Thraustochytrids: a Neglected Agent of the Marine Microbial Food Chain

Thraustochytrids: a neglected agent of the marine microbial food chain

Hiroyuki Kimura and Takeshi Naganuma* School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima 739-8528, Japan *Corresponding author: Tel: +81-824-24-7986; Fax: +81-824-22-7059; E-mail: [email protected]

Abstract

The base of the pelagic microbial food chain consists of bacterioplankton and mycoplankton (mostly thraustochytrids), the latter having drawn less attention. In a rather simplistic formulation, bacterioplankton is regarded as the harvester of autochthonous (marine) dissolved organic material exuded from phytoplankton, while mycoplankton is seen to be the scavenger of refractory dissolved organic material which is often allochthonous (riverine). This study presents a case study report on the abundance of thraustochytrids (mycoplankton) and bacterioplankton in the discharge area of River Shimanto, Northwestern Pacific, Japan. Thraustochytrid cells were stained with acriflavine and counted directly by epifluorescence microscopy. Thraustochytrids were found in the surface water at a density of 0.9 to 3.6 x 103 cells l-1, with an overall average of 2.5 x 103 cells l-1. Distribution of thraustochytrids was correlated to (or controlled by) river discharge as depicted by lowered salinity. On the other hand, abundance of bacterioplankton, 1.2 to 1.4 x 109 cells l-1, showed no significant correlation with river discharge. Thraustochytrids may grow on terrestrial organic matter in the riverine input, which is often refractory to degradation, and play a role in enhancing carbon cycling in the estuarine and coastal areas.

Keywords: Mycoplankton, bacterioplankton, abundance, riverine input

1. Introduction Miura, 1997), and thus affects the whole pelagic food web structure. Another agent of DOM utilization is mycoplankton, 1.1 The microbial food chain and which have drawn less attention from ecologists due to thraustochytrids their relatively low abundance (102-3 cells l-1 surface water; Naganuma et al., 1998; Kimura et al., 1999) com- Marine pelagic ecosystems are sustained by two pared to that of bacterioplankton (108-9 cells l-1 surface types of food chains, namely the classical grazing food water). Marine mycoplankton are largely comprised of chain and the microbial food chain (e.g., Valiela, 1995). fungoid protoctists, the thraustochytrids. The microbial food chain serves to salvage organic Thraustochytrids are a forgotten agent of the marine materials such as phytoplankton exudates and phyto-/ microbial food chain but the importance of zoodetritus from the grazing food chain (Pomeroy and thraustochytrids in pelagic secondary production has Wiebe, 1993). At the base of pelagic microbial food been increasingly noticed. Production of chains is a large pool of dissolved organic matter (DOM) thraustochytrids is likely associated with the degrada- primarily utilized by bacterioplankton (Sherr and Sherr, tion of refractory DOM that is not readily utilized by 1987). Production of bacterioplankton often accounts bacterioplankton (Bremer, 1995; Bremer and Talbot, for a considerable part of pelagic secondary production 1995). Thus, it can be simplistically formulated that (Cole et al., 1988; Naganuma, 1997; Naganuma and mycoplankton serve as the scavenger of refractory

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DOM which is often allochthonous (riverine) in nature, nutrients for many animals and for the growth of fish while bacterioplankton salvage the autochthonous (ma- larvae. rine) DOM exuded from phytoplankton. Another and more important ecological role of thraustochytrids may be the decomposition of refrac- 1.2 Taxonomy of thraustochytrids tory organic substrates in marine ecosystems (Heald and Odum, 1970). Thraustochytrids often occur in as- Modern taxonomy classifies thraustochytrids in the sociation with decaying plant material such as algal tis- phylum Heterokonta, which is in turn placed in the king- sue (Miller and Jones, 1983; Sathe-Patak et al., 1993) dom Chromista, based on 18S rDNA sequencing (Cava- and phaneroganic material (Fell and Master, 1973; lier-Smith et al., 1994). The phylum Heterokonta includes Findlay et al., 1986). It is likely that thraustochytrids 1) chromophytes such as diatoms and kelps and 2) also grow on refractory substrates of terrestrial origin, oomycetes such as thraustochytrids. Thus, such as cellulose and lignin contained in river water thraustochytrids are no longer classified in a fungal (Bremer, 1995; Bremer and Talbot, 1995). group and are not necessarily to be regarded as In addition to the capability of recycling of refrac- mycoplankton. However, as long as diatoms and kelps tory organic matter, cells of thraustochytrids may have are traditionally called phytoplankton and algae al- higher carbon contents, giving larger impact on C-cy- though they are no longer so, thraustochytrids may cling than a bacterioplankton cell does. This is evi- still be called mycoplankton. denced by the direct determination of thraustochytrid Thraustochytrids are a group of non-photosynthetic, cell C:N ratio (10.5; Kimura et al., 1999) that is higher heterotrophic, marine fungoid protoctists (Moss, 1986; than that of bacterioplankton (5.9 to 6.8; Fukuda et al., Porter, 1989). This group has a typical structure con- 1998). The low abundance of thraustochytrids (10-6 of sisting of an ectoplasmic net and a non-cellulosic, bacterioplankton) is thus compensated by the high cel- sulfurylated cell wall (Darley et al., 1973). The ectoplas- lular C:N ratio, high biovolume (103 of bacterioplankton) mic net is thought to be the site of organic degradation and high biomass (104 of bacterioplankton). using excreted enzymes. 1.4 Ecological importance 1.3 Ecology of thraustochytrids The microbial food chain is a pathway of particle- Previous studies have determined the abundance of size increments: from µm-sized bacteria to flagellates, to thraustochytrids over a wide range of habitats, includ- ciliates, and to mm-sized zooplankton, involving four ing the water column, algae, particulate detritus and trophic levels. When the chain starts from 10 µm-sized invertebrates (Raghukumar, 1990; Riemann and thraustochytrids, only three trophic levels are involved Schaumann, 1993; Frank et al., 1994; Naganuma et al., and thus the whole transfer efficiency would be ten 1998). A rapid direct-detection technique using the times higher than the chain starting from fluorogenic acriflavine dye has been developed and bacterioplankton. Planktonic thraustochytrids are prob- used for the enumeration of particle-bound (i.e., non- ably a suitable food item in terms of size for 100 µm- planktonic) thraustochytrids (Raghukumar and sized protozoans (Fenchel, 1980; Rassoulzadegan et al., Schaumann, 1993). The acriflavine fluorescence tech- 1988), heterotrophic dinoflagellates (Hansen, 1992; nique relies upon the fact that the wall and nucleus of Hansen et al., 1996) and filter-feeding bivalves thraustochytrid cell fluoresce differently in red and blue- (Jørgensen, 1996). This is another aspect of green, respectively, by blue-light excitation. This dual thraustochytrid contribution to enhancing the efficiency fluorescence distinguishes thraustochytrids from other of the marine microbial food chain. protoctists and detritus. A different aspect of thraustochytrid importance is The thraustochytrids are sized largely between 5-20 nutritional value to predators. Thraustochytrids are µm. Thus thraustochytrids are thought to serve as im- known to contain high cellular contents of the long- portant food sources for picoplankton-feeders and con- chain polyunsaturated fatty acids DHA and DPA tribute to the enhancement of pelagic secondary pro- (Nakahara et al., 1996; Nagao et al., in press). Both DHA duction (Naganuma et al., 1998). Thraustochytrids and DPA are essential fatty acids for many marine ani- uniquely produce polyunsaturated fatty acids such as mals and thus must be obtained through the diet (Fell docosapentaenoic acid (DPA) and docosahexaenoic and Newell, 1998). Thraustochytrids provide not only acid (DHA) (Nakahara et al., 1996), which are essential material high in carbon but also diets high in DHA/DPA

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Samples of surface water were collected at ten sites in the discharge area of the Shimanto River, northwestern Pacific Ocean, Japan (Figure 1) during the cruise of R/V Toyoshiomaru, Hiroshima University, February- March, 1999. At each sampling site, vertical profiles of water temperature (T), salinity (S), dissolved oxygen (DO) concentration and fluorescence intensity was mea- sured with a Sea Bird CTD. The water samples were collected with a clean plas- tic bucket that was pre-washed with distilled water. The samples were used for the determination of chlorophyll a (Chl-a) concentration and the determination of both thraustochytrid and bacterioplankton abundances. The Chl-a containing particles were collected on Whatman GF/F, and Chl-a was extracted and preserved in N,N- dimethylformamide at -20° C until fluorometric determi- Figure 1 Site of sample collection in estuary of Shimanto river nations were made (Lorenzen, 1967). flowing into the northwestern Pacific, Japan. Observed surface water temperature was between 17.3 (Site 0) and 18.4° C (Site 8). The range of surface to organisms at higher trophic levels (Lewis et al., 2000). water salinity (expressed in ‘practical salinity unit’, psu) The ecological importance of the neglected was between 34.60 (Site 0) and 34.71 (Site 7). Dissolved mycoplankton should be properly re-evaluated in terms oxygen concentration ranged from 7.1 (Site 3) to 8.5 mg O l-1 (Site 6). This DO range is common and is known to of : 1) conversion of refractory terrigenous DOM into 2 highly nutritive marine organic particles, 2) impact on have little adverse influence on many marine (aerobic) marine C-cycling, and 3) size-based enhancement of organisms. Chl-a concentrations were low, within a range -1 the transfer efficiency of the whole microbial food chain. of 0.805 (Site 5) to 0.976 µg l (Site 0), possibly due to Most of these aspects have yet to be studied. low winter light availability. As Chl-a concentration was The hypothesis to be tested in this study is: ‘abun- not correlated significantly to either thraustochytrid nor dance of thraustochytrids is controlled by river dis- bacterioplankton abundances, no further references to charge that exports riverine organic matter for the Chl- a, or to DO are given in this report. thraustochytrid diet’. This communication reports the results of an investigation conducted in the discharge 2.3 Fluorescence staining and counting of area of the Shimanto River, northwestern Pacific, Ja- thraustochytrids and bacterioplankton pan. Cells of thraustochytrid and bacterial cells in sample 2. Materials and methods waters were collected on pre-blackened polycarbonate filters (Isopore, Millipore; pore size 0.2 µm; diameter, 25 2.1 Study area mm) immediately after sampling. A surface water sample of 20 to 100 ml was filtered for collection of The Shimanto River has a large watershed area (2,270 thraustochytrid cells, and 0.5 to 1.0 ml of the same water km2) of relatively unexploited forests. Impact of civil was filtered for collecting bacterioplankton cells. and industrial sewage to the river is minimal, and it is The thraustochytrid cells on the Isopore filters were well-known as the purest and last-to-be-polluted river immediately stained according to the acriflavine direct in Japan. Studied sites were located in an offshore area detection technique (Raghukumar and Schaumann, where the river discharges (Figure 1). River discharge 1993) using 4 ml of 0.05% acriflavine (Sigma) in 0.1 M was monitored by the variation of surface water tem- citrate buffer (pH 3.0) for 4 min with subsequent rinsing perature and salinity. with 75% isopropanol. This fluorescence technique is

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based on the fact that the thraustochytrid cell wall, having sulfurylated polysaccharides, fluoresces red and the nucleus fluoresces yellow-green with blue-light excitation. This fluorescence feature serves as the benchmark for the distinguishing thraustochytrid cells from other nanoplankton of this size (5 to 20 µm). That is, nano-phytoplankton fluoresce wholly red due to Chl-a autofluorescence, and nano-sized protozoans fluoresce green due to the lack of a sulfurylated cell wall. Bacterioplankton cells on Isopore filters were immediately stained by the direct count method of Hobbie et al. (1977) and Zimmermann et al. (1978) with 0.01% acri- dine orange solution in 10 mM phosphate buffer at pH 8.0 for 1 mim. The filters were air-dried and kept at -10° C until examination with epifluorescence microscopy, as was done for thraustochytrid cells. The stained cells of thraustochytrids and bacterioplankton were counted using epifluorescence microscopy under violet-blue excitation. Ultra violet excitation was also used to differentiate thraustochytrids from Chl-a-autofluorescing nanoplankton. Figure 2 Distribution of river water-affected sites ( ; salinity [psu] < 34.68) and seawater-predominant sites ( ; salinity [psu] > 34.68) with salinity and temperature. Numbers indicate sampling site. 3. Results and discussion

3.1 River discharge and coastal hydrography 103 cells l-1 (Site 8) to a maximum of 3.6 x103 cells l-1 (Sites 0 and 2), with an overall average of 2.5 ± 1.0x 103 cells l- Surface waters of the 10 sampling sites showed dis- 1 (Figure 3). The average for the ‘river-affected’ group tinguishable variation in T and S, as shown by the T-S was 3.5 ± 0.2 x 103 cells l-1, while the average for the diagram (Figure 2) which demonstrates two distinct ‘seawater-dominant’ group was 1.8 ± 0.7 x 103 cells l-1; groups of surface waters: the ‘seawater-dominant’ the two averages were shown to be significantly differ- group (Sites 4 to 9) and the ‘river-affected’ group (Sites ent (p = 0.002) different by t-test. Therefore, abundance 0 to 3). The grouping of Site 2 was ambiguous, having of thraustochytrids in the River Shimanto discharge area intermediate T-S values; however, Site 2 was placed in was characterized as high at the ‘river-affected’ area the river-affected group for purposes of hydrographic and low in the ‘seawater-dominant’ area. This strongly continuity. The ‘seawater-dominant group’ was char- suggests that the abundance of thraustochytrids is acterized by high-T (>17.8° C) and high-S (>34.68 psu), controlled by riverine input (possibly of terrigenous while the ‘river-affected’ group was characterized by organic matter such as lignin and cellulose (Bremer, 1995; low-T (<18.2° C) and low-S (<34.68 psu). High-T values Bremer and Talbot, 1995; Valiela, 1995). were due to the warm oceanic current (Kuroshio Cur- Previous studies reported the thraustochytrid abun- rent) flowing from south to northeast around the stud- dance to average 1.0 to 1.6 x 104 cells l-1 in the coastal ied area (Hydrographic Department of Maritime Safety Seto Inland Sea and adjacent water (Naganuma et al., Agency, 1999). Thus, the discharged river plume was 1998; Kimura et al., 1999) higher than abundances in distorted NE-ward. this study. The relatively low counts may be ascribed to lesser availability of riverine organics, when compared 3.2 Abundances of thraustochytrids and with the Seto Inland Sea waters which receive more bacterioplankton river discharge. On the other hand, bacterioplankton abundances were within a narrow but relatively a high range of from Thraustochytrids were found in the surface waters 1.22 x 109 cells l-1 (Site 2) to 1.44 x 109 cells l-1 (Site 0), with at population densities ranging from a minimum of 0.9 x an overall average of 1.34 ± 0.07 x 109 cells l-1. The aver-

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Table 1 Correlation coefficients (r; n=10) for the correlations between surface water salinity (S) and abundance of thraustochytrids (Thra) or bacterioplankton (Bact). Two models of correlation equations, linear and allometric, were examined. * denotes significant at p < 0.01; ** denotes significant at p < 0.05.

S vs Thra S vs Bact

Linear equation -0.756* 0.253

Allometric equation -0.687** 0.248

picted in Figure 1 based on the T-S diagram (Figure 2). While surface water temperature did not distinguish the ‘seawater-dominant’ and ‘river-affected’ groups, surface water salinity was the sole benchmark to sepa- rate the two groups. Surface salinity was reasonably correlated to the abundances of thraustochytrids and bacterioplankton, thereby showing riverine control on thraustochytrid abundance, by two types of equations: linear (Y=aX+b) and allometric (Y=aXb). Statistical sig- nificance of the correlations was examined by the sig- nificance of the correlation coefficient (r; n=10) as shown in Table 1. Both modes of correlation yielded highly significant r values for the salinity-thraustochytrids correlation, while neither mode was significant for the salinity-bacterioplankton correlation. High r values for the salinity-thraustochytrids correlation strongly suggest that, again, the abundance of thraustochytrids is controlled by riverine input of terrigenous organic matter such as lignin and cellulose. Figure 3 Surface site variations in the abundance of planktonic This view is supported by the capability of marine fun- bacteria and thraustichytrids in coastal area of north Pacific, goid protoctists to synthesize cellulolytic enzymes Japan, in February and March 1999. Lines indicate the overall averages at river-affected and seawater-dominant sites, (Bremer and Talbot, 1995) and to digest cellulosic cell respectively. wall materials (Perkins, 1973; Bremer, 1995; Valiela, 1995). Since a significant correlation between salinity (controlled by river discharge) and thraustochytrid abun- 9 age for the ‘river-affected’ group was 1.34 ± 0.10 x 10 dance was found (while bacterioplankton abundance -1 cells l , while the average for the ‘seawater- was not correlated to salinity) the orginal hypothesis 9 -1 dominant’group was 1.34 ± 0.05 x 10 cells l ; the two was half-proved. The other part of the hypothesis averages were shown to be not significantly different (whether riverine organics are part of the thraustochytrid by t-test (p = 0.924). Abundance of bacterioplankton in diet) is to be tested elsewhere. the Shimanto River discharge area was thus concluded not to be controlled by riverine input. References

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