1
Supporting information
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Experimental procedures – Additional information and preliminary experiments
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Field collection of snails
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Six cross-sectional samples of snails, comprising the species Chlorostoma brunnea, C.
7
montereyi, and Promartynia pulligo, were collected by divers from kelp surfaces at depths of
8
approximately 10 meters (Table S2). Five of these samples were collected from kelp near
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Cambria, California, close to the mouth of Santa Rosa Creek. One sample was collected near
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Carmel, California, close to the mouth of the Carmel River. Three samples were collected when
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the nearby source of freshwater input was flowing (open), and three were collected when the
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nearby freshwater source was closed. The first five snail samples collected were used only for
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the observational component of the present project. Fecal samples collected over three days in
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the laboratory from the last sample of snails (n=81) were screened for T. gondii oocysts as part of
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the observational study, then the snails were used for the exposure experiment as described
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above.
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Detection of T. gondii in snail feces by membrane filtration and microscopy - Preliminary
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experiment
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The concentrations of separate stock suspensions of T. gondii oocysts and surrogate microspheres
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were estimated by enumeration of aliquots from each using a hemacytometer and
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epifluorescence microscopy. An aliquot of each stock suspension was then transferred to a 15 mL
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tube containing 10 mL PBS and the concentrations of these suspensions were estimated by
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vacuum filtration of 100 µL aliquots through 5 µm pore size mixed cellulose membrane filters
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and examination of the filters by epifluorescence microscopy. Aliquots of the working
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suspensions were then transferred to each of eighteen 0.1 mL samples of snail fecal homogenate
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to a final concentration of 2000 oocysts per mL (200 oocysts per sample) of each particle type.
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Nine additional 0.1 mL samples of snail fecal homogenate received no oocysts or microspheres.
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All samples were mixed by vortexing for ten seconds and immediately vacuum filtered through 5
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µm pore membrane filters. These membranes were examined by epifluorescence microscopy and
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the detection sensitivity of this method was estimated as the mean of the ratio of observed
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oocysts or microspheres in each sample to the theoretical number added to each sample (200
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particles of each type). The number of available snail digestive and respiratory organ samples
34
was not sufficient to permit assessment of detection threshold for oocysts or microspheres in
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snail digestive and respiratory organ samples.
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The mean ratio of observed to expected T. gondii oocysts on membrane filters containing
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fecal homogenate was 89/200 (44.6%, sd=13.1), and the mean ratio of observed to expected
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microspheres on the same filters was 181/200 (90.6%, sd=13.6). The difference between these
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means is statistically significant (α=0.05, t = 21.1, p <0.0001). The ratio of observed T. gondii
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oocysts to observed microspheres on those filters is 0.49. No oocysts or microspheres were
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detected in the nine negative control samples.
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43
Optimization of papain digestion for solubilization of snail tissues
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To assess the effect of the cysteine protease papain on the detection and enumeration of T. gondii
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oocysts and surrogate microspheres, nine snail digestive and respiratory organ homogenates were
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harvested from field-collected brown turban snails and stored temporarily in 1.5 mL tubes filled
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with 70% ethanol. Two papain solutions were prepared and activated by adding 0.02 or 0.2
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grams papain and 0.04 grams L-cysteine to 44 mL volumes of 0.05M buffered EDTA (pH 8).
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These solutions were incubated for 20 minutes at 40°C. The snail organ samples were transferred
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to 15 mL conical-bottom tubes, deionized water was added to each tube to a total volume of 4
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mL, and these were homogenized by repeated passage through blunt 15 gauge needles using 12
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mL syringes. Seven of these samples were then spiked with heat-inactivated T. gondii oocysts
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and surrogate microspheres to final concentrations of 375 per mL of each particle type. Aliquots
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of 11 mL of activated papain solution were added to eight of the nine samples, and 11 mL PBS
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was added to the ninth sample (Table A3). All samples were incubated at 65°C for 18 hours, and
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2 mL aliquots were removed from each sample for membrane filtration and microscopy after 1,
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2, 3, 5, 8, 12, and 18 hours of incubation. Mean particle counts for each group were plotted as
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functions of incubation time and a Mann-Kendal test (“mktrend”, Santander Meteorology
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Group), corrected for autocorrelation, in R v. 2.14.0 (R Core Development Team, 2011) was used
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to detect significant trends at the α = 0.05 level.
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Results for the papain digestion experiment are presented graphically in figures A1 and
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A2. Mean oocyst counts peaked after 5 hours incubation for samples treated with 0.033 g per mL
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papain (p = 0.016), and were highest at 18 hours incubation for samples treated with 0.003 g per
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mL papain (p = 0.368). Counts of surrogate microspheres increased steadily to a maximum at 18
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hours incubation for samples treated with 0.033 g per mL papain (p = 0.014), and showed an
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insignificant trend across incubation time points for samples treated with 0.003 g per mL papain
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(p = 0.124). Oocyst and microsphere counts in samples untreated with papain were nearly
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coincident with the curves for the low-papain groups, and did not have significant trends (p =
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0.433 and p = 0.288, respectively). No oocysts or microspheres were detected in unspiked
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negative control samples.
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The relatively flat slopes and insignificant trend scores for mean data from samples
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treated at the lower papain concentration level suggest that this treatment had minimal effect, if
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any, on the oocyst or microsphere counts obtained by membrane filtration and microscopy for
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those samples. Oocysts detected by microscopy in samples treated at the higher papain
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concentration exhibited declining intensity of autofluorescence, and oocyst counts declined, at
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time points beyond 5 hours incubation. The fluorescence of microspheres treated at the higher
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papain concentration did not seem to vary across incubation time points, and counts of surrogate
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in this treatment group increased significantly to a maximum at the maximum incubation time
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(18 hours). These results suggest that the best of the papain treatments evaluated here for snail
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digestive and respiratory organ homogenates is 5 hours incubation with 0.033 g per mL papain.
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Incubation beyond 5 hours increased surrogate microsphere detection, perhaps because it further
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reduced the viscosity and opacity of the snail tissue matrix, but it appeared to compromise oocyst
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detection. The decline in oocyst counts at incubation times beyond 5 hours was likely due to
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declining sensitivity of detection associated with a loss of oocyst autofluorescence.
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Detection of T. gondii in snail feces and organs by nested PCR
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To assess the performance of nested PCR as an alternative primary test to membrane filtration
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and epifluorescence microscopy for T. gondii oocyst detection in snail feces and organ samples,
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four 100 µl aliquots of snail fecal homogenate and four 100 µl aliquots of snail
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digestive/respiratory organ homogenate in 1.5 mL tubes were spiked with 0, 3, 30, or 300 heat-
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inactivated T. gondii oocysts and mixed by vortexing for ten seconds. Each sample was subjected
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to two freeze-thaw cycles in liquid nitrogen and 95°C water followed by column purification
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using the Qiagen DNEasy Blood & Tissue kit (Qiagen cat. no. 69504). All columns were eluted
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in 30 µl 95°C water, and each volume of eluate was divided into three 10 µL aliquots.
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Each aliquot of eluate was allocated to a PCR reaction targeting the B1, ITS1, or 529 bp
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repeat locus (Table A3). Constituents of each PCR reaction were as described above. The final
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amplicon from each reaction was separated from other constituents by electrophoresis in 2%
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agarose gels containing ethidium bromide and visualized by UV transillumination. Positive
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controls were included alongside experimental samples through all processing, amplification,
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and detection steps. Snail feces positive controls were prepared by spiking1000 heat-inactivated
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T. gondii oocysts into 200 µL snail fecal homogenate, and snail organ positive controls were
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prepared by spiking 1000 heat-inactivated T. gondii oocysts into 200 uL organ homogenate.
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Negative controls contained oocyst-free snail fecal homogenate, oocyst-free snail organ
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homogenate, and deionized water.
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The proportion of samples that were PCR positive for T. gondii were higher for fecal
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samples than for tissue samples at every spiking level, and for all primer sets (Table A5). In light
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of these data, PCR was not employed as a secondary test for T. gondii detection in snail organ
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samples collected during the two-week tank exposure experiment.
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Detection of T. gondii on membrane filters by nested PCR - Preliminary experiment
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To assess the performance of nested PCR at the B1, ITS1, and 529 bp repeat loci as a
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confirmatory test secondary to microscopy for detection of T. gondii in snail fecal samples, heat-
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inactivated T. gondii oocysts were spiked into 1 mL aliquots of snail fecal homogenate at three
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concentration levels: 300, 30, and 3 oocyst per mL. These fecal homogenate samples were
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vacuum filtered through 5 µm pore size mixed cellulose ester membrane filters (Millipore, cat.
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no. SMWP02500), which were mounted to glass slides for epifluorescence microscopy. After
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oocyst counts were obtained by microscopy for each membrane filter, acetone dissolution of the
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filter membrane was used to liberate material on the filter prior to DNA extraction as described
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above. The tubes were then flash frozen in liquid nitrogen and rapidly thawed in 95°C water.
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After a second freeze-thaw cycle, DNA within each sample was column purified using the
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Qiagen DNEasy Blood & Tissue kit (Qiagen cat. no. 69504). The columns were eluted with 30
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µL 95°C water, and each sample of eluate was divided into three 10 µL aliquots. Each aliquot
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was allocated to a PCR reaction that employed primers targeting one of three loci: B1, ITS1, or
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the 529 bp repeat locus. Positive controls and negative controls were included alongside
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experimental samples through all processing, amplification, and detection steps. Positive controls
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were prepared by spiking 1000 heat-inactivated T. gondii oocysts into 200 µL snail fecal
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homogenate, and negative controls contained putatively oocyst-free snail fecal homogenate and
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deionized water. Detection of T. gondii DNA by PCR/gel electrophoresis was performed by
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amplification at the B1, ITS1, and 529 bp repeat loci, as described above for unfiltered fecal and
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organ samples.
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During the tank exposure experiment, the quantity of fecal homogenate passed through
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each membrane filter was determined by the quantity of pigmented material it contained.
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Samples were filtered until a given filter contained enough material for efficient counting of
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particles by microscopy, but not enough material to obscure the particles of interest. This
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subjective judgment was made by monitoring the color of each filter during filtration. The
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filtration and microscopy operation continued for each fecal homogenate sample until a
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minimum of five oocysts and five microspheres were detected by microscopy, or until the
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entirety of the sample had been filtered. This experimental design did not guarantee that the
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number of filters examined by microscopy for each sample would be a multiple of three. Also, it
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was apparent from preliminary filtration experiments that homogenized fecal material settled
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rapidly. It was not possible to maintain uniform suspensions that would permit equal allocation
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of suspended material to all filters. Therefore, acetone dissolution was performed separately on
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each filter and DNA extracts were divided into three aliquots and allocated to PCR reactions for
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each primer set. In order to be consistent with the design of the exposure experiment, fecal
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homogenate samples in this preliminary PCR validation were processed the same way.
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Therefore, each PCR reaction contained a theoretical allocation of DNA equivalent to 1/3 of the
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total extracted from the corresponding filter membrane. Consequently, results for PCR and
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microscopy in this experiment are not directly comparable (Table A6).
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Tables
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Table S1: Comparison of the numbers of membranes containing filtered snail fecal material from
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the tank exposure experiment that had detectable Toxoplasma gondii oocysts via epifluorescence
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microscopy or nested PCR at the B1, ITS1, and 529 bp repetitive loci.
Outcomes
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155
Microscopy positive
Microscopy negative
Totals
B1 PCR positive
90
2
92
B1 PCR negative
76
84
160
ITS1 PCR positive
96
4
100
ITS1 PCR negative
70
82
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529 bp repeat PCR positive
70
0
70
529 bp repeat PCR negative
96
86
182
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Table S2: Six cross-sectional samples of snails were collected by divers from kelp surfaces for
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laboratory exposure to Toxoplasma gondii (Sample 6) and for observational studies (Samples 1-
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6).
Sample
1
Sampling
Sample
Nearby Source
Sample
Species
Mean
Date
Source/
of Freshwater
Size
Distribution in
Snail
location
(Open/Closed)
Sample
Mass (g)
Cambria
Santa Rosa
C. brunnea: 7
7.4
Nov. 4th,
2011
2
Apr. 23rd,
Cambria
2012
3
May 3rd,
23
Creek
C. montereyi: 15
(Closed)
P. pulligo: 1
Santa Rosa
31
Creek (Open)
Cambria
2012
Santa Rosa
C. brunnea:14
10.1
C. montereyi: 17
30
Creek (Open)
C. brunnea: 3
11.3
C .montereyi: 26
P. pulligo: 1
4
May 14th,
Cambria
2012
5
Jul. 9th,
Santa Rosa
30
Creek (Open)
Carmel
2012
Carmel River
C. brunnea: 1
9.9
C. montereyi: 29
32
(Closed)
C. brunnea: 22
9.2
C. montereyi: 2
P. pulligo: 8
6
Sept. 11th,
Cambria
2012
Total
--
--
Santa Rosa
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C. brunnea: 37
Creek
C. montereyi: 10
(Closed)
P. pulligo: 34
--
227
C. brunnea: 80
C. montereyi: 98
P. pulligo: 40
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12.8
10.7
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Table S3: Composition of samples in the papain concentration and incubation time experiment.
Sample Sample
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Papain
Oocyst
Surrogate
volume
concentration
concentration (mL-1)
microsphere
(mL)
(g/mL)
concentration (mL-1)
1
15
0.033
100
100
2
15
0.033
100
100
3
15
0.033
100
100
4
15
0.033
0
0
5
15
0.003
100
100
6
15
0.003
100
100
7
15
0.003
100
100
8
15
0.003
0
0
9
15
0
100
100
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Table S4: Sequence data for primers used for detection of Toxoplasma gondii by nested
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polymerase chain reaction (PCR).
Locus
Forward primer sequence (5’ to 3’)
Reverse primer sequence (5’ to 3’)
B1 (external)
TGTTCTGTCCTATCGCAACG
ACGGATGCACTTCCTTTCTG
B1 (internal)
TCTTCCCAGACGTGGATTTC
CTCGACAATACGCTGCTTGA
ITS1 (external)
TACCGATTGAGTGTTCCGGTG
GCAATTCACAATTGCGTTTCGC
ITS1 (internal)
CGTAACAAGGTTTCCGTAGG
TTCATCGTTGCGCGAGCCAAG
529 bp repeat
CGCTGCAGGGAGGAAGACGAAA
CGCTGCAGACACAGTGCATCT
(external)
GTTG
GGATT
529 bp repeat
AGAAGGGACAGAACTCGAAG
CTCCACTCTTCAATTCTCTCC
(internal)
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167
168
Table S5: Detection of Toxoplasma gondii via nested PCR in spiked samples of snail feces and
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organ homogenate.
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Sample matrix
Feces
Feces
Feces
Feces
Primers
B1
B1
B1
B1
Oocysts (theoretical)
100
10
1
0
Proportion positive
5/5
3/5
0/5
0/5
Percent positive
100
60
0
0
Feces
Feces
ITS1
ITS1
100
10
5/5
4/5
100
80
Feces
Feces
ITS1
ITS1
1
0
0/5
0/5
0
0
Feces
Feces
Feces
Feces
529 bp
529 bp
529 bp
529 bp
100
10
1
0
4/5
3/5
0/5
0/5
80
60
0
0
Organs
Organs
Organs
B1
B1
B1
100
10
1
2/5
0/5
0/5
40
0
0
Organs
B1
0
0/5
0
Organs
Organs
Organs
Organs
ITS1
ITS1
ITS1
ITS1
100
10
1
0
2/5
0/5
0/5
0/5
40
0
0
0
Organs
Organs
Organs
Organs
529 bp
529 bp
529 bp
529 bp
100
10
1
0
1/5
0/5
0/5
0/5
20
0
0
0
172
Table S6: Detection of Toxoplasma gondii via nested PCR in spiked samples of snail feces on
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membrane filters.
Method
Oocysts (theoretical)
Proportion positive
Percent positive
PCR: B1
100*
4/5
80
PCR: B1
10**
4/5
80
PCR: B1
1***
0/5
0
PCR: B1
0
0/5
0
PCR: ITS1
100*
5/5
100
PCR: ITS1
10**
4/5
80
PCR: ITS1
1***
0/5
0
PCR: ITS1
0
0/5
0
PCR: 529 bp
100*
3/5
60
PCR: 529 bp
10**
3/5
60
PCR: 529 bp
1***
0/5
0
PCR: 529 bp
0
0/5
0
Microscopy
300
5/5
100
Microscopy
30
5/5
100
Microscopy
3
4/5
80
Microscopy
0
0/5
0
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* Sample received 1/3 of the DNA extracted from a membrane filter containing 300 oocysts
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**Sample received 1/3 of the DNA extracted from a membrane filter containing 30 oocysts
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***Sample received 1/3 of the DNA extracted from a membrane filter containing 3 oocysts
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Figures
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180
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Figure S1: Mean Toxoplasma gondii oocyst counts for samples maintained under specific papain
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concentration and incubation times in preliminary experiments. Data trends for samples treated
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at the lower papain concentration or without papain were not significant (α = 0.05).
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Figure S2: Mean surrogate microsphere counts for samples maintained under specific papain
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concentration and incubation times in preliminary experiments. Data trends for samples treated
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at the lower papain concentration or without papain were not significant (α = 0.05).