7 μm long and 2.1 ± 0.3 μmwide,with a short anterior flagellum (6.8 ± 1.1 μm) and long posterior fla-gellum (14.1 ± 1.5 μm). Multi-functionality of a flagellum, for locomotion and adhesion, is relatively rare, andpreviously reported from genera within the Kinetoplastea, suggesting that the flagellate on M. macrocopa maybelong to this group. To combat flagellate mass occurrence inMoina cultures, we recommend a treatment with4 g L−1 sea salt.
Cladocerans of the genusMoina, andMoinamacrocopa Straus in par-ticular, are progressively important in aquaculture and ecotoxicology.Moina spp. are increasingly used as food for larval and post-larval rear-ing of crustaceans (Alam et al., 1993) and teleost fish in culture (Heet al., 2001; Ingram, 2009; Peña-Aguado et al., 2009). Due to a relativelyhigh protein and nutrient content, Moina spp. is a superior live foodcompared to Artemia (Alam et al., 1993; Loh et al., 2012). Furthermore,the use of freshwater zooplankton, such asM. macrocopa, may be moreconvenient for feeding freshwater species than is use of saltwaterArtemia (Alam et al., 1993; Loh et al., 2012).
Although Moina is widely distributed, from temperate to tropicalregions, commercial scale quantities of this cladoceran are not easilyobtained from natural habitats (Loh et al., 2013). Mass cultivation forlive feed has been successful, and Moina tolerates low oxygen and
high ammonia, reproduces rapidly, and grows rapidly on a range offood sources (Loh et al., 2013). There continues to be considerablefocus 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 widelyused model animals in ecotoxicity testing of synthetic and naturalxenobiotics. Of particular note is that Moina sp. may be used as thereplacement for Daphnia in regions where the latter does not occurnaturally (Ferrão-Filho et al., 2010; Mano et al., 2010; Sarma andNandini, 2006).
The successful and reliable culture of cladocerans as food foraquaculture species is dependent onmany factors, includingmaintenanceof healthy stocks, and effective diagnosis of disease-causing organismssuch as parasites. Cladocerans are hosts to a diversity of epibiont taxa, in-cluding filamentous bacteria, fungi, algae, peritrich ciliates, and rotifers(Ebert, 2005; Green, 1974). Heavy coatings of epibionts can be a weightburden, increase drag (Gilbert and Schröder, 2003), reduce populationgrowth (Green, 1974; Stirnadel andEbert, 1997), and those on the thorac-ic limbs can lower the resistance of their host to oxygen deficiency(Pacuad, 1939). Among the parasitic taxa infecting cladocerans are bacte-ria, fungi, microsporidia, cestodes, and nematodes, which may cause
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behavioral changes (Decaestecker et al., 2005; Makrushin, 2010) and re-duced egg production (Green, 1974; Stirnadel and Ebert, 1997).
Although flagellates have not been reported from cladocerans,they do infect copepods, another group of small freshwater crusta-ceans (Hitchen, 1974), and thus they might be found on cladocerans.Cephalothamnium cyclopum Stein (incertae sedis Kinetoplastea)forms stalked colonies on the copepod Cyclops sp.; one flagellum at-tached to a communally-secreted stalk, the other is used in foodgathering (Hitchen, 1974).
While the epibiont and parasite fauna of cladocerans is well knownfor Daphnia (Ebert, 2005), the fauna of the increasingly importantgenus Moina is little known. Since the classical study by Green (1974),identifying a variety of epibionts and parasites on M. macrocopa, suchasMegachytrium sp., Chloranigiella epizooticum Korschikoff, Pansporellaperplexa Chatton, Epistylis helenae Green, and Brachionus rubensEhrenberg, there appears to have been only one report of a parasitein Moina, namely the microsporidia Gurleya sp. in M. macrocopa(Makrushin, 2010). We now extend knowledge of pathogenic infec-tions in Moina spp. by reporting our light microscopy and scanningelectron microscopy observations on the dense infections of flagel-lates associated with mortality of cultured M. macrocopa used in xe-nobiotic exposure experiments.
2. Materials and methods
2.1. Stress ecology studies and source of Moina
The background for the present investigation was our maintenanceof cultures of M. macrocopa for stress ecology studies, in which weaimed to determine whether the heritage of cross tolerance was epige-netically controlled and based on DNA methylation in the presence ofhumic substances. To pursue this, in xenobiotic experiments, we
a
Fig. 1. Light micrographs ofMoina macrocopa and live bodonid flagellates from theM. macrocopsecond antenna of the cladoceran, arrows indicate the flagellates, (c, d) two flagellates showingthe yellow-brown refractile inclusions. Scale bars = 100 μm (a, b) and 10 μm (c, d).
pre-exposed Moina to humic substances, and then tested theircross tolerance against sea salt (following Suhett et al. (2011)). Dur-ing these xenobiotic experiments, some cultures became infectedwith flagellates, allowing us the opportunity to study them, andtheir response to DOC and salt.
M. macrocopa (Fig. 1a) is a characteristic inhabitant of small, usuallyephemeral, water bodies from temperate to tropical regions, which areoften rich in dissolved organic carbon (Petrusek, 2002). Our clone wasoriginally isolated from a puddle in Rio de Janeiro, Brazil (Elmoor-Loureiro et al., 2010), and has been successfully used since then in lifetable and cross tolerance studies in stress ecology (Hofmann et al.,2012; Suhett et al., 2011), and in a recent DNA methylation study(Menzel et al., 2011).
2.2. Maintenance of cladoceran cultures and cross tolerance experiments
The stock culture was maintained in artificial Daphnia medium(Klüttgen et al., 1994). Only neonates of the 3rd generation under iden-tical laboratory conditionswere used for the experiments.M.macrocopareproduces partheogenetically under stable laboratory conditions (con-trasting with sexual reproduction in times of stress), and all offspringfrom this asexual reproduction were female. M. macrocopa was feddaily, ad libitum, with the coccal green algae Raphidocelis subcapitata(Korshikov) Nygaard, Komárek, J. Kristiansen & O.M. Skulberg.
Each xenobiotic experiment was initiated with 10 replicates, in eachof which were 10 M. macrocopa in a 200 ml Erlenmeyer flask. Duringthe experiments, although some replicates were lost, there was alwaysaminimumof 8 replicates for each experiment; (thus thenumber of livecladocerans expected at each time point per experiment was 80, 90 or100, assuming no mortalities). The flasks were kept in a temperature-controlled room at 20 ± 1 °C, and illuminated by cool white light in a14:10 h light:dark rhythm. Every second day, the number of live and
b
c d
a cultures. (a) Healthy parthenogeneticMoina macrocopa female, (b) posterior part of thethe shortwhiplash anterior flagellumand the long posterior/recurrent flagellum, note also
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dead cladocerans was recorded, and the exposure medium was ex-changed. Dead individuals were removed immediately after counting.
2.3. Infected cultures and treatments
During the experiments, we observed that while the uninfectedcontrols remained healthy (these were the cultures with no additionof humic substances or salt), the flagellate-infected controls sufferedmortalities.
To try to reduce subsequent mortalities, infected cultures weretreated with either: (i) humic substances (as 25 mg L−1 DOC) becausethey can reduce growth and survival of aquatic parasites and pathogens(Meinelt et al., 2007, 2008), or (ii) sea salt (as 4 g L−1 sea salt), becausesalt is commonly used to treat ectoparasitic infections in fish; theconcentration was well within the previously documented tolerancerange of the M. macrocopa clone we studied (Suhett et al., 2011).During the 10 day treatment period, live and dead cladocerans werecounted every second day, and dead individuals removed, as describedabove.
2.4. Microscopy and video recordings
Light microscopy and video recordings were used to documentshape and motility of the flagellates. To slow the flagellates, a fewdrops of a 1% methyl cellulose solution were added.
Surface ultrastructure of the flagellates was observed by scanningelectron microscopy study of individuals retained on the tissues of thecladocerans. Tissues were fixed with 2.5% (v/v) glutaraldehyde and 2%(w/v) paraformaldehyde in 100 mM cacodylate buffer (pH 7.4) for30 min at room temperature. After fixation, samples were rinsedthree times for 10 minwith 100 mMcacodylate buffer, and dehydratedthrough a graded ethanol series. After washing three times withhexamethyldisilazane (Electron Microscopy Sciences), flagellates werecoated with gold and analyzed on a LEO 1430 scanning electronmicroscope. Morphometrics were determined from examination ofSEM specimens.
2.5. Data analysis
The entire lifespan of an exposure groupwas derived frommortalitydata, and the data at each time point [% of day 0 individuals still alive]were means for the replicates in each group. Differences betweengroups over the entire 10 days, were tested for statistical signifi-cance by the log-rank test, which was developed specifically forlifespan curves (Bioinformatics at the Walter and Eliza Hall Institute ofMedical Research (http://bioinf.wehi.edu.au/software/russell/logrank/)).The log-rank test has previously been applied to lifespan data forM. macrocopa (Bouchnak and Steinberg, 2014; Suhett et al., 2011).Differences were considered statistically significant when p b 0.05.
Table 1Temporal changes inmortalities in fourMoinamacrocopa cultures. Data are percent of day0 individuals that were alive at each time point (means ± SD). Each xenobiotic exposureexperiment was initiated with 10 replicates, each with 10 individual cladocerans.
In the uninfected cultures, all of the M. macrocopa were alive atday 10 (Table 1). However, in the infected and untreated cultures,approximately half of the M. macrocopa had died after one week,and only 26% remained alive at day 10. Survival in the infected,untreated cultures was significantly lower than in the uninfected cul-tures (p b 0.01).
In the infected cultures treated with 25 mg L−1 DOC, mortalitieswere high, and were not significantly different from the infectedcultures that were not treated. In contrast, in the infected culturestreated with 4 g L−1 sea salt, mortalities were arrested, with 76%of the M. macrocopa alive at day 10 (Table 1). Although survival inthis treatment was significantly higher than in the infected culturestreated with humic substances [25 mg L−1 DOC] (p b 0.01), it wassignificantly lower than in the uninfected controls (p b 0.05).
Moribund cladocerans were transparent, their digestive tractswere empty, and their motor activity was greatly reduced. In con-trast, the healthy individuals were slightly opalescent, their diges-tive tracts were full of green algae, and they moved very actively.
The addition of humic substances was ineffective against the fla-gellates affecting the Moina, although humic substances can reducegrowth and survival of some aquatic parasites and pathogens(Meinelt et al., 2007, 2008), and increase lifespan of hosts. However,the addition of 4 g L−1 sea salt, a concentration well within the toler-ance range of the clone (Suhett et al., 2011), resulted in significantly re-duced host mortality. We presume that the flagellates were sensitive tothe change in osmolarity, which in turn reduced their viability, and thustheir damaging effects to the cladocerans.
3.2. Association with cladoceran tissue
There were numerous flagellates inside the body cavity of the liveM. macrocopa, and flagellates were also seen outside the body (Fig. 1a,b). Although most of the flagellates were attached to the host tissuesby their long posterior flagellum, they could also swim freely and thenreattach to the Moina tissues. In the culture media, flagellates wereusually attached by their posterior flagellum to rigid structures,such as glass slides or a Petri-dish; free-swimming flagellates wererarely observed.
3.3. Movement
When free-swimming, the flagellatesmoved forward with awob-bling motion, frequently rotating around their long axis. The shorteranterior flagella had a whip-like action, and the longer posterior/recurrent flagella trailed (Fig. 1c–d).
When attached by the tip of their long posterior/recurrent flagellum,to the cladoceran or the culture vessel, the flagellates whirled around,and the short anterior flagellum was very active. The flagellates couldquickly attach, detach, and reattach.
Video clips showing the flagellates inside the cladoceran tissue (lowmagnification), and in the culturemedium (highmagnification), can beviewed online as supplementary data (see Appendix A).
3.4. Morphometrics
The morphometrics of the flagellates, when viewed under thescanning electron microscope were (minimum, maximum, mean,standard deviation): 5.8–8.1 μm long (mean 6.9 ± 0.7, n = 15)and 1.5–2.5 μm wide (mean = 2.1 ± 0.3, n = 12) (Fig. 2a–d). Theanterior flagellum was 5.4–8.5 μm long (mean = 6.8 ± 1.1, n =13), and the posterior/recurrent flagellum was 13.5–15.4 μm long(14.1 ± 1.5, n = 8); the twoflagella emerged together, approximately
Fig. 2. Scanning electronmicrographs of flagellates from laboratory culture of the cladoceranMoinamacrocopa. Note also the numerous rod-shapedbacteria. (a, b)Wholeflagellates show-ing their heterodynamic flagella, the short whiplash anterior flagellum and the long trailing posterior/recurrent flagellum, (c, d) longitudinal view showing the anterior of the flagellate,and the two emergent flagella, note also the two pores (visible in d), (e) flagellate undergoing longitudinal binary fission, which has begun at the anterior, note the two pores at the right,and the attachment of the flagellate by the flagellar tip, (f) surface of flagella, which is smooth, no hairs are visible [enlargement of part of panel a], and (g) surface of posterior/recurrentflagellum, longitudinal ridges appear present, no hairs are visible [enlargement of part of panel b]. Scale bars in the micrographs are 1 μm.
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1.0 μm from the anterior end of the cell (Fig. 2c, d). The surfaceof the body was smooth. In some individuals, there were twopores each approximately 0.15–0.20 μm in diameter, situated1.5 μm posterior to the emergence of the flagella (Fig. 2d, e). Inlive flagellates, we observed multiple distinct yellowish-brownrefractile inclusions, approximately 0.25–0.50 μm in diameter(Fig. 1c–d).
Individuals divided by longitudinal binary fission, which com-menced at the anterior end of the cell (Fig. 2e). In some cells, the flagella
surface was smooth (Fig. 2f), while in others there were longitudinalridges (Fig. 2g).
In our descriptions of the flagellate, we have chosen to continueto use the term “flagellum” for each of the locomotory organelles,as it is a conventional practice. However, we are aware of the propos-al, recently made by Adl et al. (2012), to refer to a eukaryotic flagel-lum as a cilium, and thus such organisms as we now describe, wouldbe considered biciliated. It is not yet clear whether the new terminol-ogy proposed by Adl et al. (2012) will be widely adopted.
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3.5. Identity of the flagellate
The flagellate in theM. macrocopa cultures is tentatively assignedto the Kinetoplastea on the basis of the behavior and morphology weobserved. Strong support for placement of theMoina flagellate with-in the Kinetoplastea comes from the observation that it can attach bythe tip of the posterior flagellum; this quality is unique to this groupof flagellates (Vickerman, 1989).
Diverse groups offlagellates contain elongate specieswith 2 unequal(heterodynamic) flagella, including the cryptomonads, euglenids,kinetoplastids, and retortamonads. The unadorned surface of theflagella of organism we have described, distinguishes it from thecryptomonds and euglenids which have hairs on the flagella, andfrom the retortamonds which have lamellae on the flagella. Fur-thermore, the flagellum of kinetoplastids has a unique paraflagellarrod, the presence of which may have been indicated in the Moinaflagellate by the longitudinal ridges (as shown in Fig. 2g).
Within the Kinetoplastea, there are numerous genera with two fla-gella. Based on our observations of live organisms, the flagellatewe now report appears most akin to Bodo, Rhynchobodo, andRhynchomonas. We did not interpret the anterior of theMoina organismas a snout (thus precluding Rhynchobodo), nor did we see creepingmo-tility (precluding Rhynchomonas). Thus the flagellate appearsmost sim-ilar to Bodo. However, to make a firm assignment to genus, additionallight microscopy and molecular characterization are needed, as de-scribed in the section on “Recommendations” below.
3.6. Pathogenicity
The evidence suggests that the flagellate was a cause, rather than aconsequence, of the poor condition of the cladocerans. In support of itsrole as a pathogenic parasite is the following: (i) strong association be-tween bodonid infection andmortality ofM.macrocopa, (ii) strong asso-ciation between sea salt treatment and reduced mortality, and (iii) noassociation between otherwise stressed Moina populations (populationdensity, starvation, age) and flagellate infection.
The present report appears to be the first documentation of flagel-lates being associated with morbidity and mortality in a population ofMoina spp. Although there are reports of the population dynamics ofMoina spp. in culture (as cited in the Introduction to this paper), andthe seasonal dynamics of M. macrocopa in natural ponds in Iran(Khalaf and Shihab, 1979), flagellate infection has not been described.We consider it highly likely that a diversity of protozoan infectionsplay a role in morbidity and mortality in cultured and wild populationsof Moina spp., but that such infections are under reported.
We consider that other organisms present in the culture medium,including bacteria and viruses, may have contributed to the morbidityand mortality we now report. Numerous rod-shaped bacteria werepresent in the cultures with the flagellate-infected M. macrocopa, asshown in the scanning electron micrographs (Fig. 2a, b).
3.7. Recommendations
To confirm the identity of flagellates from infected Moina, lightmicroscopy and molecular approaches are needed. Light microscopyshould include staining of the cells by DAPI, which would then showif there were large amounts of DNA in their mitochondrion (thusconfirming that they are kinetoplastids), and the location of the kineto-plast DNA (thereby allowing assignment to the Order Neobodonida,Parabodonida, or Eubodonida) (Adl et al., 2012). Molecular charac-terization is also needed. Molecular tools for the detection and iden-tification of kinetoplastids are rapidly advancing, particularly forpathogenic taxa such as Ichthyobodo spp. (i.e. sequencing of theSSU rDNA/18S rRNA) (Moreira et al., 2004), specific quantitativereal-time PCR targeting SSU rDNA, and novel primer sets for identifi-cation using PCR and sequencing (Isaksen et al., 2012). To facilitate
comparative studies, voucher specimens (infected Moina in ethanol,and flagellates preserved for light and electron microscopy), shouldbe deposited in museum collections.
To safeguard Moina cultures and clarify the role of the flagellates inmortality, we recommend that flagellates be removed by filteringwater through a 2 μmpore filter. If, however, heavy flagellate infectionsdo occur, they may be combatted by increasing the salinity of the cul-turemediumup to approximately 4.0 g L−1, which is below the salinityof 5.5 g L−1 that is lethal toM. macrocopa (Suhett et al., 2011).
Acknowledgment
We are pleased to thank Dr. Christian Schuetz of Johns HopkinsUniversity School of Medicine for his assistance in translating textfrom German to English. We thank Gabriele Drescher for technicalassistance with the preparation of samples for electron microscopy.All experiments were carried out in compliancewith the correspondinglaws in Germany, and the work was conducted ethically and conformsto the uniform requirements for manuscripts submitted to biomedicaljournals.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.aquaculture.2013.09.029.
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