Invest. Mar., Valparaíso, 35(2): 113-120, 2007 off the Chilean coast 113

Short Communication

New records of marine choanoflagellates off the Chilean coast

Katia Soto-Liebe1, Gloria Collantes2 & Juan Kuznar1 1Facultad de Ciencias, Universidad de Valparaíso, Casilla 5030 Valparaíso 2Facultad de Ciencias del Mar y Recursos Naturales, Universidad de Valparaíso, Casilla 5080 Reñaca, Viña del Mar, Chile

ABSTRACT. This is the first report of planktonic choanoflagellates from the Chilean coast, relating their abundance with that of and viruses. Surface water samples were taken off Antofagasta, Montemar, and Puerto Montt and samples from ballast tanks were also analyzed. The choanoflagellates were identified following morphological type descriptions. Viruses and bacteria were stained with SYBR Green and choanoflagellates, bacteria, and viruses were counted simulta- neously using red autofluorescence for the former and green fluorescence for the latter two. Six of were observed for the first time in Chilean waters:Calliacantha multispina (0.3·103 L-1), Acanthocorbis apoda, Stepha- noeca diplocostata (2.0·103 L-1), Crinolina isefiordensis, Parvicorbicula superpositus, and Pleurasiga minima (5.0·103 L-1). The concentrations of bacteria (1.1·106-4.5·10 6) and viral-like particles (VLPs) (7.9·10 6-21·10 6) agreed with those typically found in marine coastal waters. In addition, Acanthocorbis asymmetrica was found in ballast waters, where its high concentration (20·103 L-1) likely resulted from the particular physical and biological environment therein. Key words: Choanoflagellates, bacteria, virus, ballast water, epifluorescence, Chile.

Nuevos registros de coanoflagelados marinos de la costa de Chile

RESUMEN. Este es el primer reporte sobre coanoflagelados planctónicos de la costa chilena y su abundancia se relacionó con la de y virus. Se analizaron muestras de aguas superficiales obtenidas en Antofagasta, Montemar y Puerto Montt. Además se analizaron muestras adicionales de aguas provenientes de estanques de lastre. Los coanoflagelados se determinaron de acuerdo a descripciones morfológicas tipo. Virus y bacterias fueron teñidos con SYBR Green y la enumeración simultánea de coanoflagelados, bacterias y virus se determinó a través de autofluorescencia roja y fluo- rescencia verde respectivamente. Seis especies de Acanthoecidae fueron observadas por primera vez en aguas chilenas: Calliacantha multispina (0,3·103 L-1), Acanthocorbis apoda y Stephanoeca diplocostata (2,0·103 L-1), Crinolina isefior- densis, Parvicorbicula superpositus y Pleurasiga minima (5.0 · 103 L-1). Las concentraciones de bacteria (1,1·106- 4.5·10 6 6 6 ) y virus (7,9·10 – 21·10 ) son las típicamente descritas para aguas marinas costeras. En aguas de lastre se encontró Acanthocorbis asymmetrica cuya alta concentración (20·103 L-1) debiera ser consecuencia de este particular ambiente físico y biológico. Palabras clave: coanoflagelados, bacteria, virus, agua de lastre, epifluorescencia, Chile.

Corresponding author: Gloria Collantes ([email protected])

The history of choanoflagellates was delineated invariably colorless species, oval in shape, with a by Kent (1882) and Ellis (1930). Later, the current single , and encircled at the base by a col- systematic hierarchy within the order was given lar composed of fine . Many species by Norris (1965). Studies on mitochondrial (Lead- are enveloped in a composed of silica rods beater, 1974) and kinetid structures (Hibberd, 1975) and costal strips (Throndsen, 1993). Other species unequivocally clarified the phylogenetic affinity of have an organic thecae (Buck, 1981). Choanofla- the order, placing it in the protoctist Phylum Zoo- gellate is based almost solely on lorica mastigina (Buck, 1990; Snell et al., 2001). or morphology as analyzed under light and/or The Class Choanoflagellidea contains almost electron (Leadbeater, 1972a, 1972b, 114 Investigaciones Marinas, Vol. 35(2) 2007

1975; Thomsen, 1976). The Order Choanoflagel- as Synechococcus (Carrias et al., 1996). In addition lida, Kent 1880, consists of three Families that have to the species mentioned previously, the list of vi- been tentatively raised to the rank of Order. The ruses that infect aquatic is growing Order Codonosigida/Family con- continuously. Viruses have profound implications sists of naked cells or those having an investment for marine biodiversity and viral lysis can signifi- invisible under light microscopy. The cells of the cantly influence population dynam- Order Salpingoecida/Family have ics and biogeochemical cycles in aquatic habitats a hyaline close-fitting theca, are visible under light (Raven, 2006). microscopy, and are prevalent in freshwater. Final- Although many works on choanoflagellates ly, the Order /Family Acanthoecidae indicate a cosmopolitan distribution of the group, is made up of cells that are surrounded by a lorica planktonic choanoflagellates have not yet been in- (often wide) and consisting of costae constructed cluded in Chilean biodiversity (Simonetti et al., from costal strips of silica; they are conspicuous in 1995), nor is the taxon mentioned in the preliminary marine environments (Thomsen, 1992; Vørs, 1992; list of Chilean (Antezana, 2001). Throndsen, 1993). Progress has been made in the taxonomy of Stephanoeca diplocostata and Cal- We report here several species of choanoflagel- liacantha sp. through partial sequences of the 18S lates recorded for the first time along the temperate ribosomal RNA gene (Medina et al., 2003; Cava- Pacific coast of South America. In addition, a pre- lier-Smith & Chao, 2003). liminary analysis of abundances relative to bacteria and viruses was also carried Asexual in most of these species out. occurs through longitudinal fission (Thomsen & Larsen, 1992). For the Acanthoecidae, juvenile cells Surface water samples were taken in Novem- may have component costal strips associated with ber 2001 from three sites off Antofagasta (23º38'S- them when departing from the parent lorica (tec- 70º24'W), Montemar (32º57'S-71º33'W), and Puer- tiform replication). This particular strategy seems to Montt (41º41'S-72º38'W), Chile. to be species-specific. Some sedentary species re- The sampling sites were situated 100 m from produce by means of a swimming mastigote (Buck, the coast. The samples (n = 3) were collected with 1981). sterile propylene bottles (250 mL) at 50 cm depth. The apparent mode of nutrition is phagotrophy Additional triplicate samples were taken from the (Laval, 1971; Leadbeater & Morton, 1974; Fenchel, ballast tank water of a local ship that arrived in 1982; Marchant, 1985; Sherr, 1988) with ingested Puerto Montt (41º29’S–72º57'W) after a short trip material consisting of bacteria (including cyanobac- from Puerto Chacabuco (45º30'S–72º57'W). Tem- teria), nanoplanktonic , and debris. Some spe- perature and salinity were determined in situ. All cies are capable of subsisting solely on dissolved samples were filtered through a 35 µm net, main- organic matter in axenic cultures (Gold et al., 1970), tained in the dark at 4ºC, and kept in 1-ml aliquots whereas others may alternate between osmotrophic containing 5% formaldehyde. Bacteria and viruses or pinocytotic modes (Manton et al., 1981). The role were stained with SYBR Green I (Noble 2001) of choanoflagellates within the trophodynamics of after water samples were filtered through an ultra- aquatic regimes is that of an intermediary between fine filter (Whatman Anodisc, 0.02 µm Membrane primary producers and (Meyer & El- Filters; Fisher Scientific, Inc). To stain the retained Sayed, 1983). Bacteria and were rec- material, the filter was placed over a drop of SYBR ognized as important members of a microbial food Green I standard solution (Molecular Probes Inc.) web (Graham & Wilcox, 2000). Various , in- 1:1000 in Milli Q water. After 15 min in the dark at cluding algal species, amoebae, and can be room temperature, the excess stain was eliminated extremely important grazers in both fresh and ma- by washing the filter three times with Milli Q wa- rine waters. Small and ciliates can ingest ter. The filter was removed from the filtration ap- as much as 100% of their own cell volume within paratus, placed on a glass slide, and covered with a one hour, and can double their populations in about 25-mm square glass containing a drop of anti-fade three hours. In a lake study, small heterotrophic flag- mounting solution (50% PBS / 50% glycerol with ellates such as bodonids and choanoflagellates were 0.1% p-phenylenediamine). shown to account for 84% of total protistan grazing The stained samples were examined in an Olym- on the picoplankton, primarily such pus BX60 epifluorescence microscope. Choanofla- Choanoflagellates off the Chilean coast 115 gellates were identified following morphological Crinolina isefiordensis Thomsen, 1976 (Fig. 1g) type descriptions (Ellis, 1930, Throndsen, 1970; Lorica length: 25-30 µm (25 µm) Thomsen, 1976; Manton & Oates, 1979ab; Hara Lorica width: 20-30 um (20 µm) & Takahashi, 1984; Booth, 1990). The simultane- Costae: 2 transverse, 15-16 longitudinal (14 longi- ous enumeration of choanoflagellates, bacteria, and tudinal) viruses was done using the red autofluorescence Characteristic features: skirt-shaped lorica, open at of choanoflagellates and the green fluorescence of both ends. bacteria and viruses. Randomly selected fields were counted using the Image Pro Plus 4.5 (MediaCy- bernetics). Calliacantha multispina Manton & Oates, 1979a (Fig. 1h) This study revealed six species of choanoflagel- lates (Acanthoecidae) observed for the first time at Lorica length: 22-32 µm (20 µm) the three sites selected off Chile (Fig. 1, Table 1). Lorica width: (10 µm) An additional species was observed in the ballast Costae: 2 transverse, 4-5 longitudinal (2 transverse, water sample (Fig. 1, Table 1). A brief description 6 longitudinal) of type specimens is presented below: lorica sizes Characteristic features: The number of longitudinal and costa numbers for the Chilean specimens are costae decreases towards the posterior end of the given in parentheses. lorica.

Acanthocorbis apoda (Leadbeater) Hara & Taka- Acanthocorbis asymmetrica Ellis, 1930 (Figs. 1i- hashi, 1984 (Figs. 1a-1c) 1l) Lorica length: 13-18 µm (10 µm) Lorica length: 15-20 µm (15µm) Lorica width: (10 µm) Lorica width: 8 µm (7-8 µm) Costae: 1 transverse, 12-16 longitudinal (1 trans- Costae: 2 transverse, 15-17 longitudinal (2-3 trans- verse, 16 longitudinal) verse, 15-18 longitudinal) Characteristic features: typical waist-shaped lorica. Characteristic features: stalk deviates markedly from the log axis of the cell, giving it an asymmetri- cal appearance. Parvicorbicula superpositus Booth, 1990 (Fig. 1d) Lorica length: 23-25 µm (15 µm) Bacteria and viruses were visible all around Lorica width: (10 µm) and inside the choanoflagellates (Fig. 1). The abun- Costae: 2 transverse, 10 longitudinal (2 transverse, dance of choanoflagellates, bacteria, and viruses 10 longitudinal) in samples from three different sites off Chile and Characteristic features: funnel-shaped lorica. from ballast waters are shown in Table 1. Bacte- ria and VLP concentrations were as expected for Pleurasiga minima Throndsen, 1970 (Fig. 1e) coastal marine waters and VLPs always exceeded Lorica length: 10 µm (10 µm) bacterial abundance. Interestingly, choanoflagellate concentrations were 4 to 10 times higher in the bal- Lorica width: (10 µm) last water in spite of an apparently slight decrease in Costae: 2 transverse, 7-10 longitudinal (2 trans- the bacterial concentration. The temperature and the verse, 7 longitudinal) salinity of the surface waters off Puerto Montt (es- Characteristic features: all longitudinal costae meet tuarine site) were lower than those off Antofagasta at the back of the lorica. and Montemar (Table 1). The range of species found in the Chilean waters Stephaenoeca diplocostata Ellis, 1930 (Fig. 1f) was highly similar to that reported for other coasts Lorica length: 16-22 µm (6.0 µm) of the world: C. multispina from the coasts of South Lorica width: 8-10 µm (5.0 µm) Africa and southern England (Tong, 1997); A. apo- Costae: 5 transverse, 16-20 longitudinal (5 trans- da from the coasts of Norway (Leadbeater, 1972a, verse, 16 longitudinal) 1972b), Taiwan, Japan (Hara & Takahashi, 1984), Characteristic features: Paired transverse costae and southern England (Tong, 1997); S. diplocostata where the lorica diameter is greatest. from brackish waters or near-shore marine regions 116 Investigaciones Marinas, Vol. 35(2) 2007

Figure 1. Marine choanoflagellates observed by means of EFM in SYBR Green stained samples. a)Acanthocorbis apoda showing collar with retained bacterias (short arrow), b, c) Acanthocorbis apoda showing loricas and the inner protoplast, d) Parvicorvicula superpositus, e) Pleurasiga minima, f) Stephanoeca diplocostata, g) Crinolina isefiordensi, h) Calliacantha multispina, i, j, k,) Acanthocorbis asymmetrica empty loricas showing variable accu- mulation of transversal costae, l) Acanthocorbis asymmetrica showing the inner protoplast. Images are the nega- tive of the original. Scale bars: 2.5 µm. Choanoflagellates off the Chilean coast 117

Table 1. Abundances of choanoflagellates, bacteria and virus from three different localities from the coast of Chile and from ballast water.

Sampling place Choanoflagellates Choanoflagellates·L-1 Bacterias·mL-1 VLP·mL-1 T (ºC)/ Salinity Antofagasta Calliacantha multispina (23º38'S/70º24'W) 0.3·103 1.1·106 21·106 18.0 / 34.8 Montemar cove Acanthocorbis apoda (32º57'S /71º33'W) 5.0·103 2.1·106 14·106 Stephanoeca diplocostata 14.5 / 34.5 Crinolina isefiordensis Puerto Montt, El Arenal Parvicorbicula superpositus. (41°41'S/72°38'W) 2.0·103 4.5·106 7.9·106 Pleurasiga minima 13.0 / 30.5 Acanthocorbis asymmetrica Ballast water 20·103 0.8·106 18·106 in the Northern Hemisphere (Ellis, 1930) and coast environment. For example, VLPs are much more of southern England (Tong, 1997); C. isefiordensis abundant than bacteria. The latter are important from the coasts of Isefjord, Denmark (Thomsen, components of choanoflagellate food and, there- 1976) and southern England (Tong, 1997); P. su- fore, it was interesting to compare their abundances. perpositus from the coasts of the Sub-Arctic North However, no clear relatioships between these abun- Pacific (Booth, 1990) and southern England dances can be seen in Table 1. This kind of analy- (Tong, 1997); and P. minima from the coasts of Arc- sis should probably be done in deeper layers where tic waters (Throndsen, 1970) and southern England choanoflagellates increase in number. Fenchel (Tong, 1997). The species from the temperate South (1982) and Anderson & Fenchel (1985) suggested a Pacific coast reported in this study confirm that signicant influence of choanoflagellates on the bac- Acanthoecidae species have a cosmopolitan distri- terial abundance by judging correlational changes bution. Chilean choanoflagellates showed some dif- between the two. Ishiyama et al. (1993) showed no ferences in lorica sizes and number of costae. significant correlation between choanoflagellates These species are difficult to distinguish from and , indicating that the ingestion ; in addition, they are generally present only of bacterioplankton by many choanoflagellate cells in small numbers, although they reach large num- suggests that the biomass should be sufficient for bers under eutrophic conditions such as those that choanoflagellates as well as for other bacteriovores can be found in ballast waters (Table 1). Acanthoe- in the Antarctic Ocean (Fenchel, 1982; Imai & Ito, cidae species also seem to be associated with es- 1984; Buck & Garrison, 1988; Marchant & Perrin, tuarine or near-coastal sites (Leadbeater, 1972b), as 1990). shown by comparisons with species recorded from The abundance of A. asymmetrica in ballast open water sites (Buck & Garrison, 1988; Booth, water was rather high (20·103 L-1) compared to the 1990; Thomsen et al., 1990). The abundance of abundance of the choanoflagellates species from choanoflagellates sampled off Chile was rather low the field (0.3·103 L-1; 2.0·103 L-1; 5.0·103 L-1). This compared to that found in other places. In Antarctic result was expected because choanoflagellates are waters, values were found ranging from 14·103 L-1 more abundant in oxygen-deficient environments 3 -1 (surface) to 50·10 L (20 m depth) (Chen, 1994); and those richer in heterotrophic bacteria (March- this is more than ten times the values shown in ant et al., 1987). The abundance of bacteria (0.8·106 Table 1. These differences could be due to the fact mL-1) in the ballast water samples was low com- that our samples were taken from surface waters. In pared to the abundance of bacteria from the field addition, the data obtained by Chen (1994) was col- samples (1.1·106 mL-1; 2.1·106 mL-1; 4.5·106 mL-1) lected in summer, which offers the best conditions (Table 1, Fig. 1). In principle, high bacterial counts for a high level of primary production. are expected to be associated with higher levels of The abundance of planktonic species reflects the choanoflagellate cells. The environmental -condi trophic relations between them as well as with the tions of the ballast waters may have favoured het- 118 Investigaciones Marinas, Vol. 35(2) 2007 erotrophic bacterial growth, promoting an active across the ice-edge zone in the Weddell Sea, Ant- grazing of choanoflagellates. arctica. Mar. Biol., 98: 263-269. The latter case would result in a transitorily in- Buck, K.R. 1990. Phylum Zoomastigina, Class Cho- creased population of choanoflagellates and a - de anomastigotes (Choanoflagellates). In: L. Margu- creased number of bacteria. Although these findings lis, J.O. Corliss, M. Melkonian & D.J. Chapman are preliminary, casual, and based on only a few (eds.). 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Received: 23 October 2006; Accepted: 30 July 2007