DOI: 10.5061/dryad.612jm6420
title: Infectivity of the parasite Metschnikowia bicuspidata is decreased by time spent as a transmission spore, but exposure to phycotoxins in the water column has no effect

description: Transmission from one host to another is a crucial component of parasite
 fitness. For some aquatic parasites, transmission occurs via a free-living
 stage that spends time in the water, awaiting an encounter with a new
 host. These parasite transmission stages can be impacted by biotic and
 abiotic factors that influence the parasite’s ability to successfully
 infect or grow in a new host. Here we tested whether time spent in the
 water column and/or exposure to common cyanobacterial toxins impacted
 parasite transmission stages. More specifically, we tested whether the
 infectivity, within-host growth, and virulence of the fungal parasite
 Metschnikowia bicuspidata changed as a result of time spent in the water
 or from exposure to cyanotoxins in the water column. We exposed parasite
 transmission spores to different levels of one of two ecologically
 important cyanotoxins, microcystin-LR and anatoxin-a, and factorially
 manipulated the amount of time spores were incubated in water. We removed
 the toxins and used those same spores to infect one genotype of the common
 lake zooplankton Daphnia dentifera. We found that cyanotoxins did not
 impact parasite fitness (infection prevalence and spore yield per infected
 host) or virulence (host lifetime reproduction and survivorship) at the
 tested concentrations (10μg/L & 30μg/L). However, we found that
 spending longer as a transmission spore decreased a spore’s chances for
 successful infection: spores that were only incubated for 24 hours
 infected approximately 75% of exposed hosts, whereas spores incubated for
 10 days infected less than 50% of exposed hosts. We also found a negative
 relationship between the final spore yield from infected hosts and the
 proportion of hosts that became infected. In treatments where spores spent
 longer in the water column prior to encountering a host, infection
 prevalence was lower (indicating lower per spore infectivity), but each
 infected host yielded more spores at the end of infection. We hypothesize
 that this pattern may result from intraspecific parasite competition
 within the host. Overall, these results suggest that transmission spores
 of this parasite are not strongly influenced by cyanotoxins in the water
 column, but that other aspects of spending time in the water strongly
 influence parasite fitness.
description: In this study, we used the zooplankton <em>Daphnia
 dentifera</em>, which is common in stratified lakes in temperate
 North America (Tessier & Woodruff, 2002). For our experiments, we
 used the Midland 37 (MID37) genotype, which was isolated from Midland Lake
 in Greene County, Indiana, and has been used in several prior experiments
 (e.g., Auld, Hall & Duffy, 2012; Auld et al., 2014). We also used
 the common fungal parasite <em>Metschnikowia bicuspidata</em>
 (“Standard” isolate, originally isolated from Baker Lake in Barry County,
 Michigan). <em>Daphnia</em> become infected by inadvertently
 consuming transmission spores they encounter in the water column when
 feeding. By “transmission spore”, we refer to the mature, needle-shaped
 ascus that contains the ascospore (Metschnikoff, 1884; Codreanu &
 Codreanu-Balcescu, 1981). After consumption by the host, infection can
 begin if the needle-shaped spore crosses the gut barrier and is not fought
 off by a host hemocyte response (Metschnikoff, 1884; Stewart Merrill
 & Cáceres, 2018). Once infection has taken hold, the fungus
 replicates within the hemolymph of the host (Stewart Merrill &
 Cáceres, 2018). The parasite reduces the fecundity and lifespan of
 infected hosts (Auld et al., 2012). <em>Metschnikowia</em> is
 an obligate killer, meaning it must kill its host in order to transmit to
 a new host (Ebert, 2005); transmission spores are released into the
 environment after host death, after which they can be consumed by a new
 host, completing the parasite’s life cycle. The
 experiment was run in two blocks designed to address different ecological
 and methodological questions. The first experiment addressed the main
 ecological question posed in the introduction of this manuscript. Other
 studies have reported adsorption of microcystins by plastics (Hyenstrand
 et al., 2001; Moura et al., 2022), which raised the possibility that the
 results of our first experiment may have been due to toxin concentrations
 that were lower than we intended. Therefore, in the second experiment, we
 evaluated whether the plastic vessels we used in the first block adsorbed
 microcystin-LR from the water and whether using glass vs. plastic vessels
 for the incubations impacted infections. In the first
 experiment, we incubated fungal transmission spores for different lengths
 of time in filtered lake water (Pall AE filters, 1 µm pore size). The
 incubation times were 24 hours, 3 days, 5 days, 7 days, and 10 days. We
 also added two common cyanobacterial toxins, microcystin-LR and
 anatoxin-a, to the water during the incubations. We chose these
 cyanotoxins because they are commonly produced during blooms (Huisman et
 al., 2018) and because prior research suggested that they reduced
 infection prevalence (in the case of microcystin-LR) or that production of
 them can increase in the presence of <em>Metschnikowia</em>
 (in the case of anatoxin; Sánchez et al., 2019). Microcystin-LR is
 produced by some members of the genus <em>Microcystis</em>,
 which has been extensively studied due to concerns over its geographical
 expansion and capability of producing CHABs in both marine and freshwater
 ecosystems (Huisman et al., 2018). Anatoxin-a is produced by some members
 of the genus <em>Anabaena</em>. Microcystin-LR is a
 hepatotoxin while anatoxin-a is considered a potent neurotoxin in
 vertebrate models (Christensen & Khan, 2020). Both toxins are also
 produced by other genera of cyanobacteria such as
 <em>Plantktothrix</em>, <em>Oscillatoria, Aphanizomenon,
 Cylindrospermum</em>, and <em>Dolichospermum</em>, all
 capable of producing CHABs (Huisman et al., 2018). We also included two
 types of negative controls: a solvent control of 0.01% acetic acid (see
 toxin preparation for explanation) and a negative control with no toxin or
 solvent added. The incubation times were crossed factorially with the
 toxin treatments, as described below. After the appropriate incubation
 time, we carried out infection assays in which we exposed
 <em>Daphnia</em> hosts to these spores and measured infection,
 spore production, host reproduction, and host mortality over
 time. <em><strong>Toxin
 preparation</strong></em> Pure
 microcystin-LR standard was purchased from Cayman Chemical (Ann Arbor, MI)
 and suspended in 1mL of nano-pure water for a concentration of 1 mg/mL.
 Anatoxin-a standard was acquired from Abraxis (Warminster, PA). The
 anatoxin-a comes in a solution of 3:1 water and methanol and 0.01% acetic
 acid. We placed 1.5 mL of the anatoxin solution in a 2 mL Eppendorf tube
 and evaporated the methanol using an Eppendorf Vacufuge (Eppendorf,
 Hamburg, Germany) at 23 °C. Once evaporation had occurred, we added
 nanopure water to restore to the original volume/concentration. Toxins
 were frozen in between uses during the exposure period.
 <em><strong>Spore
 preparation</strong></em> Spores for the
 experiment were grown in vivo by infection of <em>Daphnia
 dentifera</em> (“Standard” genotype) in the laboratory. Infected
 animals with well-developed late-stage infections were collected from
 laboratory cultures and placed in 2mL Eppendorf tubes with 100-500 uL of
 milliQ water, then stored in the refrigerator at 4 °C. For this
 experiment, we used spores from animals that had been stored in the
 refrigerator for 2 weeks. To generate the spore slurry for experimental
 infections, we crushed infected animals to release spores, and then
 determined the density of mature ascospores using a hemocytometer (Hausser
 Scientific 3100, Horsham, PA, USA) and a compound microscope (Olympus
 BX53, Center Valley, PA, USA) at 400X magnification. Each incubation
 treatment was initiated with a new spore slurry (made from infected
 animals that were harvested live from the laboratory cultures, then stored
 in the refrigerator for 2 weeks) because we know that
 <em>Metschnikowia</em> spores lose infectivity over time, even
 in the refrigerator (Duffy & Hunsberger, 2019).
 <em><strong>Toxin exposure to transmission spores
 </strong></em> On the first day of the
 experiment (“day 1”), we initiated the longest incubation treatment (10
 days) by placing 5000 mature transmission spores of
 <em>Metschnikowia bicuspidata</em> in 15 mL Falcon tubes
 filled with 10 mL of lake water that had been filtered through a Pall AE
 filter (Pall Corporation, Port Washington, NY). We then added
 microcystin-LR, anatoxin-a, acetic acid (negative control), or no
 toxin/chemical; for both microcystin-LR and anatoxin-a, we had two toxin
 levels: 10 and 30 µg/L. The concentrations we chose for this study are
 below and/or well within the range observed during natural CHABs blooms
 (Park et al., 1998; Pawlik-Skowrońska et al., 2004; Ibelings et al., 2005;
 Ha, Hidaka & Tsuno, 2009), and therefore ecologically relevant.
 These treatment doses are below the LC50s reported in previous
 <em>Daphnia</em> toxicology experiments for microcystins and
 anatoxin-a (DeMott, Zhang & Carmichael, 1991; Pawlik-Skowrońska,
 Toporowska & Mazur-Marzec, 2019). Even though we did not expose
 hosts to these toxins, we chose these concentrations because they should
 not cause high levels of stress and mortality in
 <em>Daphnia</em>, so any impact to pathogens could have a
 substantial impact on parasite-host interactions. While these
 concentrations are likely on the high side of what spores are likely to
 encounter in nature, if there is no impact of the cyanotoxins at these
 levels, it suggests that they are unlikely to significantly impact the
 free-living stages of this parasite in the water column. There were 10
 replicates of each treatment (including the negative control treatment of
 acetic acid), with the exception of the no toxin controls (0 µg/L), which
 had 20 replicates; this yielded a total of 70 experimental units per
 incubation time treatment. The tubes with spores were left uncapped for 10
 days inside a large plastic tote covered with a lid at 20 °C with a 16:8
 L:D photoperiod. On day 4, the same procedure from day 1 was repeated. In
 this treatment, spores were incubated for 7 days at 20 °C with a 16:8 L:D
 photoperiod, yielding the 7-day incubation treatment. On day 6, the same
 procedure was used to initiate the 5-day incubation treatment.
 Additionally, on this day, adult <em>Daphnia</em> were set up
 so that we could use their offspring in the infection assays. Adult
 <em>Daphnia</em> of the MID37 genotype were placed in 150 mL
 beakers (5 adults per beaker) filled with 100 mL of filtered lake water.
 Beakers were placed in incubators at 20 °C with a 16:8 L:D photoperiod for
 24 hours. On day 7, we collected neonates (0-24 hours old) produced from
 mothers that had been set up the previous day. We placed 10 neonates per
 beaker in 250 mL beakers with 150 mL filtered lake water for a total of
 400 animals. Each beaker received 2 mg C per L of <em>Ankistrodesmus
 falcatus</em> food and was placed in incubators at 20 °C with a 16:8
 L:D photoperiod. After that, 2 mg C per mL of
 <em>Ankistrodesmus</em> was added to each beaker each day
 until the day the experimental animals were exposed to parasites. On days
 8 and 10, we used the same procedure as described above to initiate the 3-
 and 1-day incubation treatments, respectively.
 <strong><em>Infection
 assays</em></strong> On day 11, we carried
 out infection assays, in which we exposed <em>Daphnia</em> to
 transmission spores that had been incubated for different time lengths and
 with varying levels of exposure to toxins/chemicals. All Falcon tubes,
 containing spores with different exposure times and toxin levels, were
 collected and placed in a centrifuge (Sorvall ST 16, Thermo Scientific,
 Waltham, MA, USA) to spin down the spores. The tubes were spun at 3000 rpm
 for 10 min. We decanted the tubes using a 10mL pipette without disrupting
 the spore pellet at the bottom. In a pilot experiment, we confirmed that
 the original concentration of spores was recovered from vials after a
 24-hour incubation followed by centrifugation. After the water was removed
 (which also removed the toxin), we resuspended the spores by adding 10 mL
 of filtered lake water and disturbing the pellet by vigorously pipetting
 the water in the tube. Then, we placed one 6-7-day old
 <em>Daphnia</em> in each Falcon tube and allowed the tubes to
 incubate again at 20 °C with 16:8 L:D photoperiod. Because we had
 originally placed 5000 mature transmission spores in each tube, this
 yielded an exposure dose of 500 spores/mL. Hosts were fed 1 mg C per mL of
 <em>Ankistrodesmus</em> on this day; using this lower level of
 food on the day of exposure is common in infection assays because it
 promotes infection. After 24 hours (that is, on day 12), each
 <em>Daphnia</em> individual was removed from the tubes with
 spores and placed in a 50 mL beaker filled with 30 mL filtered lake water
 that did not contain spores (one animal per beaker). Animals were fed 2
 mg/L C of <em>Ankistrodesmus falcatus ad libidum</em> for the
 rest of the experiment (20 days post-infection, 32 days from day 1 of the
 entire experiment). During those 20 days, we tracked
 mortality in each of the beakers five days per week (Mondays through
 Fridays). Water changes were done twice a week; during these, we counted
 offspring in each beaker; offspring were removed from beakers and
 discarded. Any animals that died during the trial were placed in 2 mL
 Eppendorf tubes with 100 µL of nanopure water and stored at 4 °C for later
 spore counts. At the end of the experiment (20 days post-infection, 32
 days from day 1 of the entire experiment), any remaining live animals (130
 total) were placed in a 2mL Eppendorf tube with 100 µL of nanopure water.
 Animals were then ground to release spores, and spores in the ascus stage
 were counted using a hemocytometer under a compound microscope at 400X
 magnification. In the second experiment, we incubated
 fungal transmission spores using glass conical tubes and plastic falcon
 tubes. For this experiment, we used 24-hour & 3-day incubation
 times. The primary concern that prompted this experiment is that, in a
 prior study, microcystin-LR was found to adsorb to plastic (Hyenstrand et
 al., 2001). Therefore, in this experiment, we focused on microcystin-LR,
 using the same concentrations as in the first experiment: 30 µg/L, 10 µg/L
 and a control with no toxin. In total there were nine treatments in this
 experiment (Figure S1). There were three microcystin-LR concentrations and
 two incubation lengths, crossed factorially. Most of the incubations were
 done in glass tubes. In addition, for the 24-hr incubation lengths in the
 30 µg/L microcystin treatment, we also factorially crossed vessel type
 (glass vs. plastic tubes, so that we could test for an effect of vessel
 type) and presence/absence of spores (in order to compare the level of
 microcystin-LR in glass vs. plastic tubes, which was done using water from
 the spore-free incubations).  This second experiment
 was run in a similar manner to the first experiment. Spores were incubated
 with their corresponding toxin treatment for 24 hours or 3 days. We
 focused on these shorter incubation treatments for the following reasons.
 First, in the earlier study, the authors detected adsorption rapidly
 (within seconds); thus, this should have been ample time for adsorption to
 occur. Second, if microcystin-LR adsorbed to the plastic we used in our
 experiment and reduces infectivity, we would expect to see a reduction in
 the infectiousness of spores incubated in glass vs. plastic. Thus, we
 would best be able to detect this at the incubation lengths that had
 yielded the highest infection levels (24 hours and 3 days).
 Treatments with spores had spores added to them in a similar
 manner to what was described for the first experiment; for treatments with
 no spores, we added toxin to the appropriate vessel with filtered lake
 water but no spores. After the incubation period had elapsed, we
 centrifuged tubes to spin spores down to the bottom of the tubes; for
 consistency, we spun all tubes, regardless of whether they were from a
 treatment with spores present or absent. After centrifugation, we
 collected 1mL water sample from each tube; we placed these samples in 2mL
 glass vials and stored them at -80F for later quantification of toxin
 concentration in the water. The rest of the water was decanted without
 disturbing the spore pellet in the bottom. After spores were resuspended
 in new filtered lake water, 6–7-day old <em>Daphnia dentifera
 </em>(MID37 clone) were placed in each tube and allowed to incubate
 with spores (or no spores) for 24 hours to allow time for infection. After
 24 hours, all animals were removed from tubes and placed in 50mL beakers
 with 30mL of filtered lake water. Animals were then fed ad-libitum for 20
 days. Animals that died within 20 days of exposure were preserved in 2mL
 Eppendorf tubes with 100 µL of milliQ water and stored at 4C. At 20 days
 post-exposure, any remaining live animals were placed in a 2mL Eppendorf
 tube with 100 µL of nanopure water, then stored at 4C. Later, animals were
 removed from cold storage and ground to release spores; spores in the
 ascus stage were counted using a hemocytometer under a compound microscope
 at 400X magnification. <em><strong>Data
 analysis </strong></em> For all our models,
 we analyzed data from the microcystin-LR and anatoxin-a treatments
 separately. This means that for each analysis described below, there was
 one performed for the microcystin-LR relevant data and another for the
 anatoxin-a data. The same no-toxin control data (0 µg/L; 20 replicates per
 incubation time) were used for the two sets of data (microcystin-LR and
 anatoxin-a). When analyzing the anatoxin-a data, the acetic acid
 treatments were included in the analyses (as an additional negative
 control) and were treated in our analyses as a low concentration (0.01%);
 statistical analysis did not find differences between the no toxin
 controls and the acetic acid controls (data not shown). Prior to analyses,
 the data were checked for normality using the Shapiro-Wilk test. Data that
 did not meet normality were analyzed using appropriate family error
 distribution link functions and checking for overdispersion. All analyses
 were carried out in R Studio Version 1.2.1335 using the stats v3.6.1
 package. We assessed environmental effects on the
 parasite’s ability to infect by comparing differences in the number of
 animals that developed terminal infections (that is, infections that
 produced asci (Stewart Merrill & Cáceres, 2018)). In this
 analysis, our response variable had two outcomes (terminal infection or
 not). We performed a generalized linear model (GLM) with incubation time
 and toxin concentration as explanatory variables, using a binomial family
 error distribution. We also evaluated if toxin
 concentration and incubation time (that is, the time spent as a
 transmission spore in the water) affected the number of mature
 transmission spores produced per infected host individual. For this
 analysis, we ran a GLM; here the number of spores was our response
 variable and, similarly to the analysis of infections, toxin concentration
 and incubation time were used as the explanatory variables. In this model,
 we used a Gaussian error distribution. To evaluate the
 effects of incubation time and toxin exposure on the parasite’s virulence,
 we measured host offspring production, host survival over the 20-day
 experiment, and the proportion of hosts who died within 20 days of
 parasite exposure. For these analyses, we ran GLMs with toxin
 concentration and time of exposure as explanatory variables; for the two
 former response variables—the number of offspring each host individual
 produced and the number of days each host survived (up to 20 days
 post-infection)—we used a Poisson family error distribution. For the
 proportion of dead hosts at the end of the experiment, we used a binomial
 family distribution. We ran two linear models analyzing
 the relationship between the proportion of infected individuals and the
 mean number of spores produced at the end of infection. In the first, we
 averaged across the different toxin exposure treatments, yielding one
 value for each incubation time (n = 5). In the second, we averaged within
 the toxin treatments, yielding five values per toxin treatment (one per
 incubation time treatment, total n = 20). For the
 second set of experiments, we evaluated the data in two ways. First, we
 evaluated the same response variables as explained above for the first
 experiment and evaluated whether the results were qualitatively similar or
 different. Second, we merged the data sets from the first and second
 experiments, using data only from the microcystin treatments, and, in the
 case of the data from the second experiment, we used both plastic and
 glass data adding "vessel" as part of the model. We then used a
 generalized linear mixed model (GLMM) with the same set-up as the first
 experiment in terms of response and treatment variables and using “first”
 or “second” experiment as a random block variable.
 Finally, using data from the second experiment, we also evaluated
 whether dissolved toxin concentration was different when using different
 exposure vessels after 24 hours by bootstrapping, and whether there were
 differences in infection prevalence, spore production and offspring
 reproduction when using different vessels for the exposure of spores. More
 details on these analyses and results can be found in the supplement.
 Results were qualitatively the same regardless of whether exposures were
 done in glass vs. plastic and toxin levels did not significantly differ
 between the two vessel types (see supplement), leading us to conclude that
 the effects seen in our first experiment were not altered by the use of
 plastic tubes. Therefore, in the results in the main text, we present only
 the results from experiment 1, because this had all incubation lengths and
 both toxins. 
description: File can be open with Microsoft Excel or any software capable of
 opening excel or csv files. Data files contain a notes column for special
 cases in particular data points.