COMPARISON OF ROSE BENGAL AND CELLTRACKER GREEN STAINING FOR IDENTIFICATION OF LIVE SALT-MARSH FORAMINIFERA

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1 Journal of Foraminiferal Research, v. 42, no. 3, p , July 2012 COMPARISON OF ROSE BENGAL AND CELLTRACKER GREEN STAINING FOR IDENTIFICATION OF LIVE SALT-MARSH FORAMINIFERA BRIGIDA O. FIGUEIRA 1,2,4,HUGH R. GRENFELL 2,BRUCE W. HAYWARD 2 AND ANDREA C. ALFARO 3 ABSTRACT Modern analog faunal distributions are increasingly being used in fossil foraminiferal studies to provide quantitative estimates of past environmental conditions, requiring an accurate assessment of modern taphonomic assemblages. A fundamental issue with such an approach is the differentiation of live versus dead foraminifera in the modern assemblage. The effectiveness of various biological staining techniques for this purpose has long been debated. In this study, the reliability of the stain rose Bengal, which has been widely used for over 50 years, was compared to that of the modern fluorogenic probe CellTracker TM Green in identifying live agglutinated salt-marsh foraminifera from two locations on the South Island, New Zealand. Cored samples within these locations yielded low diversity assemblages, dominated by Trochamminita salsa, an important high tidal salt-marsh species in the Southern Hemisphere. Parametric statistical analysis of replicate data shows that there is no significant difference between the ability of the two techniques to discriminate between live and dead foraminifera. INTRODUCTION Differentiating between live and dead foraminifera has been an important issue for micropaleontologists for over 50 years. For many paleoenvironmental studies, including investigations of climate and sea-level reconstruction, pollution effects, and other anthropogenic impacts (e.g., Alve, 1995; Cann and others, 2000; Barbosa and others, 2005; Southall and others, 2006; Vilela and others, 2007; Gehrels and others, 2008; Berkeley and others, 2009), it is necessary to identify live and dead foraminifera for taphonomic reasons (Murray, 2000). Biological staining techniques are used to label live individuals and appropriately include or exclude them from analyses (e.g., Patterson and others, 2000; Edwards and others, 2004; Barbosa and others, 2005). In addition, some studies need to identify an appropriate cm-sampling depth that best represents the proportion of live and dead individuals within a given modern assemblage for later comparison with fossil assemblages (e.g., Goldstein and Harben, 1993; Leorri and Martin, 2009). Bernhard (2000) reviewed a wide variety of possible techniques for identifying live versus dead foraminifera. Both terminal and non-terminal techniques have been used to identify live cells. Terminal techniques include biological stains, such as rose Bengal (rb) and Sudan Black 1 School of Environment, University of Auckland, Auckland 1142, New Zealand 2 Geomarine Research, 49 Swainston Road, St. Johns, Auckland 1072, New Zealand 3 School of Applied Sciences, Auckland University of Technology, Auckland 1142, New Zealand 4 Correspondence author. b.figueira@auckland.ac.nz B, biochemical assays, ultrastructural studies, and life position in sediment. Non-terminal techniques include cytoplasmic color, presence of an apertural bolus, densitygradient centrifugation, negative geotaxis, cytoplasmic streaming, reticulopodial networks, and fluorogenic probes. However, perhaps the easiest and most cost effective technique is the use of stains such as rb and Sudan Black B to detect living tissue (Walker and others, 1974; Bernhard, 1988, 2000). More recently fluorogenic probes have been successfully utilized (Bernhard, 2000; Bernhard and others, 2006, 2010; Pucci and others, 2009; Borrelli and others, 2011). De Nooijer and others (2006) tested the efficacy of the yellow tetrazolium salt MTT to determine live versus dead in two North Atlantic calcareous species. All of these techniques have advantages and disadvantages, and a general consensus on the best one to use is still lacking. This is primarily because studies do not test one staining technique against another or statistically analyze the data (e.g., Borrelli and others, 2011). Rose Bengal is a lipophilic fluorescent dye, which has been used extensively as a standard method to distinguish live foraminifera for over the past 50 years (Walton, 1952; Horobin, 1997; Murray, 2006). This non-vital biological stain absorbs onto live or dead proteins in the major cytoplasmic components and organic lining of tests, and produces a deep rose color (Walker and others, 1974; Bernhard, 2000). Thus, this stain can be problematic because recently dead individuals (up to months) will also be stained, giving false positive results (Bernhard and Bowser, 1996; Murray and Bowser, 2000; Bernhard and others, 2006). In addition, erroneous results can arise when there are bacteria or symbiotic microalgae on or within the tests of dead foraminifera (Walker and others, 1974; Bernhard and others, 2006). Although these observations question the effectiveness of rb as a staining technique, its major advantages are that, in contrast to fluorogenic probes such as CellTracker TM Green CMFDA (CTG), it is quick, inexpensive, and can be performed easily in the field. Sudan Black B has been cited in the literature as a possible replacement stain for the rb technique. Walker and others (1974) showed that heated acetylated Sudan Black B and heated saturated solutions of Sudan Black B could be used more reliably than rb when staining cultured foraminifera. However, Bernhard (1988) demonstrated that rb and Sudan Black B achieved similarly poor results compared to ATP (adenosine-59-triphosphate) biochemical tests to identify live foraminifera. Rose Bengal appears to be as reliable as other non-vital stains, and the results are not statistically different from those obtained with vital staining techniques (Murray and Bowser, 2000). Alve and Bernhard (1995) found that in studies requiring numerical abundances of foraminifera, rb is more reliable than ATP assays, since the former is not time dependent. An estimate of fluorescently labeled foraminiferal density with that 206

2 COMPARISON OF ROSE BENGAL AND CTG 207 derived from rb was roughly comparable (Bernhard and others, 1997). The fluorogenic probe CTG belongs to a group of CellTracker TM reagents, which are fluorescent chloromethyl derivatives. Initially, it is a non-fluorescent compound, but after enzymatic electrolysis CTG yields a fluorescent product which accumulates intracellularly and highlights live tissue (Bernhard and others, 2006). CellTracker Green TM exhibits bright, green fluorescence in the cytoplasm at all physiological ph levels (Invitrogen, 2009). Unlike rb, this methodology is considered to reliably stain only living tissue and not give false positives for dead protoplasm. While a number of papers have compared different staining techniques, only one to date has compared rb and CTG stains. Bernhard and others (2006) investigated deepwater ( m depth) benthic foraminifera and found that CTG was more reliable than rb, as the latter stain over-estimated the abundance of living specimens by 47%. Although CTG is a promising technique to differentiate live from dead deep-sea calcareous foraminifera, it is an expensive (NZD $ for 1 mg CTG), time-consuming method, requiring wet-picking of specimens and access to relatively expensive fluorescence microscopes (Bernhard and others, 2006). A dissecting fluorescence microscope could speed up wet-picking (J. Bernhard communication, 2012). CellTracker Green is not a technique that can be applied easily in the field away from laboratory facilities, because in our case the field areas were remote and keeping the CTG solution and samples chilled was a problem. It is also unclear whether this technique can be applied to shallow-water agglutinated foraminifera, whose thicker tests make it difficult to see the stain (Bernhard and others, 2006). This is the first study to compare the use of rb and CTG on agglutinated salt-marsh foraminiferal faunas. For our frequent analysis of modern faunas, we needed to determine whether the complex CTG method provides significantly better results than the simple rb method. The most abundant foraminifera found in the salt marshes is Trochamminita salsa (Cushman and Brönnimann), which comprises.50% of the total assemblages and dominates the upper salt marsh close to the spring highwater level, where it preferentially lives within the top 10 cm of sediment. This species is commonly found in New Zealand, Tasmania, and Chile (e.g., Jennings and others, 1995; Hayward and others, 1999; Southall and others, 2006; Gehrels and others, 2008; Callard and others, 2011), and thus considered of vital importance to studies using modern analog techniques (e.g., sea-level change). METHODS AND MATERIALS FIELD SAMPLING Study areas at Waikawa Harbour (WKW) in south Otago and Whanganui Inlet (WH) in NW Nelson on the South Island, New Zealand (Fig.1; Table 1), were sampled in May 2008 and June 2009, respectively. At each location we selected two sites, from each of which two cores (50 cm long, 6-cm diameter) were taken with an Eijkelkamp Gouge Auger, one for rb staining and a second parallel core (10 cm away) for treatment with CTG stain. The eight cores were then sampled at 4 5-cm depth intervals (Table 1) from the surface, and 36 samples collected, each,14 ml in volume. Waikawa Harbour At the Waikawa Harbour salt marsh the site WKW1 and the replicate WKW5 (2.3 m away) were sampled in the same vegetation zone (Selliera radicans Cavanilles) and at the same elevation,,2.2 m above mean sea level (MSL) at spring high-water level (Fig. 1). Cores WKW1 CT and WKW5 CT were used to test the CTG technique, and the results were compared with samples stained with rb from cores WKW1 RB and WKW5 RB (Table 1). Because no live foraminifera were found deeper than 13 cm, only four intervals were studied at this location. In core WKW1 CT, 3.8%, buffered formaldehyde was used as a preservative, and 4% paraformaldehyde PFA in WKW5 CT (see below). Whanganui Inlet The sites WHA and WHB at Whanganui Inlet were 300 meters apart (Fig. 1). Although there was a greater distance between them than at the sites in Waikawa Harbour, WHA and WHB were cored within the same vegetation zone (Juncus kraussii subsp. australiensis (Buchenau) Snogerup) and at the same elevation (,2 m above MSL at spring high-water level). The study sites are within a national park, arguably in one of the most pristine, undisturbed salt marshes in New Zealand. Cores WHA CT and WHB CT were used to test the CTG technique, which was compared with rb from the WHA RB and WHB RB cores (Table 1). Only 3.8% buffered formaldehyde was used as a preservative for CTG (see below). STAINING TECHNIQUES Staining with rb followed the methods described by Murray (1991). A 20-ml solution (1g rb/l distilled water) was added to each of the samples (i.e., depth intervals), which were then left for 24 hours to allow time for the stain to penetrate the tests of the foraminifera. The samples were subsequently washed over a 63-mm sieve to remove the stain solution and mud fraction, preserved in 70% ethanol, and refrigerated. Cores treated with the CTG method were processed following Bernhard and others (2006). Specifically, a 50-mg aliquot of CTG was dissolved in dimethyl sulfoxide (DMSO), resulting in a 10-mM solution that was kept chilled until required for use. In the field, the CTG solution was diluted with 100 ml of preservative to give the necessary 1-mM concentration. All the treated samples were refrigerated until they could be analyzed in the laboratory. The preservative commonly used for CTG studies is 4% paraformaldehyde (J. Bernhard communication, 2008), but it has a short shelf life of about two weeks, degrading over time into a higher concentration of methanol. Buffered formaldehyde (3.8%) can be used but it also contains methanol which can result in autofluorescence (J. Ross communication, 2010). We wanted to determine any potential performance difference between the two preservatives for the CTG samples. For the Waikawa Harbour study,,20 ml of preservative was added to each sample from WKW1A (formaldehyde) and WKW5A (paraformal-

3 208 FIGUEIRA AND OTHERS FIGURE 1. Map of the two study locations at Waikawa Harbour, south Otago, and Whanganui Inlet, NW Nelson, South Island, New Zealand. TABLE 1. Core names, grid references (NZMS 260 map series, 1:50,000 scale), location, and depths of the studied samples. WKW 5 Waikawa Harbour; WH 5 Whanganui Inlet. Cores Grid reference Location Samples depths (cm) WKW1 CT*** and WKW1 RB* G47/ WKW 0 1, 4 5, 8 9, WKW5 CT** and WKW5 RB* G47/ WKW 0 1, 4 5, 8 9, WHA CT** and WHA RB* M25/ WH 0 1, 4 5, 8 9, 12 13, WHB CT** and WHB RB* M25/ WH 0 1, 4 5, 8 9, 12 13, Preservatives used: * 5 alcohol, ** 5 formaldehyde, *** 5 paraformaldehyde.

4 COMPARISON OF ROSE BENGAL AND CTG 209 FIGURE 2. Fluorescent and transmitted light photomicrographs of live or dead specimens. Dead: A, B Haplophragmoides wilberti; J, K Partrochammina bartrami. Live: C, D Haplophragmoides wilberti; E I Trochammina inflata; L S Trochamminita salsa. Specimens range in size from mm. Fluorescence filter used for specimens A, C, E, G, I, J, L, N, P, and R. Transmitted light used for specimens B, D, F, H, M, O, Q, and S. Image exposures vary due to the wall density of some of the agglutinated specimens.

5 210 FIGUEIRA AND OTHERS FIGURE 3. Mean abundance (6SD) of live and dead foraminifera as a percentage of the total fauna. Ts 5 Trochamminita salsa, Ti 5 Trochammina inflata, Hw 5 Haplophragmoides wilberti, Jm 5 Jadammina macrescens, and Mf 5 Miliammina fusca stained with rose Bengal and CellTracker TM Green for four depth intervals in the replicate cores at Waikawa Harbour. dehyde). Because no significant difference in the degree of fluorescence and performance was observed for the two preservatives under the microscope, the subsequent Whanganui Inlet study used only formaldehyde. FORAMINIFERAL ANALYSES The wet-splitting method described by Scott and Hermelin (1993) was used for all samples because CTG samples must be examined wet to see any fluorescence. The.63 mm fraction was split to a proportion from which at least 100 specimens could be counted on a Bergeroff tray (a cm piece of transparent acrylic with a V-shaped, spiral groove cut into it). Many studies use picks of specimens to obtain statistically accurate estimates of the relative abundances of different species (e.g., Edwards and others, 2004; Southall and others, 2006). In our salt-marsh faunas the species richness ranged between 1 7 species, commonly with one species comprising.70% of the fauna. Previous studies have shown that in such low diversity faunas a census of 100 specimens still provides results that are statistically accurate (Boltovskoy and Wright, 1976; Patterson and Fishbein, 1989; Quinn and Keough, 2002; Gehrels and others, 2005). The specimens stained with rb were wet-picked under a Nikon SMZ645 binocular microscope and then mounted on micropaleontological slides for reference. Those showing rb staining around the aperture and in the last chamber were considered to be live, and this was confirmed by breaking some specimens. Other authors have used different criteria in

6 COMPARISON OF ROSE BENGAL AND CTG 211 TABLE 2. Statistical analyses (three-way ANOVAs) of foraminiferal species collected from different substrate depth layers at Waikawa Harbour. All foraminifera were stained with one of two vital stains (rose Bengal and CellTracker TM Green) to identify the number of dead and live individuals. All data were arcsine-transformed prior to the analyses. Significant tests are in bold. df 5 degrees of freedom, MS 5 mean square, F 5 Fteststatistic,p5thepvalue. Waikawa Harbor Dead Live Source df MS F p MS F p Species Stain Depth Species 3 Stain Species 3 Depth Stain 3 Depth Species 3 Stain 3 Depth Error different settings (e.g., deep sea, Fontanier and others, 2002), but our approach is particular to our setting. The specimens treated with CTG were first picked under a Nikon SMZ645 binocular microscope, and then viewed under a Nikon TE2000E inverted fluorescence microscope, where the fluorescence of live specimens usually could be seen inside the final chamber but often in several earlier chambers. Specimens were considered live when at least half of the chambers were fluorescing (Fig. 2; Bernhard and others, 2001, 2006). When fluorescence was seen on the outside of the shell only, the specimens were not considered to be alive since other live organisms, such as diatoms or bacteria, could have been attached to the outside wall, giving a false positive (Fig. 2). STATISTICAL ANALYSES At Waikawa Harbour the nature of the data collected dictated that a conservative statistical approach be taken. Parametric statistics were used to compare the two staining techniques for various foraminiferal species within samples at different sediment depths. The Waikawa raw data were first tested for parametric assumptions, and then arcsinetransformed. Individual three-way ANOVAs (species, stain, and depth as fixed factors) were used to identify differences among percent abundances. The study is the first comparison of these two stains being used on agglutinated taxa. In some experimental studies statistical analyses are often conducted on abundance per unit weight, volume, or area. This may be possible in deep-sea investigations (e.g., Fontanier and others, 2002), where environmental changes are typically very slow and a 20-cm-long core could be uniform in lithology and foraminiferal distribution. In shallow marine, estuarine environments this is seldom the case. In our cores the mineral and organic content of the down-core samples is highly variable, with foraminiferal abundances higher in the more organic-rich samples compared to those with low organic and high mineral content (see Appendix 1 for abundance and raw data). At Whanganui Inlet the data were not analyzed statistically, but a graphical comparison is made between the sites (Figs. 4, 5). RESULTS At Waikawa Harbour comparisons between the two staining methods (rb and CTG) resulted in no significant differences of dead and stained specimens across locations, sediment depths, or species (Fig. 3; Table 2). The species found were Trochamminita salsa (Cushman and Brönnimann), Trochammina inflata (Montagu), Haplophragmoides wilberti Andersen, Jadammina macrescens (Brady), Paratrochammina cf. P. bartrami (Hedley, Hurdle, and Burdett), Miliammina fusca (Brady), and Elphidium sp. At all sediment depths, T. salsa was the dominant live and dead foraminifera (Fig. 3), followed by T. inflata. For T. salsa, the mean percent abundance (6 SD) of dead foraminifera (highest mean abundance of % in the bottom layer at cm depth) was significantly higher than that of live individuals (highest mean abundance of % in the top layer at 0 1-cm depth). In addition, there was an inverse relationship between dead and live T. salsa with increasing core depth, with higher abundances of live individuals at the surface and higher abundances of dead individuals at the bottom (Fig. 3). A two-way ANOVA comparing live versus dead (Status factor) T. salsa among sediment layers (Depth factor) resulted in significant differences between live and dead individuals, and nonsignificant differences between core depths (Table 3). An important interaction between status and depth was highly significant, reflecting the inverse relationship in abundance between status and depth. The data from Whanganui Inlet were not suitable for the parametric statistical analysis used above. However, a comparison of the live-dead percentages from WHA and WHB (Figs. 4, 5) shows that both stains produce similar results that are within the margin of error. The results also show that the percentage of live foraminifera decreases down core, as would TABLE 3. Statistical analyses (two-way ANOVAs) of T. salsa abundance of dead and live individuals (Status factor) collected from different substrate depth layers (Depth factor) at Waikawa Harbour. All foraminifera were stained with one of two vital stains (rose Bengal and CellTracker TM Green) to identify the number of dead and live individuals. All data were arcsine-transformed prior to the analyses. Significant tests are in bold. df 5 degrees of freedom, MS 5 mean square, F 5 F test statistic, p 5 the p value. Waikawa Harbor Source df MS F p Status Depth Status 3 Depth Error

7 212 FIGUEIRA AND OTHERS FIGURE 4. Whanganui Inlet core WHA mean abundance (6 95% confidence limits) of live and dead foraminifera as a percentage of the total fauna. Ts 5 Trochamminita salsa, Ti 5 Trochammina inflata, Hw 5 Haplophragmoides wilberti, and Jm 5 Jadammina macrescens stained with rose Bengal and CellTracker TM Green for five depth intervals.

8 COMPARISON OF ROSE BENGAL AND CTG 213 FIGURE 5. Whanganui Inlet core WHB mean abundance (6 95% confidence limits) of live and dead foraminifera as a percentage of the total fauna. Ts 5 Trochamminita salsa, Ti 5 Trochammina inflata, Hw 5 Haplophragmoides wilberti, and Jm 5 Jadammina macrescens stained with rose Bengal and CellTracker TM Green for five depth intervals.

9 214 FIGUEIRA AND OTHERS be expected. In the deepest samples (WHA and WHB, cm), the percentage for live foraminifera was much lower than for dead (e.g., 5 10% vs. #90% in T. salsa). The dominant species (both live and dead) was again T. salsa with either H wilberti or J. macrescens subdominant. DISCUSSION This study presents the first evidence that the fluorogenic probe CTG technique can be used effectively to identify live agglutinated foraminifera from salt-marsh environments, as Bernhard and others (2006) have demonstrated for deep-sea calcareous foraminifera. However, Bernhard (2000) cautioned that live individuals with opaque shells could not be easily detected with this technique. Our results indicate that CTG is effective with agglutinated salt-marsh species, which have thicker and more opaque shells than calcareous deepsea species. Bernhard and others (2006) also suggested that CTG was a superior method to the commonly used rb technique, which over-estimated live deep-sea calcareous foraminifera. Contrary to their findings, our results indicate that rb is just as effective as CTG for the identification of live agglutinated salt-marsh foraminifera. A possible explanation for the difference in previous studies could be that the cytoplasm of dead foraminifera takes longer to degrade in the lower temperatures of deep-sea environments, compared to warmer salt-marsh habitats. Additionally, periods of aridity may lower the water table in the salt marsh, promoting oxidation and degradation of cytoplasm. Thus, the rb staining technique appears to work well with shallowwater foraminifera, and may provide equally reliable counts of live individuals. In our study, foraminifera were picked wet, which makes it easier to identify the stain within the specimens. Thus, it is unlikely that CTG-stained individuals would have been missed to bias our results. Since both rb and CTG techniques provided similar findings, the cheaper and easier-to-use rb method may be the technique of choice to identify live salt-marsh foraminifera in future studies. Of the seven species found in this study, Trochamminita salsa dominated all sediment layers at both locations. This species is resistant to desiccation and low salinity, being found in the salt marsh close to the level of extreme high water (Hayward and Hollis 1994; Hollis and others, 1995; Hayward and others, 2007; Gehrels and others, 2008). The absolute and relative abundances of this species are higher in the high salt marsh than in the low salt marsh (Jennings and others, 1995; Hayward and others, 1999; Southall and others, 2006; Callard and others, 2011). This distribution pattern enables T. salsa to be used for sea-level reconstruction studies, where limited elevational-range species are ideal for the modern analog technique. For modern analog techniques it is crucial to know the depth in the sediment and the tidal elevation in the salt marsh where the constraining species are found. This enables identification of the assemblage which best represents the fossil assemblage, and determination of which species have the smallest elevational range. In the present study, live and dead T. salsa exhibited an inverse relationship with sediment depth. Live specimens were more abundant at shallow depths, and their abundance decreased with increasing depth. This distribution appears to indicate that the optimum depth for live T. salsa is the top 10 cm of sediment, which is similar to the distribution of many other foraminiferal species throughout the world that normally inhabit this depth range (Goldstein and others, 1995; Ozarko and others, 1997; Hippensteel and others, 2000; Leorri and Martin, 2009). CONCLUSIONS When used with care, rose Bengal performed as well as CellTracker Green in differentiating live from dead specimens in New Zealand salt-marsh agglutinated foraminiferal faunas. Rose Bengal stain can be confidently utilized in salt-marsh modern analog studies and also to determine the appropriate depth to sample modern faunas along transects. Nevertheless, we do not advocate that rb can be universally used to identify live specimens in all environments, and some ecological studies may have to carry out specific additional testing of techniques. 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T., 1974, Sudan Black B; a superior stain to Rose Bengal for distinguishing living from non-living foraminifera: Journal of Foraminiferal Research, v. 4, p WALTON, W., 1952, Techniques for recognition of living foraminifera: Contributions from the Cushman Foundation for Foraminiferal Research, v. 7, p Received 20 February 2011 Accepted 24 April 2012 APPENDIX 1 Foraminiferal counts from Waikawa Harbour and Whanganui Inlet, South Island, New Zealand This table can be found on the Cushman Foundation website in the JFR Article Data Repository ( as item number JFR - DR