Sample collection, shipment and handling
Water samples were collected by personnel from the Salton Sea Wildlife Refuge, as defined by the needs of the project, the work schedule for the Salton Sea Wildlife Refuge personnel and at times weather and other logistical conditions. Collections started in November 1999 and ended in April 2001. Sampling dates varied but samples were received from every month except May, June and July 2000. Twenty shipments were received over this 18-month period. Fifteen of the shipments contained water and plankton samples while six contained grebe tissue samples (one shipment contained both water and tissue samples). Grebe necropsies and tissue shipments were done by SS Refuge personnel and personnel at the National Wildlife Health Center-USGS in Madison, Wisconsin (Chris Franson) after collection by Salton Sea personnel. Control grebe tissue samples were arranged by Chris Franson and were collected at Mono Lake California during October 2000.
Sample Kits used to collect and ship the toxigenic algae samples, contained:
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(1) shipping cooler
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large liner bag (for lining inside of cooler) and cable-tie for securing it closed
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500 ml-Nalgene sample bottles
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zip-lock bag (for enclosing sample report form)
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ice-pack
Sample collection locations
During the time that shipments 1–3 were made a transect system was used to collect samples. These transects were set by Salton Sea personnel and covered river inlets, open waters and near shore areas. Later it was determined that most phytoplankton was to be found near areas of river inlets where salinity was lowest. This was also the areas where bird mortalities were typically highest. This resulted in 4 sample sites: A=Alamo River, N=New River, O=open water and 1 location in the north, W=Whitewater River. Figure 5 gives the overall view of sample sites on the Salton Sea for this project.
Sample receipt and processing
1. Samples were logged in and then stored at 4°C for phytoplankton analyses and at -80°C in preparation for lyophilization, extraction and toxin analyses. A typical treatment regime for these samples was as follows: 1) Samples were logged in and sample location and conditions were noted. 2) Samples were split, with most of the sample being lyophilized and a small amount kept back for phytoplankton examination and identification of major cyanobacteria present. 3) Since most of the dry weight of a sample was salt, only visible green or green brown samples were used for microcystin analyses. 4) All samples that had identifiable cyanobacteria in them, by microscopic observation, were placed into either tubes with liquid culture (CT medium plus salt) or onto agar plates containing CT medium plus salts. Other cyanobacteria growth media were tested initially including BG-11, ASM-1 and Z-8 but the CT media plus salt was found to give the best overall growth results.
Taxonomy of Salton Sea cyanobacteria isolates
Cultured isolates were examined on a Nikon Optiphot phase microscope with phase and fluorescence optics. Taxonomy, based on morphological characters, was determined from reference to Komarek and Anagnostidis [18], Komarek and Anagnostidis [19], Desikachary [20] and Carpenter [21].
Culture of laboratory isolates
Isolates were made by streaking water or sediment sample on plates composed of CT+ medium and agar. Colonies were visually grouped by observation with a dissecting scope and 2–5 representatives of each type of colony were lifted from a given plate via micropipette and transferred to liquid CT+ medium in a 10 ml test tube. From the test tube cultures, representatives of each type were transferred to a 4L flask of CT+, aerated and maintained under 24 hour illumination. Cultures were harvested before senescence, spun down in a Sorvall centrifuge at 5000 rpm, the medium was poured off and the pellet was rinsed with dionized water to help remove salt. The pellet was transferred to a stainless steel pan, frozen and subsequently freeze-dried. CT medium content is as given by Watanabe and Ichimura [22]. The + refers to the addition of NaCl to the medium (7 g/L).
Detection of microcystins
The most common of the cyanotoxins, likely to be found in this study, are the cyclic peptide hepatotoxic microcystins and nodularins. Rapid and sensitive methods now exist for detecting and monitoring these toxins in environmental samples (water, cells, sediment and animal tissue). This includes a sensitive polyclonal antibody immunoassay, developed by FS Chu at the Univ. of Wisconsin, and adapted by An and Carmichael [23] and Carmichael and An [24]. This enzyme-linked immunosorbant assay (ELISA) is sensitive to ppb and the antibody cross reacts with most of the known microcystins. This chemical assay is complimented by a colorimetric enzyme activity assay [23] that measures the inhibition of microcystin against protein phosphatase 1 and 2A. Inhibition of PP1 and 2A is the specific mechanism of action for microcystins and is directly related to microcystins toxicity. These two assays can be used to monitor and quantitate microcystins in all the various studies outlined in this proposal.
ELISA assay for the cyclic peptide microcystins (MCYST) and nodularin (NODLN). The method is based upon the polyclonal antibody method described by Chu et al. 1989, 1990 and as adapted by An and Carmichael [23]and Carmichael and An [24]. The level of sensitivity for microcystin/nodularin using this method is about 0.5 ng/ml. Values below or near this level are not considered significant. Fifty microliters (50 μl) of sample containing 500 μg of algae is used for the assay. Serial dilutions of 10-1-10-4 (in duplicate) are used to run the assay.
Samples are run on a PP1 or 2A inhibition assay. The cyclic peptide liver toxins, microcystin and nodularin, have been shown to be specific and potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). Inhibition of these enzymes has been shown to be correlated with the ability of these toxins to be tumor promoters, especially liver tumor promotion. The assay is therefore useful in combination with the ELISA assay (which tests for presence of the compounds – not all of which are bioactive) as an activity assay (to measure actual toxic effect). The assay is about 1000 times more sensitive than the HPLC or mouse bioassay. Assay of PP activity was done by measuring the rate of color formation from the liberation of P-nitrophenol from P-nitrophenol phosphate using a Molecular Devices Corp., Vmax kinetic microplate reader, Palo Alto, CA. [25].
Preparation of samples for ELISA and LC/MS
Microcystin analysis
Freeze dried cells were extracted in methanol at a ratio of 1 g dry weight cells to 50 mL methanol. Extracts were sonicated for 30 s and placed on a rotating table overnight. After filtration through a glass fiber filter (1.6 μm pore size), the supernatants were evaporated to dryness in a Speedvac. Extracts were resuspended in 5 mL of reagent grade water and passed through a solid phase extraction column (Isolute, IST, Glamorgan, UK) containing 500 mg C18(EC). The column was washed with 5 mL of 20% (v/v) methanol and microcystins were eluted with 10 mL of 80% (v/v) methanol. The later fraction was evaporated to dryness in a Speedvac, resuspended in 1 mL of 10% (v/v) methanol, and subjected to ELISA and LC/MS analysis.
Extraction of tissues
Liver or intestine samples (0.5–1.0 g) were homogenized in 10 mL of hexane (Power Gen 125 tissue homogenizer, Fisher Scientific Pennsylvania, USA) at 15000 rpm using a 7 mm saw tooth generator probe (Fisher Scientific, Pennsylvania, USA). After homogenization approximately 1 ml pf ethanol was added to the sample to break up emulsion formation. The sample was slowly shaken for about 0.5 hr on a orbital shaker. After this time the hexane layer was removed, dried with a stream of air or nitrogen and reconstituted in 0.5 ml of 35% MeOH. Preparation of this sample for separation of the microcystin-containing fraction was by Isolute C18 packing/3 ml reservoir cartridge. The 100% MeOH fraction from this cartridge was dried under a stream of air and reconstituted in 1 ml of 5% MeOH. This fraction was used in the ELISA and LC/MS analyses [26].
LC/ESI-MS conditions
Column: MetaChem Monochrom C18, 2 × 50 mm, 5 micron particle size
Mobile Phase: A) 0.1% formic acid in water B) 0.1% formic acid in acetonitrile
Gradient: 25 % B to 50% B in 5 minutes, with first minute diverted to waste
(Purge 20 column volumes with 50% B at end of run; equilibrate with 20 column volumes prior to run)
Temperature: 35 deg C (column heater to stabilize temperature)
Flow: 0.25 mL/min
Injection Volume: 20 μl
Selected Reaction Monitoring (MS/MS) Scan Experiments (minimum 3 replicates)
Limit of Detection: 300–500 pg (on column)
Limit of Quantification: 0.5–1.0 ng (on column); high ppm (ng/g -ug/g) for samples; precision ≤ 15% (trace analysis for tissue samples); ≤ 5% for algae samples
Extraction Efficiency (SPE sample prep): 90%
Ionization Suppression from Tissue Matrix: 35–40% suppression in signal response
Combined reduction in recovery/response: 50%
Expected Weight of Tissue Samples: 1–2 gr
Baseline resolution of analytes, RT repeatability +/- 0.5%
Solid phase extraction/sample preparation
Function:
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removal of interferences and column killers
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desalting
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concentration or trace enrichment of analyte
Capacity of SPE cartridge: 10–20 mg analyte+interferences/g sorbent
SPE Cartridge Volume: 1 μl solvent/1 mg sorbent
PCR of cyanobacteria isolates
DNA extraction
Ten mg of lyophilized or fresh cells (harvested at exponential phase and washed three times with distilled water) were mixed with microbeads in a 2 mL screwtop polypropylene vial (1:1 with volume), and broken with a Mini-Beadbeater (Biospec Products, USA) at 5000 rpm for 1 min, The solution was then suspended in 0.5 ml of a lysing solution containing archromopeptidase 0.5 mg + lysozyme 0.75 mg/mL of 10 mM Tris-HCl buffer, pH 8.0. The samples were incubated at 37°C for 30 min. Fifty μl of 10% Tris-SDS solution (SDS in 1 M Tris, w/v) was added and the solution was well mixed. Samples were then incubated at 60°C for 5 min. Lysates were then extracted twice with 0.2 mL of water-saturated phenol and 0.2 mL of chloroform. After centrifugation (15,000 rpm, 10 min) the upper layer was removed and 50 μL of RNase solution (RNase 1 mg + RNase T1 400 units)/mL of 50 mM Tris-HCl, pH 7.5 was added. This was kept at 37°C for 20 min. This was followed by a treatment with 50 μL of proteinase K solution (Proteinase K (Sigma), 4 mg/mL in 50 mM Tris-HCl, pH 7.5) at 37°C for 10 min. The samples were treated again with 0.2 mL of phenol solution plus 0.2 mL of chloroform, and centrifuged at 15, 000 rpm for 10 min. DNA was precipitated with 0.1 volume of 3 M NaOAC and 2.5 volumes of cold 100% ethanol. Precipitated DNA was pelleted by centrifugation for 15 min at 15,000 rpm, washed with 70% ethanol, 100% ethanol, dried and stored at -20°C.
Primer designation and polymerase chain reaction amplification
Based on cyanobacterial 16S rRNA gene sequences the two primers for amplification and sequence were; F1 (5' TAACACATGCAAGTCGAA3'), and newly designed R4N(5' CCTACCTTAGGCATCCCC 3'). The latter has a sequence showing high specificity to the family Nostocaceae, which was checked using a BLAST database search [26]. Polymerase chain reaction (PCR) amplification was done in a 80 μl reaction mixture using 10–20 ng genomic DNA, 0.05 units/μl Ampli Taq DNA polymerase, 10 × buffer containing 1.5 mM MgCl2, 0.2 mM dNTPs, and 0.05 μM of primers. The reaction was run in a Techne Thermal Cycler (Progene, UK) with one cycle of 94°C for 5 min.; 30 cycles of 94°C for 30s, 50°C for 30s, 70°C for 1 min, and finally 72°C for 3 min.
Sequence analysis
PCR products were purified by applying the QIA quick DNA Remove Kit (QIAGEN, USA). This was used as the template in sequencing reactions using an Applied Biosystems; PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit supplied with Ampli Taq DNA polymerase. The primers used for the sequencing reaction were the same as for amplification. Products of sequencing reactions were analyzed on an Applied Biosystem 310 DNA sequencer.
Alignment and phylogenetic analyses
Cyanobacterial 16S rRNA gene sequences available from GenBank and those found in the present study were aligned using CLUSTAL W version 1.6 [27]. This was followed by conversion to a distance matrix. The distance matrix was converted to a phylogenetic tree using the neighbor-joining (NJ) algorithm of CLUSTAL W version 1.6, with multiple substitutions corrected and positions with gaps excluded. The seed number for random number generation and number of bootstrap trials were set to 111 and 1000, respectively.
