- Email: [email protected]
A thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses
Fish & Shellfish Immunology Fish & Shellfish Immunology 15 (2003) 183–194 www.elsevier.com/locate/fsi
A thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses Robert S. Anderson a*, Brenda S. Kraus a, Sharon E. McGladdery b, Kimberly S. Reece c, Nancy A. Stokes d a
Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, MD 20688, USA b Shellfish Pathology Research and Diagnostics, Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton NB, Canada E1C 9B6 c Department of Fisheries Science, Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA d Department of Fisheries Science, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA Received 13 August 2002; accepted 23 September 2002
Abstract A previously undescribed thraustochytrid protist, designated C9G, was isolated from the gills of a clam, Mercenaria mercenaria, collected from the Bay of Fundy, Canada. Sequence data analysis showed C9G to be related to the clam pathogen QPX, quahog parasite unknown; however, it is not enveloped by secreted mucoid material as is the case for QPX. Clam hemocytes recognized and phagocytized C9G in vitro in the absence of plasma recognition factors. Hemocytes were also capable of killing ingested C9G, as shown by the use of a tetrazolium reduction viability assay. The mechanisms underlying intracellular antimicrobial activity are not yet established, but no detectable cytotoxic reactive oxygen species were generated during phagocytosis of C9G. Clam plasma proteins were shown to inhibit C9G growth at concentrations similar to those in unfractionated hemolymph. 2003 Elsevier Ltd. All rights reserved. Keywords: Mercenaria mercenaria; Immunity; Defense responses; Parasite; QPX; C9G; Small subunit ribosomal DNA; Thraustochytrid; Antibacterial responses
1. Introduction Pathogenic thraustochytrids have been isolated from various molluscan species [1–4], including Mercenaria mercenaria, in which QPX has been associated with mortalities of cultured clams mainly in * Corresponding author. Tel.: +1-410-326-7274; fax: +1-410-326-7210 E-mail address: [email protected] (R.S. Anderson). 1050-4648/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(02)00157-2
184
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Canada [5] and Massachusetts [6]. The pathogenicity of this protist, C9G, has not been established; however, it seems to be phylogenetically closely related to QPX and was isolated from a clam in a population infected with QPX. A medium originally designed for propagation of QPX [7] also supported culture of C9G, thus enabling a constant supply for the current study. In culture, C9G exists as loosely adherent cells without any evidence of the mucoid secretions typical of QPX. The secretions have been postulated to be virulence factors in that they participate in inflammatory responses via chemoattractant activity [8], as well as protection of QPX from phagocytic hemocytes [6] and humoral antimicrobial agents [9]. It is likely that C9G would be more directly exposed in vivo to host defense mechanisms than QPX because of its lack of secretory material. In this article, preliminary characterization of some cellular and humoral responses of M. mercenaria to C9G is reported. This is the first in vitro study of clam innate immune mechanisms evoked by a thraustochytrid species. 2. Methods 2.1. Culture of C9G This microorganism was isolated from gill tissues of QPX-infected M. mercenaria collected in January 2000 from Sam Orr Pond, near St. Andrews, NB (Bay of Fundy). C9G was maintained in the medium described by Kleinschuster et al. [7] at 24 (C, pH 7.2. The cultures were kept in canted-neck tissue culture flasks and subcultured every 7 days under sterile conditions. The cells tended to be adherent, so they were removed from the bottom of the flasks with a sterile cell scraper prior to subculturing. 2.2. Molecular characterization of C9G Genomic DNA was extracted from cultured C9G cells as previously described for Perkinsus cells [10]. Portions of the small subunit ribosomal DNA (SSU rDNA) were targeted for PCR amplification using QPX-specific primers, QPX-F and QPX-R2 and the Labyrinthulomycota-specific primers, LABY-A and LABY-Y [11]. PCR reaction mixtures and cycling conditions for QPX-F + QPX-R2 and for LABY-A + LABY-Y have been described [11]. The nearly full-length SSU rRNA gene was amplified by PCR using universal primers for eukaryotic organisms [12], without the 5# polylinker bases. The SSU reaction mixtures and cycling conditions were conducted as previously described [11]. An aliquot of each reaction (10% of reaction volume) was checked by agarose gel electrophoresis for the expected product sizes of 665 bp with the QPX-specific primers, approximately 435 bp with Labyrinthulomycota-specific primers, and approximately 1800 bp with the universal SSU rRNA gene primers. Triplicate PCR reactions of the SSU rRNA gene were pooled and ligated into the plasmid vector pCR2.1 (Invitrogen). The ligations were transformed into Escherichia coli INVF# cells and plasmid DNA prepared from clones with inserts. Ten clones were cycle sequenced via simultaneous bidirectional sequencing using M13 forward and reverse primers labeled with the dyes IRD-700 and IRD-800 (LI-COR). Sequencing reactions were electrophoresed on 66 cm 4% polyacrylamide gels in a LI-COR Model 4200 automated sequencer. A consensus sequence for C9G SSU rDNA was obtained using the MacVector software package (Oxford Molecular) and deposited into GenBank (accession # AF474172). 2.3. Phylogenetic analysis For phylogenetic analyses the C9G sequence was aligned with SSU rDNA sequences of QPX and 32 other labyrinthulomycetes (Fig. 1). Sequences in GenBank that were used for the phylogenetic analyses include: Aplanochytrium kerguelense (AB022103), Japonochytrium sp. (AB022104), Labyrinthula sp. (AF265330, AF265332, AB022105), L. yorkensis (AF265333), L. zosterae (AF265334, AF265335), Labyrinthuloides haliotidis (U21338), L. minuta (L27634, AF265339), QPX (AY052644, AF155209, AF261664),
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
185
Fig. 1. Phylogenetic tree generated by parsimony jackknife analysis of labyrinthulomycete SSU rDNA sequences. Two other stramenophiles, A. bisexualis and O. danica were used as outgroup taxa. Parsimony jackknife support values above 50 are shown on branches. Analysis was conducted with 100 jackknife replicates of 100 random additions and gaps were treated as missing data. The TPG and LPG described by Honda et al. [19] as well as a Labyrinthula species group described by Stokes et al. [11], are indicated.
186
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Schizochytrium aggregatum (AB022106, AF265336), S. limacinum (AB022107, AF265339), S. minutum (AB022108), Thraustochytrium aggregatum (AB022109), T. aureum (AB022110), T. kinnei (L34668), T. motivum (AF265337), T. multirudimentale (AB022111), T. pachydermum (AB022113), T. striatum (AB022112, AF265338), Thraustochytrium sp. (AB052556), Ulkenia profunda (L34054, AB022114), U. radiata (AB022115), U. visurgensis (AB022116), and several sequences simply designated Thraustochytriidae ssp. (AF2577314, AF257315, AF257316, AF257317). Two other stramenophiles, Achlya bisexualis (M32705) and Ochromonas danica (M32704) were used as outgroup taxa. Sequences were aligned using the CLUSTAL-W algorithm [13] in the MacVector 7.0 DNA Sequence Analysis Software package. Parsimony jackknife analysis was performed using PAUP* 4b8.0 [14] with 100 bootstrap resamplings of 100 random addition replicates treating gaps as missing data. 2.4. Phagocytosis The phagocytosis assay was based on that of Hed [15]. A fluorescein succinimidyl ester solution was made on the day of the experiment (0.1 mg in 50 µl dimethylsulfoxide). C9G from 3 to 5 day (logarithmic growth phase) cultures were centrifuged (160g, 10 min, 20 (C) from w40 ml medium containing 2–8107 cells/ml cell density. The C9G were resuspended in 0.5 ml 33 ppt NaCl and 50 µl fluorescein solution added; this mixture was continuously mixed for 1 h at 20 (C. The labeled C9G were washed three times by centrifugation (160g, 10 min, 20 (C) and resuspended in 0.5 ml 33 ppt NaCl. Cell numbers were quantified with a hemocytometer and the density adjusted to 2107 C9G/ml. An aliquot of this suspension was centrifuged because the cell-free supernatant contained a low concentration of unbound fluorescein, this solution was used to control subsequent hemocyte fluorescence not associated with labeled C9G. This labeling procedure did not kill the C9G, which remained w90% viable by the trypan blue exclusion assay. For comparative assays, yeast cells (Saccharomyces cerevisiae) were fluorescein-labeled by the same method as C9G cells. M. mercenaria were obtained from the Ware River Va., through a local commercial source. Hemolymph was collected from the adductor muscle and held on ice in polypropylene tubes, cell density determined, and that volume required to contain 105 cells/well added to two microtiter plate wells. The plates containing the hemocyte suspensions were centrifuged (160g, 10 min, 20 (C) and the supernatants removed. The suspension of fluorescein-labeled test particles was immediately added to one of the hemocyte-containing wells and to an empty well. For each sample, three basic situations (A:hemocytes+particles; B:hemocytes+particle supernatant; and C:particles alone) were carried out with three replicates for each hemolymph sample. All wells contained a final volume of 50 µl. The plates were centrifuged again (160g, 10 min, 20 (C) to facilitate particle–hemocyte contact and incubated in the dark (1 h, 20 (C). A trypan blue fluorescence-quenching solution (0.25 mg/ml in 0.02 M citrate buffer) was made fresh daily and 50 µl added to all wells, and the plate was incubated in the dark (5 min, 20 (C). This treatment was intended to quench the fluorescence of any free fluorescein and unphagocytosed test particles. The plates were then read on a fluorescence concentration analyzer (IDEXX, 485/535, 25 gain) to obtain relative fluorescence units (RFU). The RFU of labeled particles phagocytosed was calculated by RFU A(RFU B + RFU C). The number of particles represented by this RFU value was estimated from a standard curve of fluorescence vs. particle count, as determined by hemocytometer. Every batch of labeled particles required construction of a new standard curve to account for the different batch-to-batch labeling efficiencies routinely encountered. 2.5. Anti-C9G activity of hemocytes This procedure was adapted from a tetrazolium dye assay used to assess the antimicrobial activity of oyster hemocytes vs. the protozoan parasite Perkinsus marinus [16]. Aliquots from 3 to 5 day C9G cultures were washed and resuspended in w0.1 original volume 25 ppt Instant Ocean (IO). The C9G density in this suspension was determined in a hemocytometer and adjusted to 6106 cells/ml. Hemolymph was collected
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
187
from M. mercenaria and held on ice in polypropylene tubes, its cell density determined, and the volume required for 105 cells added to the appropriate microtiter plate wells. The plate was centrifuged (160g, 10 min, 20 (C), and the cell-free hemolymph removed. The plate was set up as follows: row 1, 50 µl IO; row 2, hemocytes overlaid with 50 µl IO; row 3, hemocytes overlaid with 50 µl C9G suspension adjusted to the desired concentration; and row 4, 50 µl C9G suspension. Each row had four replicate wells. The plate was incubated (3 h, 26 (C) to permit cellular killing of the C9G. All wells then received 100 µl of a grow-out medium for the surviving C9G (Sigma MEM 3149, minimal essential medium without phenol red) and the plate was incubated (16 h, 26 (C). Then 20 µl MTS/PMS solution (3-[4,5-dimethylthiazol-2yl]-5-[3carboxymethoxyphenol]-2-[4-sulfophenyl]-2H-tetrazolium and phenazine methosulfate) was added to all wells, following the directions for the cell Titer 96 Aqueous Cell Proliferation Assay Kit (Promega Co.). The plate was vortexed and incubated for an additional 2 h at 26 (C. The absorbance of the wells was measured at 490 nm on a SpectraCount Plate Reader (Packard). The absorbance values of the reagent blanks (row 1) were subtracted from the readings of the wells in rows 2–4 to give corrected (corr.) values, and a killing index (KI) was calculated by the formula KI⫽1ⳮ
S
Abs row 3 corr.ⳮAbs row 2 corr. Abs row 4 corr.
D
⳯ 100
2.6. Anti-C9G activity of plasma M. mercenaria plasma was prepared by centrifuging whole hemolymph (300g, 10 min, 4 (C), sterilized by filtration (0.2 µm pore size), and assayed for protein content (BCA protein assay kit, Pierce Co.). C9G cells from 3 to 5 day cultures were washed three times with 25 ppt IO, as previously described, and the cell density adjusted to 7105 cells/ml. The cell suspension (100 µl) was mixed in a microcentrifuge tube with 1 ml of plasma protein (200 or 400 µg/ml) or 1 ml IO, and the tubes incubated 2.5 h at 20 (C (all determinations performed in triplicate). The C9G cells were subsequently recovered (300g, 10 min, 20 (C), washed two times with 25 ppt IO, and resuspended in 100 µl IO. These cell suspensions were then added to 2.5 ml MEM and incubated 7 days at 24 (C. The C9G cells were again washed twice and resuspended in 2.5 ml IO, and the C9G cell density determined by spectrophotometry at 560 nm, using an appropriate standard curve. 2.7. Chemiluminescence (CL) assay for reactive oxygen species (ROS) This assay was carried out as described by Bramble and Anderson [17], with a few modifications. The hemocyte count in a sample of pooled hemolymph was determined and 2106 hemocytes, suspended in hemolymph, were added to each CL reaction vial (final volume 2.5 ml). All aspects of the experiment were carried out under dim red illumination. Lucigenin (10,10#-9,9#-dimethylbiacridinium nitrate), a CL probe specific for superoxide, was added to the vials (150 µM, final concentration) and allowed to equilibrate for w20 min. Hemocytes were then exposed to C9G (1 hemocyte:20 C9G), or to the classical macrophage CL stimuli phorbol myristate acetate (PMA) or zymosan (25 µM or 3.89 mg/ml, respectively). CL was quantified for 2 h by a liquid scintillation counter (Packard Tri-Carb 1900 CA) adjusted for single photon counting. 3. Results 3.1. Culture characteristics of C9G The maximal final cell density attained was 6.80.4106 C9G cells/ml, reached after 3–4 days, regardless of initial seeding densities, which ranged from w2 to 7105 C9G cells/ml. The numbers of C9G
188
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
cells/ml were determined turbidometrically from a standard curve relating absorbance (OD560 nm) of cell suspensions to actual numbers as quantified in a hemocytometer. 3.2. Sequence of C9G and phylogenetic relationship to QPX C9G genomic DNA did not amplify with the QPX-specific primers, QPX-F+R2, but did generate a 441 bp product with the labyrinthulomycete primers, LABY-A+LABY-Y. Since this culture was initially thought to be QPX, the nearly complete SSU RNA gene was amplified using universal primers as described above and the sequence was determined for comparison with SSU rDNA sequences of QPX and other labyrinthulomycetes. The C9G SSU rDNA fragment was 1770 bp in length. BLAST [18] searches of GenBank suggested that C9G was indeed a thraustochytrid that was closely related to QPX and T. pachydermum. Phylogenetic analysis supported placement of C9G into the thraustochytrid phylogenetic group (TPG) as described by Honda et al. [19] within the phylum Labyrinthulomycota. Parsimony analysis placed C9G as a sister taxon to T. pachydermum with a parsimony jackknife support value of 98 (Fig. 1). In addition, the T. pachydermum and C9G sequences grouped with the QPX sequences with a jackknife support value of 100. Results of analyses conducted for this study were consistent with earlier studies placing QPX, T. pachydermum (and C9G) within the TPG [11,19], rather than into the other major phylogenetic group of the Labyrinthulomycota, the labyrinthulid phylogenetic group (LPG) [19]. Examination of SSU rDNA sequences in the V3 and V8 regions also supported placement of C9G in the TPG. As shown in Fig. 2 the C9G SSU rDNA sequence contains the thraustochytrid group signature sequences [19]. 3.3. Phagocytosis of C9G When the C9G cells were fluorescein-labeled, the results indicated that a fluorescent phagocytosis method would be useful with this protist, as was shown previously to be the case with labeled yeast (data not shown). This method gave unambiguous, dose-dependent data on C9G uptake by the hemocytes (Fig. 3). In this study, 105 hemocytes/well were incubated with increasing numbers of fluorescently labeled C9G. Subsequently a fluorescence quencher was added to the medium to reduce the signal of any extracellular C9G. The uptake of labeled C9G was proportional to the C9G:hemocyte ratio, after correcting for background fluorescence of hemocytes, and unquenched fluorescence of extracellular C9G. The apparent fluorescence of the hemocytes was probably caused by bleeding of label from the C9G added to the mixture. The overall quenching efficiency of extracellular C9G decreased with the C9G:hemocytic ratio because quencher concentration was held constant for all C9G:hemocyte ratios tested. Additional phagocytosis experiments were carried out using a C9G:hemocyte ratio of 10:1 because this concentration repeatedly gave good results, and because higher ratios required excessive numbers of C9G. When the RFU data from the 10:1 studies were converted to numbers of C9G cells phagocytosed, it was found that the mean uptake was 16279 C9G cells/100 hemocytes, based on data from four individual pools. Attempts to opsonize yeast or C9G with autologous M. mercenaria serum were unsuccessful (data not presented); test particles were incubated with the highest non-agglutinating serum dilutions (full strength for C9G and 1:128 for yeast) for 60 min at 21 (C, and washed prior to being used in the phagocytosis assay. 3.4. Anti-C9G activities of hemocytes and plasma Hemocyte samples taken from three separate M. mercenaria hemolymph pools were tested for anti-C9G activity using the MTS/PMS assay. During the 3-h incubation period the hemocytes killed 39.112.7% of the administered dose. Suspension of C9G were exposed to 0, 181 or 363 µg/ml plasma protein for 2.5 h, washed, resuspended in MEM, and allowed to grow out for 7 days before quantifying their final numbers. The treatment with 181 µg/ml plasma protein resulted in 60.411.5% (n⫽3) reduction in C9G in culture;
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Fig. 2. Signature sequences of the TPG and LPG in the V3 and V8 regions of the SSU rDNA [19]. Sequences in these regions are shown for L. minuta, of the LPG, and for QPX and T. pachydermum, of the TPG. C9G SSU rDNA contains the signature sequences of the TPG. Numbers in parentheses indicate bases of organism’s SSU rDNA highlighted in bold.
189
190
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Fig. 3. Fluorescence data in thousands of RFU from a representative phagocytosis experiment. The same pool of hemocytes, same batch of labeled C9G, and the same concentration of quenching solution were used throughout. The bars equal the mean RFUSD for three interexperiment replicates.
doubling the concentration produced a similar reduction of 60.616.1% (n⫽3). Full-strength hemolymph plasma was found to contain 411108 µg/ml protein (n⫽3). 3.5. Chemiluminescence response to C9G In an attempt to elucidate a possible mechanism underlying the observed ability of hemocytes to kill C9G, the production of cytotoxic oxygen species by the cells was measured by CL. Fig. 4 shows representative CL results with the probe lucigenin used to quantify the generation of superoxide anions, the first step in the ROS pathway. The exposure of clam hemocytes to medium containing PMA or zymosan produced minor, transient increments in CL activity, as compared with the CL responses stimulated by these agents in oyster hemocytes (data not shown). In fact, addition of comparable volumes of agent-free medium elicited a slight increase in CL. Addition of C9G to the hemocytes at ratios %20:1 stimulated no significant superoxide production. 4. Discussion Sequence data analysis showed that C9G is a thraustochytrid member of phylum Labyrinthulomycota. This group is composed mainly of non-pathogenic marine and estuarine protists associated with detrital
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
191
Fig. 4. Production of superoxide by M. mercenaria hemocytes, as measured by lucigenin-dependent CL. Hemocytes were suspended in medium containing lucigenin (150 µM) and treated with 3.9 mg/ml zymosan, 25 µM PMA, or C9G (20 particles:1 hemocyte) and the resultant CL monitored for about 2.5 h. The various agents were added after the cells had equilibrated, as indicated by the arrow.
sediments, algae and plants [20]; however, it is known to contain pathogens [1–6]. The pathogenicity of C9G for M. mercenaria has not been established, but it is a potential pathogen because of its phylogenetic affinity to QPX and the fact that it is associated in the field with QPX infections. Unlike QPX, it secretes little or no mucoid material; consequently, it may be directly exposed in vivo to host defense mechanisms mediated by hemocytes and serum antimicrobial molecules. Phagocytic hemocytes of bivalves are thought to be important effector cells in host defense systems by virtue of their ability to ingest and destroy microorganisms [21,22]. To better understand the phagocytic activity of M. mercenaria hemocytes and their response to C9G, a fluorescent-based phagocytosis system was developed using a 96-well microtiter plate format. When the particle:hemocyte ratio was increased, the total number of phagocytosed particles increased concomitantly. The rate of phagocytosis was rapid for the first 15–20 min and reached a plateau at w90 min. Although interaction with soluble hemolymph factors, particularly natural agglutinins, can facilitate recognition and internalization of foreign particulates by certain bivalves [23], treatment with M. mercenaria serum did not appear to opsonize C9G. Not only could hemocytic internalization of C9G be quantified, but also the ability of the cells to inhibit C9G was demonstrated, using a classical assay (MTS/PMS). Under the conditions and hemocyte:C9G ratios used in this study, we found that a 3-h killing period, followed by a 16 h grow-out period for the surviving C9G provided useful and reproducible data. Microscopic examination of the size composition of C9G cells prior to exposure to the hemocytes, as well as after the grow-out period, suggests uniformity. This is an important
192
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
consideration when converting Ab490 to cell numbers because the MTS/PMS assay gives an indication of viable cell mass. One may assume that these indications of in vitro anti-C9G activity of hemocytes mimic the in vivo situation, but a caveat must be considered. When this assay was used to characterize the capacity of oyster hemocytes to destroy cells of its pathogenic protozoan P. marinus, 25–90% of P. marinus cells tested were killed [16]. This percentage killed seems quite high since in vivo the parasite apparently often survives phagocytosis and may actually take advantage of circulating hemocytes for dispersal throughout the oyster. It is possible that P. marinus loses its virulence during culture as a result of repeated transfers and lack of contact with oyster tissues [24]; at this time, the effects of continuous in vitro culture of C9G on its phagocytosis and killing by clam hemocytes is unknown. Cell-free clam hemolymph (plasma) was shown to contain naturally occurring anti-C9G activity. Full-strength plasma (w400 µg protein/ml) inhibited growth of C9G cultures by w60%, as did samples 50% diluted. Although the EC50 for this activity was not determined, it is evident that clam plasma contains an active anti-C9G factor, which probably acts in concert with hemocyte-mediated mechanisms to control or resolve C9G infections. Anti-protistan plasma constituents have been described in other bivalves. Hubert et al. [25] reported a 320 kDa protein in Mytilus galloprovincialis that was cytotoxic for the oyster parasite Bonamia ostreae. Antimicrobial peptides have been found in several mussels and oysters [26], but they have been little studied in other bivalves, such as clams. Anti-protistan peptides against bivalve parasites, such as P. marinus and B. ostreae have also been reported in non-bivalve species [26,27]. Isolation and characterization of anti-C9G proteins and/or peptides from M. mercenaria plasma needs to be carried out. The generation of cytotoxic ROS by phagocytes via activation of NADPH oxidase is thought to be important in the killing of phagocytosed microorganisms. This system has been demonstrated in oyster and mussel hemocytes [28,29], although the significance of its role in host defense in these bivalves has been questioned [17,30,31]. In a previous study, M. mercenaria hemocytes failed to produce ROS when exposed to ROS-stimulating agents that triggered ROS generation by oyster hemocytes [32]. In that study, the ROS response was measured by luminol-dependent CL, predominantly a means to study the myeloperoxidaseH2O2-halide pathway of hypohalous acid production [33]. In this study, the ability of M. mercenaria hemocytes to respond to classical ROS stimulators zymosan and PMA was studied using lucigeninaugmented CL, as was the response to C9G. Lucigenin-augmented CL is thought to primarily measure production of superoxide [34], the first ROS product made via NADPH oxidase. Results from the use of lucigenin support the previous finding of very low ability of M. mercenaria cells to mount a ROS response to putative stimuli, including C9G. Clearly, the hemocytes can kill ingested C9G; however, the data suggest that this activity does not depend on oxygen-dependent mechanisms. Intrahemocytic killing of C9G may be a function of lysosomal hydrolases, such as lysozyme [35], and/or antimicrobial peptides [26]. In addition to providing intracellular defense activity, it is likely that both lysosomal enzymes and antimicrobial peptides are released into the plasma during degranulation of hemocytes after microbial challenge [36,37]. Acknowledgements This study was supported in part by the Maryland Sea Grant College, by NOAA Grant No. NA06RG0101. We wish to thank Mr Greg MacCallum for his technical assistance in isolating and providing the initial cultures of C9G. The technical contributions of Ms Kathleen Apakupakul (Chesapeake Biological Laboratory Contribution No. 3636-CBL; Virginia Institute of Marine Science Contribution No. 2489) were also appreciated. References [1] McLean N, Porter D. The yellow-spot disease of Tritonia diomeda, Bergh, 1894 (Mollusca: Gastropoda: Nudibranchia): encapsulation of the thraustochytriaceous parasites by host amoebocytes. J Parasitol 1982;68:243–52.
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
193
[2] Bower SM. Labyrinthuloides haliotidis n.sp. (Protozoa: Labyrinthomorpha), a pathogenic parasite of small juvenile abalone in a British Columbia mariculture facility. Can J Zool 1987;65:1996–2007. [3] Jones GM, O’Dor RK. Ultrastructural observations on a thraustochytrid fungus parasitic in the gills of squid (Ilex illecebrosus, Lesueur). J Parasitol 1983;69:903–11. [4] Hanlon RT, Forsythe JW. Diseases caused by microorganisms. In: Kinne O, editor. Diseases of marine animals. Hamburg: Biologische Anstalt Helogoland; 1990, pp. 40–3. [5] Whyte SK, Cawthorn RJ, McGladdery SE. QPX (quahaug parasite X), a pathogen of northern quahaug Mercenaria mercenaria from the Gulf of St. Lawrence, Canada. Dis Aquat Organ 1994;19:129–36. [6] Smolowitz R, Leavitt D, Perkins F. Observations of a protistan disease similar to QPX in Mercenaria mercenaria (hard clams) from the coast of Massachusetts. J Invertebr Pathol 1998;71:9–25. [7] Kleinschuster SJ, Smolowitz R, Parent J. In vitro life-cycle and propogation of quahaug parasite unknown. J Shellfish Res 1998; 17:75–8. [8] Decker C-S, Anderson RS. Chemotaxis of haemocytes of the hard clam, Mercenaria mercenaria to quahog parasite unknown (QPX) and other microorganisms. J Shellfish Res 2002;21:404. [9] Anderson RS, Kraus BS, McGladdery S, Smolowitz R. Mucoid secretions protect QPX from antimicrobial agents. J Shellfish Res 2002;21:404. [10] Reece KS, Siddall ME, Burreson EM, Graves JE. Phylogenetic analysis of Perkinsus based on actin gene sequences. J Parasitol 1997;83:417–23. [11] Stokes NA, Ragone Calvo LM, Reece KS, Burreson EM. Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam, Mercenaria mercenaria. Dis Aquat Organ 2002;52:233–47. [12] Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 1988;71:491–9. [13] Thompson JD, Higgins DG, Gibson TJ. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Resour 1994;22:4673–80. [14] Swofford DL. PAUP*, Phylogenetic analysis using parsimony (*and other methods). Sunderland: Sinauer Associates, 2001. [15] Hed J. Methods for distinguishing ingested from adhering particles. Methods Enzymol 1986;132:198–204. [16] Volety AK, Fisher WS. In vitro killing of Perkinsus marinus by hemocytes of oysters Crassostrea virginica. J Shellfish Res 2000; 19:827–34. [17] Bramble LH, Anderson RS. Lack of involvement of reactive oxygen species in the bactericidal activity of Crassostrea virginica haemocytes in contrast to Morone saxatilis phagocytes. Fish Shellfish Immunol 1999;9:109–23. [18] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJJ. Basic local alignment search tool. Mol Biol 1990;215:403–10. [19] Honda D, Yokochi T, Nakahara T, Raghukumar S, Nakagiri A, Schaumann K et al. Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene. J Eukaryot Microbiol 1999;46:637–47. [20] Porter D. Phylum Labyrinthulomycota. In: Margulis L, Corbiss JO, Melkonian M, Chapman DI, editors. Handbook of Protoctista. Boston: Jones and Bartlett; 1989, pp. 388–97. [21] Cheng TC. Bivalves. In: Ratcliffe NA, Rowley AF, editors. Invertebrate blood cells. London: Academic Press; 1981, pp. 233–300. [22] Fisher WS. Structure and function of oyster hemocytes. In: Brehelin M, editor. Immunity in invertebrates. Berlin: Springer; 1986, pp. 25–35. [23] Vasta GR. The multiple biological roles of invertebrate lectins: their participation in nonself recognition mechanisms. In: Warr GW, Cohen N, editors. Phylogenesis of immune functions. Boca Raton: CRC Press; 1991, pp. 73–101. [24] Chintala MM, Alcox KA, Ford SE, Bushek D. Cultured cells and natural Perkinsus marinus cells: a possible mechanism for virulence differences. J Shellfish Res 1995;14:240. [25] Hubert F, van der Knapp W, Noe¨l T, Roch P. Cytotoxic and antibacterial properties of Mytilus galloprovincialis, Ostrea edulis and Crassostrea gigas (bivalve molluscs) hemolymph. Aquat Living Resour 1996;9:115–24. [26] Roch P. Defense mechanisms and disease prevention in farmed invertebrates. Aquaculture 1999;172:125–45. [27] Morvan A, Bache`re E, Da Silva PP, Pimenta P, Mialhe E. In vitro activity of the antimicrobial peptide magainin 1 against Bonamia ostrea, the intrahemocytic parasite of the flat oyster Ostrea edulis. Mol Marine Biol Biotechnol 1994;3:327–33. [28] Adema CM, van Deutekom-Mulder EC, van der Knapp WPW, Smina T. NADPH-oxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnea stagnalis. J Leukoc Biol 1993;54:379–83. [29] Anderson RS. Reactive oxygen species and antimicrobial defenses of invertebrates: a bivalve model. In: Beck G, Sugumaran M, Cooper EL, editors. Phylogenetic perspectives on the vertebrate immune system. New York: Kluwer Academic/Plenum Publishers; 2001, pp. 131–40. [30] Volety AK, Chu F-LE. Acid phosphatase activity in Perkinsus marinus, the protistan parasite of the American oyster, Crassostrea virginica. J Parasitol 1997;83:1093–8. [31] Anderson RS. Lack of hemocyte chemiluminescence stimulation by Perkinsus marinus in oysters Crassostrea virginica with dermo disease. J Aquat Anim Health 1999;11:179–82.
194
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
[32] Anderson RS. Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev Comp Immunol 1994; 18:89–96. [33] Dahlgren C, Stendahl O. Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983;39:736–41. [34] Stevens P, Hong D. The role of myeloperoxidase and superoxide anion in the luminol- and lucigenin-dependent chemiluminescence of human neutrophils. Microchem J 1984;30:135–46. [35] Cheng TC, Rodrick GE. Lysosomal and other enzymes in the hemolymph of Crassostrea virginica and Mercenaria mercenaria. Comp Biochem Physiol 1975;52B:443–7. [36] Cheng TC, Rodrick GE, Foley DA, Koehler SA. Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis. J Invertebr Pathol 1975;25:261–5. [37] Mitta G, Vandenbulcke F, Hubert F, Roch P. Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge. J Cell Sci 1999;112:4233–42.
A thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses Robert S. Anderson a*, Brenda S. Kraus a, Sharon E. McGladdery b, Kimberly S. Reece c, Nancy A. Stokes d a
Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, MD 20688, USA b Shellfish Pathology Research and Diagnostics, Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton NB, Canada E1C 9B6 c Department of Fisheries Science, Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA d Department of Fisheries Science, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA Received 13 August 2002; accepted 23 September 2002
Abstract A previously undescribed thraustochytrid protist, designated C9G, was isolated from the gills of a clam, Mercenaria mercenaria, collected from the Bay of Fundy, Canada. Sequence data analysis showed C9G to be related to the clam pathogen QPX, quahog parasite unknown; however, it is not enveloped by secreted mucoid material as is the case for QPX. Clam hemocytes recognized and phagocytized C9G in vitro in the absence of plasma recognition factors. Hemocytes were also capable of killing ingested C9G, as shown by the use of a tetrazolium reduction viability assay. The mechanisms underlying intracellular antimicrobial activity are not yet established, but no detectable cytotoxic reactive oxygen species were generated during phagocytosis of C9G. Clam plasma proteins were shown to inhibit C9G growth at concentrations similar to those in unfractionated hemolymph. 2003 Elsevier Ltd. All rights reserved. Keywords: Mercenaria mercenaria; Immunity; Defense responses; Parasite; QPX; C9G; Small subunit ribosomal DNA; Thraustochytrid; Antibacterial responses
1. Introduction Pathogenic thraustochytrids have been isolated from various molluscan species [1–4], including Mercenaria mercenaria, in which QPX has been associated with mortalities of cultured clams mainly in * Corresponding author. Tel.: +1-410-326-7274; fax: +1-410-326-7210 E-mail address: [email protected] (R.S. Anderson). 1050-4648/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(02)00157-2
184
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Canada [5] and Massachusetts [6]. The pathogenicity of this protist, C9G, has not been established; however, it seems to be phylogenetically closely related to QPX and was isolated from a clam in a population infected with QPX. A medium originally designed for propagation of QPX [7] also supported culture of C9G, thus enabling a constant supply for the current study. In culture, C9G exists as loosely adherent cells without any evidence of the mucoid secretions typical of QPX. The secretions have been postulated to be virulence factors in that they participate in inflammatory responses via chemoattractant activity [8], as well as protection of QPX from phagocytic hemocytes [6] and humoral antimicrobial agents [9]. It is likely that C9G would be more directly exposed in vivo to host defense mechanisms than QPX because of its lack of secretory material. In this article, preliminary characterization of some cellular and humoral responses of M. mercenaria to C9G is reported. This is the first in vitro study of clam innate immune mechanisms evoked by a thraustochytrid species. 2. Methods 2.1. Culture of C9G This microorganism was isolated from gill tissues of QPX-infected M. mercenaria collected in January 2000 from Sam Orr Pond, near St. Andrews, NB (Bay of Fundy). C9G was maintained in the medium described by Kleinschuster et al. [7] at 24 (C, pH 7.2. The cultures were kept in canted-neck tissue culture flasks and subcultured every 7 days under sterile conditions. The cells tended to be adherent, so they were removed from the bottom of the flasks with a sterile cell scraper prior to subculturing. 2.2. Molecular characterization of C9G Genomic DNA was extracted from cultured C9G cells as previously described for Perkinsus cells [10]. Portions of the small subunit ribosomal DNA (SSU rDNA) were targeted for PCR amplification using QPX-specific primers, QPX-F and QPX-R2 and the Labyrinthulomycota-specific primers, LABY-A and LABY-Y [11]. PCR reaction mixtures and cycling conditions for QPX-F + QPX-R2 and for LABY-A + LABY-Y have been described [11]. The nearly full-length SSU rRNA gene was amplified by PCR using universal primers for eukaryotic organisms [12], without the 5# polylinker bases. The SSU reaction mixtures and cycling conditions were conducted as previously described [11]. An aliquot of each reaction (10% of reaction volume) was checked by agarose gel electrophoresis for the expected product sizes of 665 bp with the QPX-specific primers, approximately 435 bp with Labyrinthulomycota-specific primers, and approximately 1800 bp with the universal SSU rRNA gene primers. Triplicate PCR reactions of the SSU rRNA gene were pooled and ligated into the plasmid vector pCR2.1 (Invitrogen). The ligations were transformed into Escherichia coli INVF# cells and plasmid DNA prepared from clones with inserts. Ten clones were cycle sequenced via simultaneous bidirectional sequencing using M13 forward and reverse primers labeled with the dyes IRD-700 and IRD-800 (LI-COR). Sequencing reactions were electrophoresed on 66 cm 4% polyacrylamide gels in a LI-COR Model 4200 automated sequencer. A consensus sequence for C9G SSU rDNA was obtained using the MacVector software package (Oxford Molecular) and deposited into GenBank (accession # AF474172). 2.3. Phylogenetic analysis For phylogenetic analyses the C9G sequence was aligned with SSU rDNA sequences of QPX and 32 other labyrinthulomycetes (Fig. 1). Sequences in GenBank that were used for the phylogenetic analyses include: Aplanochytrium kerguelense (AB022103), Japonochytrium sp. (AB022104), Labyrinthula sp. (AF265330, AF265332, AB022105), L. yorkensis (AF265333), L. zosterae (AF265334, AF265335), Labyrinthuloides haliotidis (U21338), L. minuta (L27634, AF265339), QPX (AY052644, AF155209, AF261664),
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
185
Fig. 1. Phylogenetic tree generated by parsimony jackknife analysis of labyrinthulomycete SSU rDNA sequences. Two other stramenophiles, A. bisexualis and O. danica were used as outgroup taxa. Parsimony jackknife support values above 50 are shown on branches. Analysis was conducted with 100 jackknife replicates of 100 random additions and gaps were treated as missing data. The TPG and LPG described by Honda et al. [19] as well as a Labyrinthula species group described by Stokes et al. [11], are indicated.
186
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Schizochytrium aggregatum (AB022106, AF265336), S. limacinum (AB022107, AF265339), S. minutum (AB022108), Thraustochytrium aggregatum (AB022109), T. aureum (AB022110), T. kinnei (L34668), T. motivum (AF265337), T. multirudimentale (AB022111), T. pachydermum (AB022113), T. striatum (AB022112, AF265338), Thraustochytrium sp. (AB052556), Ulkenia profunda (L34054, AB022114), U. radiata (AB022115), U. visurgensis (AB022116), and several sequences simply designated Thraustochytriidae ssp. (AF2577314, AF257315, AF257316, AF257317). Two other stramenophiles, Achlya bisexualis (M32705) and Ochromonas danica (M32704) were used as outgroup taxa. Sequences were aligned using the CLUSTAL-W algorithm [13] in the MacVector 7.0 DNA Sequence Analysis Software package. Parsimony jackknife analysis was performed using PAUP* 4b8.0 [14] with 100 bootstrap resamplings of 100 random addition replicates treating gaps as missing data. 2.4. Phagocytosis The phagocytosis assay was based on that of Hed [15]. A fluorescein succinimidyl ester solution was made on the day of the experiment (0.1 mg in 50 µl dimethylsulfoxide). C9G from 3 to 5 day (logarithmic growth phase) cultures were centrifuged (160g, 10 min, 20 (C) from w40 ml medium containing 2–8107 cells/ml cell density. The C9G were resuspended in 0.5 ml 33 ppt NaCl and 50 µl fluorescein solution added; this mixture was continuously mixed for 1 h at 20 (C. The labeled C9G were washed three times by centrifugation (160g, 10 min, 20 (C) and resuspended in 0.5 ml 33 ppt NaCl. Cell numbers were quantified with a hemocytometer and the density adjusted to 2107 C9G/ml. An aliquot of this suspension was centrifuged because the cell-free supernatant contained a low concentration of unbound fluorescein, this solution was used to control subsequent hemocyte fluorescence not associated with labeled C9G. This labeling procedure did not kill the C9G, which remained w90% viable by the trypan blue exclusion assay. For comparative assays, yeast cells (Saccharomyces cerevisiae) were fluorescein-labeled by the same method as C9G cells. M. mercenaria were obtained from the Ware River Va., through a local commercial source. Hemolymph was collected from the adductor muscle and held on ice in polypropylene tubes, cell density determined, and that volume required to contain 105 cells/well added to two microtiter plate wells. The plates containing the hemocyte suspensions were centrifuged (160g, 10 min, 20 (C) and the supernatants removed. The suspension of fluorescein-labeled test particles was immediately added to one of the hemocyte-containing wells and to an empty well. For each sample, three basic situations (A:hemocytes+particles; B:hemocytes+particle supernatant; and C:particles alone) were carried out with three replicates for each hemolymph sample. All wells contained a final volume of 50 µl. The plates were centrifuged again (160g, 10 min, 20 (C) to facilitate particle–hemocyte contact and incubated in the dark (1 h, 20 (C). A trypan blue fluorescence-quenching solution (0.25 mg/ml in 0.02 M citrate buffer) was made fresh daily and 50 µl added to all wells, and the plate was incubated in the dark (5 min, 20 (C). This treatment was intended to quench the fluorescence of any free fluorescein and unphagocytosed test particles. The plates were then read on a fluorescence concentration analyzer (IDEXX, 485/535, 25 gain) to obtain relative fluorescence units (RFU). The RFU of labeled particles phagocytosed was calculated by RFU A(RFU B + RFU C). The number of particles represented by this RFU value was estimated from a standard curve of fluorescence vs. particle count, as determined by hemocytometer. Every batch of labeled particles required construction of a new standard curve to account for the different batch-to-batch labeling efficiencies routinely encountered. 2.5. Anti-C9G activity of hemocytes This procedure was adapted from a tetrazolium dye assay used to assess the antimicrobial activity of oyster hemocytes vs. the protozoan parasite Perkinsus marinus [16]. Aliquots from 3 to 5 day C9G cultures were washed and resuspended in w0.1 original volume 25 ppt Instant Ocean (IO). The C9G density in this suspension was determined in a hemocytometer and adjusted to 6106 cells/ml. Hemolymph was collected
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
187
from M. mercenaria and held on ice in polypropylene tubes, its cell density determined, and the volume required for 105 cells added to the appropriate microtiter plate wells. The plate was centrifuged (160g, 10 min, 20 (C), and the cell-free hemolymph removed. The plate was set up as follows: row 1, 50 µl IO; row 2, hemocytes overlaid with 50 µl IO; row 3, hemocytes overlaid with 50 µl C9G suspension adjusted to the desired concentration; and row 4, 50 µl C9G suspension. Each row had four replicate wells. The plate was incubated (3 h, 26 (C) to permit cellular killing of the C9G. All wells then received 100 µl of a grow-out medium for the surviving C9G (Sigma MEM 3149, minimal essential medium without phenol red) and the plate was incubated (16 h, 26 (C). Then 20 µl MTS/PMS solution (3-[4,5-dimethylthiazol-2yl]-5-[3carboxymethoxyphenol]-2-[4-sulfophenyl]-2H-tetrazolium and phenazine methosulfate) was added to all wells, following the directions for the cell Titer 96 Aqueous Cell Proliferation Assay Kit (Promega Co.). The plate was vortexed and incubated for an additional 2 h at 26 (C. The absorbance of the wells was measured at 490 nm on a SpectraCount Plate Reader (Packard). The absorbance values of the reagent blanks (row 1) were subtracted from the readings of the wells in rows 2–4 to give corrected (corr.) values, and a killing index (KI) was calculated by the formula KI⫽1ⳮ
S
Abs row 3 corr.ⳮAbs row 2 corr. Abs row 4 corr.
D
⳯ 100
2.6. Anti-C9G activity of plasma M. mercenaria plasma was prepared by centrifuging whole hemolymph (300g, 10 min, 4 (C), sterilized by filtration (0.2 µm pore size), and assayed for protein content (BCA protein assay kit, Pierce Co.). C9G cells from 3 to 5 day cultures were washed three times with 25 ppt IO, as previously described, and the cell density adjusted to 7105 cells/ml. The cell suspension (100 µl) was mixed in a microcentrifuge tube with 1 ml of plasma protein (200 or 400 µg/ml) or 1 ml IO, and the tubes incubated 2.5 h at 20 (C (all determinations performed in triplicate). The C9G cells were subsequently recovered (300g, 10 min, 20 (C), washed two times with 25 ppt IO, and resuspended in 100 µl IO. These cell suspensions were then added to 2.5 ml MEM and incubated 7 days at 24 (C. The C9G cells were again washed twice and resuspended in 2.5 ml IO, and the C9G cell density determined by spectrophotometry at 560 nm, using an appropriate standard curve. 2.7. Chemiluminescence (CL) assay for reactive oxygen species (ROS) This assay was carried out as described by Bramble and Anderson [17], with a few modifications. The hemocyte count in a sample of pooled hemolymph was determined and 2106 hemocytes, suspended in hemolymph, were added to each CL reaction vial (final volume 2.5 ml). All aspects of the experiment were carried out under dim red illumination. Lucigenin (10,10#-9,9#-dimethylbiacridinium nitrate), a CL probe specific for superoxide, was added to the vials (150 µM, final concentration) and allowed to equilibrate for w20 min. Hemocytes were then exposed to C9G (1 hemocyte:20 C9G), or to the classical macrophage CL stimuli phorbol myristate acetate (PMA) or zymosan (25 µM or 3.89 mg/ml, respectively). CL was quantified for 2 h by a liquid scintillation counter (Packard Tri-Carb 1900 CA) adjusted for single photon counting. 3. Results 3.1. Culture characteristics of C9G The maximal final cell density attained was 6.80.4106 C9G cells/ml, reached after 3–4 days, regardless of initial seeding densities, which ranged from w2 to 7105 C9G cells/ml. The numbers of C9G
188
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
cells/ml were determined turbidometrically from a standard curve relating absorbance (OD560 nm) of cell suspensions to actual numbers as quantified in a hemocytometer. 3.2. Sequence of C9G and phylogenetic relationship to QPX C9G genomic DNA did not amplify with the QPX-specific primers, QPX-F+R2, but did generate a 441 bp product with the labyrinthulomycete primers, LABY-A+LABY-Y. Since this culture was initially thought to be QPX, the nearly complete SSU RNA gene was amplified using universal primers as described above and the sequence was determined for comparison with SSU rDNA sequences of QPX and other labyrinthulomycetes. The C9G SSU rDNA fragment was 1770 bp in length. BLAST [18] searches of GenBank suggested that C9G was indeed a thraustochytrid that was closely related to QPX and T. pachydermum. Phylogenetic analysis supported placement of C9G into the thraustochytrid phylogenetic group (TPG) as described by Honda et al. [19] within the phylum Labyrinthulomycota. Parsimony analysis placed C9G as a sister taxon to T. pachydermum with a parsimony jackknife support value of 98 (Fig. 1). In addition, the T. pachydermum and C9G sequences grouped with the QPX sequences with a jackknife support value of 100. Results of analyses conducted for this study were consistent with earlier studies placing QPX, T. pachydermum (and C9G) within the TPG [11,19], rather than into the other major phylogenetic group of the Labyrinthulomycota, the labyrinthulid phylogenetic group (LPG) [19]. Examination of SSU rDNA sequences in the V3 and V8 regions also supported placement of C9G in the TPG. As shown in Fig. 2 the C9G SSU rDNA sequence contains the thraustochytrid group signature sequences [19]. 3.3. Phagocytosis of C9G When the C9G cells were fluorescein-labeled, the results indicated that a fluorescent phagocytosis method would be useful with this protist, as was shown previously to be the case with labeled yeast (data not shown). This method gave unambiguous, dose-dependent data on C9G uptake by the hemocytes (Fig. 3). In this study, 105 hemocytes/well were incubated with increasing numbers of fluorescently labeled C9G. Subsequently a fluorescence quencher was added to the medium to reduce the signal of any extracellular C9G. The uptake of labeled C9G was proportional to the C9G:hemocyte ratio, after correcting for background fluorescence of hemocytes, and unquenched fluorescence of extracellular C9G. The apparent fluorescence of the hemocytes was probably caused by bleeding of label from the C9G added to the mixture. The overall quenching efficiency of extracellular C9G decreased with the C9G:hemocytic ratio because quencher concentration was held constant for all C9G:hemocyte ratios tested. Additional phagocytosis experiments were carried out using a C9G:hemocyte ratio of 10:1 because this concentration repeatedly gave good results, and because higher ratios required excessive numbers of C9G. When the RFU data from the 10:1 studies were converted to numbers of C9G cells phagocytosed, it was found that the mean uptake was 16279 C9G cells/100 hemocytes, based on data from four individual pools. Attempts to opsonize yeast or C9G with autologous M. mercenaria serum were unsuccessful (data not presented); test particles were incubated with the highest non-agglutinating serum dilutions (full strength for C9G and 1:128 for yeast) for 60 min at 21 (C, and washed prior to being used in the phagocytosis assay. 3.4. Anti-C9G activities of hemocytes and plasma Hemocyte samples taken from three separate M. mercenaria hemolymph pools were tested for anti-C9G activity using the MTS/PMS assay. During the 3-h incubation period the hemocytes killed 39.112.7% of the administered dose. Suspension of C9G were exposed to 0, 181 or 363 µg/ml plasma protein for 2.5 h, washed, resuspended in MEM, and allowed to grow out for 7 days before quantifying their final numbers. The treatment with 181 µg/ml plasma protein resulted in 60.411.5% (n⫽3) reduction in C9G in culture;
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Fig. 2. Signature sequences of the TPG and LPG in the V3 and V8 regions of the SSU rDNA [19]. Sequences in these regions are shown for L. minuta, of the LPG, and for QPX and T. pachydermum, of the TPG. C9G SSU rDNA contains the signature sequences of the TPG. Numbers in parentheses indicate bases of organism’s SSU rDNA highlighted in bold.
189
190
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
Fig. 3. Fluorescence data in thousands of RFU from a representative phagocytosis experiment. The same pool of hemocytes, same batch of labeled C9G, and the same concentration of quenching solution were used throughout. The bars equal the mean RFUSD for three interexperiment replicates.
doubling the concentration produced a similar reduction of 60.616.1% (n⫽3). Full-strength hemolymph plasma was found to contain 411108 µg/ml protein (n⫽3). 3.5. Chemiluminescence response to C9G In an attempt to elucidate a possible mechanism underlying the observed ability of hemocytes to kill C9G, the production of cytotoxic oxygen species by the cells was measured by CL. Fig. 4 shows representative CL results with the probe lucigenin used to quantify the generation of superoxide anions, the first step in the ROS pathway. The exposure of clam hemocytes to medium containing PMA or zymosan produced minor, transient increments in CL activity, as compared with the CL responses stimulated by these agents in oyster hemocytes (data not shown). In fact, addition of comparable volumes of agent-free medium elicited a slight increase in CL. Addition of C9G to the hemocytes at ratios %20:1 stimulated no significant superoxide production. 4. Discussion Sequence data analysis showed that C9G is a thraustochytrid member of phylum Labyrinthulomycota. This group is composed mainly of non-pathogenic marine and estuarine protists associated with detrital
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
191
Fig. 4. Production of superoxide by M. mercenaria hemocytes, as measured by lucigenin-dependent CL. Hemocytes were suspended in medium containing lucigenin (150 µM) and treated with 3.9 mg/ml zymosan, 25 µM PMA, or C9G (20 particles:1 hemocyte) and the resultant CL monitored for about 2.5 h. The various agents were added after the cells had equilibrated, as indicated by the arrow.
sediments, algae and plants [20]; however, it is known to contain pathogens [1–6]. The pathogenicity of C9G for M. mercenaria has not been established, but it is a potential pathogen because of its phylogenetic affinity to QPX and the fact that it is associated in the field with QPX infections. Unlike QPX, it secretes little or no mucoid material; consequently, it may be directly exposed in vivo to host defense mechanisms mediated by hemocytes and serum antimicrobial molecules. Phagocytic hemocytes of bivalves are thought to be important effector cells in host defense systems by virtue of their ability to ingest and destroy microorganisms [21,22]. To better understand the phagocytic activity of M. mercenaria hemocytes and their response to C9G, a fluorescent-based phagocytosis system was developed using a 96-well microtiter plate format. When the particle:hemocyte ratio was increased, the total number of phagocytosed particles increased concomitantly. The rate of phagocytosis was rapid for the first 15–20 min and reached a plateau at w90 min. Although interaction with soluble hemolymph factors, particularly natural agglutinins, can facilitate recognition and internalization of foreign particulates by certain bivalves [23], treatment with M. mercenaria serum did not appear to opsonize C9G. Not only could hemocytic internalization of C9G be quantified, but also the ability of the cells to inhibit C9G was demonstrated, using a classical assay (MTS/PMS). Under the conditions and hemocyte:C9G ratios used in this study, we found that a 3-h killing period, followed by a 16 h grow-out period for the surviving C9G provided useful and reproducible data. Microscopic examination of the size composition of C9G cells prior to exposure to the hemocytes, as well as after the grow-out period, suggests uniformity. This is an important
192
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
consideration when converting Ab490 to cell numbers because the MTS/PMS assay gives an indication of viable cell mass. One may assume that these indications of in vitro anti-C9G activity of hemocytes mimic the in vivo situation, but a caveat must be considered. When this assay was used to characterize the capacity of oyster hemocytes to destroy cells of its pathogenic protozoan P. marinus, 25–90% of P. marinus cells tested were killed [16]. This percentage killed seems quite high since in vivo the parasite apparently often survives phagocytosis and may actually take advantage of circulating hemocytes for dispersal throughout the oyster. It is possible that P. marinus loses its virulence during culture as a result of repeated transfers and lack of contact with oyster tissues [24]; at this time, the effects of continuous in vitro culture of C9G on its phagocytosis and killing by clam hemocytes is unknown. Cell-free clam hemolymph (plasma) was shown to contain naturally occurring anti-C9G activity. Full-strength plasma (w400 µg protein/ml) inhibited growth of C9G cultures by w60%, as did samples 50% diluted. Although the EC50 for this activity was not determined, it is evident that clam plasma contains an active anti-C9G factor, which probably acts in concert with hemocyte-mediated mechanisms to control or resolve C9G infections. Anti-protistan plasma constituents have been described in other bivalves. Hubert et al. [25] reported a 320 kDa protein in Mytilus galloprovincialis that was cytotoxic for the oyster parasite Bonamia ostreae. Antimicrobial peptides have been found in several mussels and oysters [26], but they have been little studied in other bivalves, such as clams. Anti-protistan peptides against bivalve parasites, such as P. marinus and B. ostreae have also been reported in non-bivalve species [26,27]. Isolation and characterization of anti-C9G proteins and/or peptides from M. mercenaria plasma needs to be carried out. The generation of cytotoxic ROS by phagocytes via activation of NADPH oxidase is thought to be important in the killing of phagocytosed microorganisms. This system has been demonstrated in oyster and mussel hemocytes [28,29], although the significance of its role in host defense in these bivalves has been questioned [17,30,31]. In a previous study, M. mercenaria hemocytes failed to produce ROS when exposed to ROS-stimulating agents that triggered ROS generation by oyster hemocytes [32]. In that study, the ROS response was measured by luminol-dependent CL, predominantly a means to study the myeloperoxidaseH2O2-halide pathway of hypohalous acid production [33]. In this study, the ability of M. mercenaria hemocytes to respond to classical ROS stimulators zymosan and PMA was studied using lucigeninaugmented CL, as was the response to C9G. Lucigenin-augmented CL is thought to primarily measure production of superoxide [34], the first ROS product made via NADPH oxidase. Results from the use of lucigenin support the previous finding of very low ability of M. mercenaria cells to mount a ROS response to putative stimuli, including C9G. Clearly, the hemocytes can kill ingested C9G; however, the data suggest that this activity does not depend on oxygen-dependent mechanisms. Intrahemocytic killing of C9G may be a function of lysosomal hydrolases, such as lysozyme [35], and/or antimicrobial peptides [26]. In addition to providing intracellular defense activity, it is likely that both lysosomal enzymes and antimicrobial peptides are released into the plasma during degranulation of hemocytes after microbial challenge [36,37]. Acknowledgements This study was supported in part by the Maryland Sea Grant College, by NOAA Grant No. NA06RG0101. We wish to thank Mr Greg MacCallum for his technical assistance in isolating and providing the initial cultures of C9G. The technical contributions of Ms Kathleen Apakupakul (Chesapeake Biological Laboratory Contribution No. 3636-CBL; Virginia Institute of Marine Science Contribution No. 2489) were also appreciated. References [1] McLean N, Porter D. The yellow-spot disease of Tritonia diomeda, Bergh, 1894 (Mollusca: Gastropoda: Nudibranchia): encapsulation of the thraustochytriaceous parasites by host amoebocytes. J Parasitol 1982;68:243–52.
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
193
[2] Bower SM. Labyrinthuloides haliotidis n.sp. (Protozoa: Labyrinthomorpha), a pathogenic parasite of small juvenile abalone in a British Columbia mariculture facility. Can J Zool 1987;65:1996–2007. [3] Jones GM, O’Dor RK. Ultrastructural observations on a thraustochytrid fungus parasitic in the gills of squid (Ilex illecebrosus, Lesueur). J Parasitol 1983;69:903–11. [4] Hanlon RT, Forsythe JW. Diseases caused by microorganisms. In: Kinne O, editor. Diseases of marine animals. Hamburg: Biologische Anstalt Helogoland; 1990, pp. 40–3. [5] Whyte SK, Cawthorn RJ, McGladdery SE. QPX (quahaug parasite X), a pathogen of northern quahaug Mercenaria mercenaria from the Gulf of St. Lawrence, Canada. Dis Aquat Organ 1994;19:129–36. [6] Smolowitz R, Leavitt D, Perkins F. Observations of a protistan disease similar to QPX in Mercenaria mercenaria (hard clams) from the coast of Massachusetts. J Invertebr Pathol 1998;71:9–25. [7] Kleinschuster SJ, Smolowitz R, Parent J. In vitro life-cycle and propogation of quahaug parasite unknown. J Shellfish Res 1998; 17:75–8. [8] Decker C-S, Anderson RS. Chemotaxis of haemocytes of the hard clam, Mercenaria mercenaria to quahog parasite unknown (QPX) and other microorganisms. J Shellfish Res 2002;21:404. [9] Anderson RS, Kraus BS, McGladdery S, Smolowitz R. Mucoid secretions protect QPX from antimicrobial agents. J Shellfish Res 2002;21:404. [10] Reece KS, Siddall ME, Burreson EM, Graves JE. Phylogenetic analysis of Perkinsus based on actin gene sequences. J Parasitol 1997;83:417–23. [11] Stokes NA, Ragone Calvo LM, Reece KS, Burreson EM. Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam, Mercenaria mercenaria. Dis Aquat Organ 2002;52:233–47. [12] Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 1988;71:491–9. [13] Thompson JD, Higgins DG, Gibson TJ. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Resour 1994;22:4673–80. [14] Swofford DL. PAUP*, Phylogenetic analysis using parsimony (*and other methods). Sunderland: Sinauer Associates, 2001. [15] Hed J. Methods for distinguishing ingested from adhering particles. Methods Enzymol 1986;132:198–204. [16] Volety AK, Fisher WS. In vitro killing of Perkinsus marinus by hemocytes of oysters Crassostrea virginica. J Shellfish Res 2000; 19:827–34. [17] Bramble LH, Anderson RS. Lack of involvement of reactive oxygen species in the bactericidal activity of Crassostrea virginica haemocytes in contrast to Morone saxatilis phagocytes. Fish Shellfish Immunol 1999;9:109–23. [18] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJJ. Basic local alignment search tool. Mol Biol 1990;215:403–10. [19] Honda D, Yokochi T, Nakahara T, Raghukumar S, Nakagiri A, Schaumann K et al. Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene. J Eukaryot Microbiol 1999;46:637–47. [20] Porter D. Phylum Labyrinthulomycota. In: Margulis L, Corbiss JO, Melkonian M, Chapman DI, editors. Handbook of Protoctista. Boston: Jones and Bartlett; 1989, pp. 388–97. [21] Cheng TC. Bivalves. In: Ratcliffe NA, Rowley AF, editors. Invertebrate blood cells. London: Academic Press; 1981, pp. 233–300. [22] Fisher WS. Structure and function of oyster hemocytes. In: Brehelin M, editor. Immunity in invertebrates. Berlin: Springer; 1986, pp. 25–35. [23] Vasta GR. The multiple biological roles of invertebrate lectins: their participation in nonself recognition mechanisms. In: Warr GW, Cohen N, editors. Phylogenesis of immune functions. Boca Raton: CRC Press; 1991, pp. 73–101. [24] Chintala MM, Alcox KA, Ford SE, Bushek D. Cultured cells and natural Perkinsus marinus cells: a possible mechanism for virulence differences. J Shellfish Res 1995;14:240. [25] Hubert F, van der Knapp W, Noe¨l T, Roch P. Cytotoxic and antibacterial properties of Mytilus galloprovincialis, Ostrea edulis and Crassostrea gigas (bivalve molluscs) hemolymph. Aquat Living Resour 1996;9:115–24. [26] Roch P. Defense mechanisms and disease prevention in farmed invertebrates. Aquaculture 1999;172:125–45. [27] Morvan A, Bache`re E, Da Silva PP, Pimenta P, Mialhe E. In vitro activity of the antimicrobial peptide magainin 1 against Bonamia ostrea, the intrahemocytic parasite of the flat oyster Ostrea edulis. Mol Marine Biol Biotechnol 1994;3:327–33. [28] Adema CM, van Deutekom-Mulder EC, van der Knapp WPW, Smina T. NADPH-oxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnea stagnalis. J Leukoc Biol 1993;54:379–83. [29] Anderson RS. Reactive oxygen species and antimicrobial defenses of invertebrates: a bivalve model. In: Beck G, Sugumaran M, Cooper EL, editors. Phylogenetic perspectives on the vertebrate immune system. New York: Kluwer Academic/Plenum Publishers; 2001, pp. 131–40. [30] Volety AK, Chu F-LE. Acid phosphatase activity in Perkinsus marinus, the protistan parasite of the American oyster, Crassostrea virginica. J Parasitol 1997;83:1093–8. [31] Anderson RS. Lack of hemocyte chemiluminescence stimulation by Perkinsus marinus in oysters Crassostrea virginica with dermo disease. J Aquat Anim Health 1999;11:179–82.
194
R.S. Anderson et al. / Fish & Shellfish Immunology 15 (2003) 183–194
[32] Anderson RS. Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev Comp Immunol 1994; 18:89–96. [33] Dahlgren C, Stendahl O. Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983;39:736–41. [34] Stevens P, Hong D. The role of myeloperoxidase and superoxide anion in the luminol- and lucigenin-dependent chemiluminescence of human neutrophils. Microchem J 1984;30:135–46. [35] Cheng TC, Rodrick GE. Lysosomal and other enzymes in the hemolymph of Crassostrea virginica and Mercenaria mercenaria. Comp Biochem Physiol 1975;52B:443–7. [36] Cheng TC, Rodrick GE, Foley DA, Koehler SA. Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis. J Invertebr Pathol 1975;25:261–5. [37] Mitta G, Vandenbulcke F, Hubert F, Roch P. Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge. J Cell Sci 1999;112:4233–42.