J Comp Physiol B (2014) 184:415–423 DOI 10.1007/s00360-014-0818-z
Original Paper
Ionic regulation in the Antarctic nematode Panagrolaimus davidi, measured using electron probe X‑ray microanalysis David A. Wharton
Received: 7 November 2013 / Revised: 30 January 2014 / Accepted: 12 February 2014 / Published online: 7 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract The element composition of the pseudocoelomic fluid of the Antarctic nematode Panagrolaimus davidi was analysed by electron probe X-ray microanalysis after absorbing the fluid into Sephadex G-25 beads, and after producing calibration curves by analysing various concentrations of elements of interest absorbed into beads. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. When external concentrations of specific ions were elevated there was evidence for the regulation of internal concentrations of sodium, potassium, magnesium and chlorine. The time course of changes in response to exposure to elevated levels of KCl shows an increase in internal concentrations of potassium and chlorine up to 2 h after exposure, followed by a decline. This is consistent with a model of ionic regulation proposed for Caenorhabditis elegans which suggests that high concentrations of ionic osmolytes are replaced by compatible organic osmolytes. Keywords Ionic regulation · X-ray microanalysis · Pseudocoelomic fluid · Antarctic · Nematode Abbreviations EPMA Electron probe X-ray microanalysis BSS Balanced salt solution CI Confidence interval MQ Milli-Q SEM Scanning electron microscope Communicated by I.D. Hume. D. A. Wharton (*) Department of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand e-mail: [email protected]
Introduction Free-living soil nematodes face increases in the ionic concentration of the soil water surrounding them as water is lost by the soil. The resulting stress may be particularly severe in terrestrial Antarctic habitats and results from both desiccation and freezing, as water becomes locked up in snow and ice (Wharton 2003, #2145). Panagrolaimus davidi Timm 1971 was isolated from coastal areas of Ross Island, Antarctica and has been grown in culture since 1988 (Wharton and Brown 1989). This culture strain was later designated P. davidi CB1 (Lewis et al. 2009). More recently it has been established that the field strain of P. davidi is a different species to P. davidi CB1 (Raymond et al. 2014). However, the two species are morphologically very similar and both are currently referred to as P. davidi. Both species are included in this study. The field strain of P. davidi is associated with bird colonies, particularly Adelie Penguins, and with a wide range of salinities. It is the only nematode found within penguin rookeries, where both salinity and organic matter content are high (Raymond et al. 2013). The culture strain P. davidi CB1 also tolerates high osmotic stress (Wharton 2010) and has the freezing and desiccation stress resistance that might be expected in an Antarctic species (Wharton 2011). The two species might thus be expected to have similar physiological responses, the ability to tolerate a wide range of ionic conditions and to be good models to study ionic regulations in nematodes. Ionic regulation in nematodes has been little studied (Wharton and Perry 2011). Measurements of the composition of their pseudocoelomic fluid are few and have been restricted to a single large parasitic species, due to the difficulty of extracting fluid from small free-living species. The only such measurements date from the 1950s (Hobson
13
416
et al. 1952a, b), using classical analytical methods with limited accuracy compared with modern instrumental techniques. Element concentrations can be measured in a whole nematodes or in small samples of nematodes using flame emission spectroscopy, atomic absorption spectroscopy and radioassay methods (Wright and Newall 1980) but there are few published studies using these techniques. It has been suggested that electron probe X-ray microanalysis (EPMA) could be used to determine element concentrations in different nematode compartments (Wharton 1986; Wright and Newall 1980) but to date there are no published studies on ionic regulation in nematodes using this technique. The EPMA technique has been used to demonstrate the presence of lead in Panagrolaimus spp. exposed to elevated concentrations of this element (Williams and Seraphin 1998) and the response of individual Xiphinema vuittenezi to elevated concentrations of copper (Savoly et al. 2012). Total reflection X-ray fluorescence spectroscopy has also been used to determine element concentrations in single nematodes (Savoly et al. 2012), but this technique involves ashing the specimen. In this paper EPMA is used to determine the elemental composition of the pseudocoelomic fluid of P. davidi extracted from single nematodes and to determine the ability of this nematode to regulate its composition in the face of changes in the external concentrations of elements. I adapted a technique developed to sample the elemental composition of the surface fluids of the human airways (Kozlova et al. 2006; Nilsson et al. 2004).
Materials and methods Sample collection P. davidi CB1 was grown on balanced salt solution (BSS) in agar plates as described by Wharton (2010). The nematodes were subcultured at weekly intervals and used within 7 days for experiments. Antarctic soil samples were collected from Cape Royds, Ross Island during November 2011 and were stored frozen at −20 °C. A modified Baermann technique (Hooper 1986) was used to extract nematodes at 20 °C overnight from a 20 g soil sample with the addition of 80 g of Milli-Q (MQ) water (Millipore, Milli-Q Water Purifying System, Billerica, MA, USA). The nematodes were concentrated by centrifuging and a sample of soil water was taken from the supernatant. Electron probe X‑ray microanalysis: calibration Standard solutions of sodium, potassium, magnesium and calcium were made by adding the appropriate concentration of their chloride salt to BSS to raise the concentration
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J Comp Physiol B (2014) 184:415–423
of the element to 50, 100, 150 or 200 mM. For KCl the resulting concentration of chlorine was calculated. The standards were absorbed into Sephadex beads (G-25, GE Healthcare, Pittsburgh, PA, USA) by adding a small quantity of beads to ~1 ml of the solution in a watch glass. The beads were washed several times in the standard solution to remove beads that had air bubbles trapped within them. After 30 min the remaining solution was removed with a fine pipette and the beads were immediately covered with Xiameter PMX-200 Silicone Fluid (Dow-Corning Co., Midland, MI, USA). The beads were transferred to a small square of aluminium foil, most of the silicone fluid removed with a fine pipette and the remaining silicone fluid allowed to evaporate. The sample was stored over silica gel at room temperature before mounting on aluminium stubs, coating with carbon and observation by a scanning electron microscope (SEM). X-ray spectra were acquired from about 15 beads per sample on a Jeol FE-SEM 6700 Scanning Electron Microscope with a Jeol 2300F EDS system. The microscope was operated at an accelerating voltage of 20 kV, with an emission current of 10 μA, a working distance of 15 mm, a beam size of 100 nm, a magnification of 2,000×, a live count time of 100 s and a count rate of ~1,000 counts s−1. Similar sized beads were selected, not shielded from the X-ray detector and a point analysis performed centred on the surface of the bead. Carbon and oxygen (mainly from the Sephadex bead) and aluminium (from the foil) gave the strongest signals but clear signals from the other elements of interest were obtained. Silicon was either not detected or at low counts, indicating that the silicone fluid was removed during processing. Measurements of nematode pseudocoelomic fluid and soil water Nematodes (adults and late 4th-stage larvae) were transferred using a mounted eyelash onto the surface of 1 % Bacto™ agar made up with MQ water, BSS or BSS with chloride salts added to elevate the concentration of the element of interest to 100 or 200 mM. Element concentrations in MQ agar were calculated from the manufacturer’s analyses and in BSS agar from the further additions of salts (Piggott et al. 2000). After 24 h at 20 °C the nematodes were transferred to two drops of mineral oil (Cargille’s A, Cedar Grove, NJ, USA) and their cuticle punctured with a fine glass knife. Sephadex beads were added to the mineral oil and a single bead manoeuvred into contact with the clear liquid leaking from each nematode, which was rapidly absorbed into the bead. This process was also photographed on a Zeiss Axiophot photomicroscope (Carl Zeiss Inc., Thornwood, NY, USA), using Differential Interference Contrast optics. After 30 min the mineral oil was removed
J Comp Physiol B (2014) 184:415–423
with several washes of silicone fluid and the nematodes and attached beads were transferred to a small square of aluminium foil. The samples were further processed as for the standards and analysed using the same conditions. The time course of the uptake of potassium and chlorine was followed by transferring nematodes to the surface of BSS agar with KCl added to elevate the concentration of potassium to 200 mM. Nematodes were exposed for various periods (0, 0.5, 1, 2, 4 and 24 h) before absorption into Sephadex beads and analysis, as before. Nematodes freshly extracted from Antarctic soil were transferred to a watch glass, the surface water removed using a fine pipette and filter paper spills and then covered with two drops of mineral oil. Pseudocoelomic fluid was collected and analysed as above. Antarctic soil water (from the extraction) was absorbed into Sephadex beads and analysed as for standard solutions above.
Results Electron probe X‑ray microanalysis: calibration Despite attempting to keep the count rate constant between beads it varied between 650 and 1,650 counts s−1. To standardise the counts for the element being measured the counts for the element were plotted against the total counts for all elements in the spectra. An example is shown in Fig. 1. After the removal of any obvious outliers, the equation for the regression line was used to calculate the element counts (and the 95 % CI) for a total count of 70,000 in each analysis. This gave the smallest 95 % CIs, compared with total counts of 50,000, 60,000 and 80,000. The relationship between counts of the element of interest and its concentration was linear for sodium, potassium and chlorine, but those of magnesium and calcium departed from linearity at the higher concentrations (Fig. 2). Measurements of nematode pseudocoelomic fluid and soil water Fluid leaking from the cut surface of a nematode could clearly be seen to be absorbed by a Sephadex bead brought into contact with it, with the bead swelling and changing in appearance during this process (Fig. 3). After transfer to the SEM some beads remained attached to their nematode (Fig. 4). Some were detached but could be distinguished from any beads which had not absorbed fluid by their EPMA spectra (the latter had only carbon, oxygen and aluminium peaks). Elements detected in pseudocoelomic fluid of P. davidi exposed on the surface of 1 % agar made up in MQ water or BSS and of nematodes freshly extracted from Antarctic
417
Fig. 1 Regression of potassium counts against the total counts for all elements detected in a 200 mM potassium standard absorbed into Sephadex beads. N = 16, the dashed lines are the 95 % CI
soil (from Cape Royds) are shown in Table 1 and a spectrum for pseudocoelomic fluid from Cape Royds nematodes in Fig. 5. The concentrations calculated in the agars and measured in Cape Royds soil water are also shown in Table 1. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. Chlorine had a less consistent pattern with higher concentrations in the pseudocoelomic fluid of P. davidi than the external medium for Cape Royds nematodes and nematodes on the surface of MQ agar but lower concentrations on the surface of BSS agar. Element concentrations of pseudocoelomic fluid after 24 h on the surface of MQ agar, BSS agar or BSS agar with chloride salts added to elevate the concentration of the element of interest to 100 or 200 mM are shown in Fig. 6. The concentrations of sodium and potassium in the pseudocoelomic fluid of P. davidi were above that of their external concentrations for MQ agar and BSS agar (Table 1; Fig. 6) but below that of agar containing 100 or 200 mM of the element (Fig. 6). Magnesium was not detected in pseudocoelomic fluid from nematodes exposed to MQ agar or BSS agar but was detected after exposure to agar with a magnesium concentration of 100 or 200 mM, but at a lower level than the external concentration of this element. Calcium does not appear to be taken up into the pseudocoelomic fluid at any external concentration tested. Chlorine concentration in the pseudocoelomic fluid of P. davidi was above that of the external concentration of MQ agar but below that of the other external concentrations tested (Table 1; Fig. 6). The potassium concentration in the pseudocoelomic fluid of P. davidi changes after various times of exposure to agar containing 200 mM potassium (Fig. 7). The concentration in the pseudocoelomic fluid of P. davidi peaks
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J Comp Physiol B (2014) 184:415–423
Fig. 2 Calibration curves for sodium, potassium, magnesium, calcium and chlorine. The Y-axis shows the counts for the element of interest at the level of total counts of 70,000 counts per 100 s, calculated from a regression of counts for the element against total counts for the beads. Number of beads analysed 13–17; error bars are 95 % CI. The regression lines for Mg and Ca exclude the data for 200 mM. The slope of the regression line is significantly different from zero in each case (p
Original Paper
Ionic regulation in the Antarctic nematode Panagrolaimus davidi, measured using electron probe X‑ray microanalysis David A. Wharton
Received: 7 November 2013 / Revised: 30 January 2014 / Accepted: 12 February 2014 / Published online: 7 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract The element composition of the pseudocoelomic fluid of the Antarctic nematode Panagrolaimus davidi was analysed by electron probe X-ray microanalysis after absorbing the fluid into Sephadex G-25 beads, and after producing calibration curves by analysing various concentrations of elements of interest absorbed into beads. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. When external concentrations of specific ions were elevated there was evidence for the regulation of internal concentrations of sodium, potassium, magnesium and chlorine. The time course of changes in response to exposure to elevated levels of KCl shows an increase in internal concentrations of potassium and chlorine up to 2 h after exposure, followed by a decline. This is consistent with a model of ionic regulation proposed for Caenorhabditis elegans which suggests that high concentrations of ionic osmolytes are replaced by compatible organic osmolytes. Keywords Ionic regulation · X-ray microanalysis · Pseudocoelomic fluid · Antarctic · Nematode Abbreviations EPMA Electron probe X-ray microanalysis BSS Balanced salt solution CI Confidence interval MQ Milli-Q SEM Scanning electron microscope Communicated by I.D. Hume. D. A. Wharton (*) Department of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand e-mail: [email protected]
Introduction Free-living soil nematodes face increases in the ionic concentration of the soil water surrounding them as water is lost by the soil. The resulting stress may be particularly severe in terrestrial Antarctic habitats and results from both desiccation and freezing, as water becomes locked up in snow and ice (Wharton 2003, #2145). Panagrolaimus davidi Timm 1971 was isolated from coastal areas of Ross Island, Antarctica and has been grown in culture since 1988 (Wharton and Brown 1989). This culture strain was later designated P. davidi CB1 (Lewis et al. 2009). More recently it has been established that the field strain of P. davidi is a different species to P. davidi CB1 (Raymond et al. 2014). However, the two species are morphologically very similar and both are currently referred to as P. davidi. Both species are included in this study. The field strain of P. davidi is associated with bird colonies, particularly Adelie Penguins, and with a wide range of salinities. It is the only nematode found within penguin rookeries, where both salinity and organic matter content are high (Raymond et al. 2013). The culture strain P. davidi CB1 also tolerates high osmotic stress (Wharton 2010) and has the freezing and desiccation stress resistance that might be expected in an Antarctic species (Wharton 2011). The two species might thus be expected to have similar physiological responses, the ability to tolerate a wide range of ionic conditions and to be good models to study ionic regulations in nematodes. Ionic regulation in nematodes has been little studied (Wharton and Perry 2011). Measurements of the composition of their pseudocoelomic fluid are few and have been restricted to a single large parasitic species, due to the difficulty of extracting fluid from small free-living species. The only such measurements date from the 1950s (Hobson
13
416
et al. 1952a, b), using classical analytical methods with limited accuracy compared with modern instrumental techniques. Element concentrations can be measured in a whole nematodes or in small samples of nematodes using flame emission spectroscopy, atomic absorption spectroscopy and radioassay methods (Wright and Newall 1980) but there are few published studies using these techniques. It has been suggested that electron probe X-ray microanalysis (EPMA) could be used to determine element concentrations in different nematode compartments (Wharton 1986; Wright and Newall 1980) but to date there are no published studies on ionic regulation in nematodes using this technique. The EPMA technique has been used to demonstrate the presence of lead in Panagrolaimus spp. exposed to elevated concentrations of this element (Williams and Seraphin 1998) and the response of individual Xiphinema vuittenezi to elevated concentrations of copper (Savoly et al. 2012). Total reflection X-ray fluorescence spectroscopy has also been used to determine element concentrations in single nematodes (Savoly et al. 2012), but this technique involves ashing the specimen. In this paper EPMA is used to determine the elemental composition of the pseudocoelomic fluid of P. davidi extracted from single nematodes and to determine the ability of this nematode to regulate its composition in the face of changes in the external concentrations of elements. I adapted a technique developed to sample the elemental composition of the surface fluids of the human airways (Kozlova et al. 2006; Nilsson et al. 2004).
Materials and methods Sample collection P. davidi CB1 was grown on balanced salt solution (BSS) in agar plates as described by Wharton (2010). The nematodes were subcultured at weekly intervals and used within 7 days for experiments. Antarctic soil samples were collected from Cape Royds, Ross Island during November 2011 and were stored frozen at −20 °C. A modified Baermann technique (Hooper 1986) was used to extract nematodes at 20 °C overnight from a 20 g soil sample with the addition of 80 g of Milli-Q (MQ) water (Millipore, Milli-Q Water Purifying System, Billerica, MA, USA). The nematodes were concentrated by centrifuging and a sample of soil water was taken from the supernatant. Electron probe X‑ray microanalysis: calibration Standard solutions of sodium, potassium, magnesium and calcium were made by adding the appropriate concentration of their chloride salt to BSS to raise the concentration
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J Comp Physiol B (2014) 184:415–423
of the element to 50, 100, 150 or 200 mM. For KCl the resulting concentration of chlorine was calculated. The standards were absorbed into Sephadex beads (G-25, GE Healthcare, Pittsburgh, PA, USA) by adding a small quantity of beads to ~1 ml of the solution in a watch glass. The beads were washed several times in the standard solution to remove beads that had air bubbles trapped within them. After 30 min the remaining solution was removed with a fine pipette and the beads were immediately covered with Xiameter PMX-200 Silicone Fluid (Dow-Corning Co., Midland, MI, USA). The beads were transferred to a small square of aluminium foil, most of the silicone fluid removed with a fine pipette and the remaining silicone fluid allowed to evaporate. The sample was stored over silica gel at room temperature before mounting on aluminium stubs, coating with carbon and observation by a scanning electron microscope (SEM). X-ray spectra were acquired from about 15 beads per sample on a Jeol FE-SEM 6700 Scanning Electron Microscope with a Jeol 2300F EDS system. The microscope was operated at an accelerating voltage of 20 kV, with an emission current of 10 μA, a working distance of 15 mm, a beam size of 100 nm, a magnification of 2,000×, a live count time of 100 s and a count rate of ~1,000 counts s−1. Similar sized beads were selected, not shielded from the X-ray detector and a point analysis performed centred on the surface of the bead. Carbon and oxygen (mainly from the Sephadex bead) and aluminium (from the foil) gave the strongest signals but clear signals from the other elements of interest were obtained. Silicon was either not detected or at low counts, indicating that the silicone fluid was removed during processing. Measurements of nematode pseudocoelomic fluid and soil water Nematodes (adults and late 4th-stage larvae) were transferred using a mounted eyelash onto the surface of 1 % Bacto™ agar made up with MQ water, BSS or BSS with chloride salts added to elevate the concentration of the element of interest to 100 or 200 mM. Element concentrations in MQ agar were calculated from the manufacturer’s analyses and in BSS agar from the further additions of salts (Piggott et al. 2000). After 24 h at 20 °C the nematodes were transferred to two drops of mineral oil (Cargille’s A, Cedar Grove, NJ, USA) and their cuticle punctured with a fine glass knife. Sephadex beads were added to the mineral oil and a single bead manoeuvred into contact with the clear liquid leaking from each nematode, which was rapidly absorbed into the bead. This process was also photographed on a Zeiss Axiophot photomicroscope (Carl Zeiss Inc., Thornwood, NY, USA), using Differential Interference Contrast optics. After 30 min the mineral oil was removed
J Comp Physiol B (2014) 184:415–423
with several washes of silicone fluid and the nematodes and attached beads were transferred to a small square of aluminium foil. The samples were further processed as for the standards and analysed using the same conditions. The time course of the uptake of potassium and chlorine was followed by transferring nematodes to the surface of BSS agar with KCl added to elevate the concentration of potassium to 200 mM. Nematodes were exposed for various periods (0, 0.5, 1, 2, 4 and 24 h) before absorption into Sephadex beads and analysis, as before. Nematodes freshly extracted from Antarctic soil were transferred to a watch glass, the surface water removed using a fine pipette and filter paper spills and then covered with two drops of mineral oil. Pseudocoelomic fluid was collected and analysed as above. Antarctic soil water (from the extraction) was absorbed into Sephadex beads and analysed as for standard solutions above.
Results Electron probe X‑ray microanalysis: calibration Despite attempting to keep the count rate constant between beads it varied between 650 and 1,650 counts s−1. To standardise the counts for the element being measured the counts for the element were plotted against the total counts for all elements in the spectra. An example is shown in Fig. 1. After the removal of any obvious outliers, the equation for the regression line was used to calculate the element counts (and the 95 % CI) for a total count of 70,000 in each analysis. This gave the smallest 95 % CIs, compared with total counts of 50,000, 60,000 and 80,000. The relationship between counts of the element of interest and its concentration was linear for sodium, potassium and chlorine, but those of magnesium and calcium departed from linearity at the higher concentrations (Fig. 2). Measurements of nematode pseudocoelomic fluid and soil water Fluid leaking from the cut surface of a nematode could clearly be seen to be absorbed by a Sephadex bead brought into contact with it, with the bead swelling and changing in appearance during this process (Fig. 3). After transfer to the SEM some beads remained attached to their nematode (Fig. 4). Some were detached but could be distinguished from any beads which had not absorbed fluid by their EPMA spectra (the latter had only carbon, oxygen and aluminium peaks). Elements detected in pseudocoelomic fluid of P. davidi exposed on the surface of 1 % agar made up in MQ water or BSS and of nematodes freshly extracted from Antarctic
417
Fig. 1 Regression of potassium counts against the total counts for all elements detected in a 200 mM potassium standard absorbed into Sephadex beads. N = 16, the dashed lines are the 95 % CI
soil (from Cape Royds) are shown in Table 1 and a spectrum for pseudocoelomic fluid from Cape Royds nematodes in Fig. 5. The concentrations calculated in the agars and measured in Cape Royds soil water are also shown in Table 1. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. Chlorine had a less consistent pattern with higher concentrations in the pseudocoelomic fluid of P. davidi than the external medium for Cape Royds nematodes and nematodes on the surface of MQ agar but lower concentrations on the surface of BSS agar. Element concentrations of pseudocoelomic fluid after 24 h on the surface of MQ agar, BSS agar or BSS agar with chloride salts added to elevate the concentration of the element of interest to 100 or 200 mM are shown in Fig. 6. The concentrations of sodium and potassium in the pseudocoelomic fluid of P. davidi were above that of their external concentrations for MQ agar and BSS agar (Table 1; Fig. 6) but below that of agar containing 100 or 200 mM of the element (Fig. 6). Magnesium was not detected in pseudocoelomic fluid from nematodes exposed to MQ agar or BSS agar but was detected after exposure to agar with a magnesium concentration of 100 or 200 mM, but at a lower level than the external concentration of this element. Calcium does not appear to be taken up into the pseudocoelomic fluid at any external concentration tested. Chlorine concentration in the pseudocoelomic fluid of P. davidi was above that of the external concentration of MQ agar but below that of the other external concentrations tested (Table 1; Fig. 6). The potassium concentration in the pseudocoelomic fluid of P. davidi changes after various times of exposure to agar containing 200 mM potassium (Fig. 7). The concentration in the pseudocoelomic fluid of P. davidi peaks
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J Comp Physiol B (2014) 184:415–423
Fig. 2 Calibration curves for sodium, potassium, magnesium, calcium and chlorine. The Y-axis shows the counts for the element of interest at the level of total counts of 70,000 counts per 100 s, calculated from a regression of counts for the element against total counts for the beads. Number of beads analysed 13–17; error bars are 95 % CI. The regression lines for Mg and Ca exclude the data for 200 mM. The slope of the regression line is significantly different from zero in each case (p
