DOI: 10.5061/dryad.nzs7h44xq
title: Inhibition of gut digestive proteases by cyanobacterial diets decreases infection in a Daphnia host-parasite system

description: Secondary metabolites produced by primary producers have a wide range of
 functions as well as indirect effects outside the scope of their direct
 target. Research suggests that protease inhibitors produced by
 cyanobacteria influence grazing by herbivores and may also protect against
 parasites of cyanobacteria. In this study we asked whether those same
 protease inhibitors produced by cyanobacteria also can influence
 interactions of herbivores with their parasites.  We used
 the Daphnia-Metschnikowia zooplankton host-fungal parasite system
 to address this question because it is well-documented that cyanobacteria
 protease inhibitors suppress trypsin and chymotrypsin in the gut of
 Daphnia, and because it is known that Metschnikowia infects via the gut.
 We tested the hypothesis that Daphnia gut proteases are necessary for
 Metschnikowia spores to be released from their asci. We then also tested
 whether diets that decrease trypsin and chymotrypsin activity in the guts
 of Daphnia lead to lower levels of infection. Our results show that
 chymotrypsin promotes release of the fungal spores from their asci.
 Moreover, a diet that strongly inhibited chymotrypsin activity
 in Daphnia decreased infection levels, particularly in the most
 susceptible Daphnia clones. Our results support the growing literature
 that cyanobacterial diets can be beneficial to zooplankton hosts when
 challenged by parasites and uncover a mechanism that contributes to the
 protective effect of cyanobacterial diets. Specifically, we demonstrate
 that host chymotrypsin enzymes promote dehiscence
 of Metschnikowia spores; when cyanobacteria inhibit activity of
 chymotrypsin in hosts, this most likely traps the spore inside the ascus,
 preventing the parasite from puncturing the gut and beginning the
 infection process, and reduced the proportion of Daphnia infected. This
 study illustrates how secondary metabolites of phytoplankton can protect
 herbivores against their own enemies.
description: <em>Host-parasite system</em> Our
 study used 11 clones of <em>Daphnia magna, </em>which is a
 common species in ponds and lakes, especially in Europe. These clones come
 from different populations and have been used in studies focusing on the
 impact of cyanobacterial diets on <em>Daphnia</em> gut
 digestive enzymes (e.g., Schwarzenberger <em>et al.</em>,
 2021; see supplementary material and Table S1 for additional information
 about these clones). We 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 after consuming transmission
 spores they encounter in the water column when feeding. Infection takes
 place when the needle-shaped spore crosses the gut barrier and is not
 fought off by a host hemocyte response (Metschnikoff, 1884; Stewart
 Merrill and Cáceres, 2018). Once infection has taken hold, the fungus
 replicates within the hemolymph of the host (Stewart Merrill and Cáceres,
 2018). The parasite reduces the fecundity and lifespan of infected hosts
 (Auld, Hall, and Duffy, 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.
 <em>Cultivation and preparation of phytoplankton
 food</em> We cultivated five strains of
 phytoplankton: one green alga and four cyanobacteria. We obtained the
 green alga <em>Scenedesmus obliquus</em> UTEX 3155 from the
 Culture Collection of Algae at the University of Texas at Austin. The
 cyanobacteria <em>Microcystis aeruginosa</em> strain CYA160/1
 and strain CYA43 came from the Norwegian Institute for Water Research.
 Finally, the <em>Microcystis aeruginosa</em> strains wild type
 (WT) PCC7806 and mutant (MT) PCC7806 -mycB were obtained from the Pasteur
 Culture Collection of Cyanobacteria at the Institute Pasteur. The
 <em>Microcystis</em> strains used here vary in their
 production of microcystin and also their protease inhibitors (some
 effective at inhibiting chymotrypsin and others at inhibiting trypsin in
 the gut of <em>Daphnia</em> (Agrawal <em>et
 al.</em>, 2005; Schwarzenberger, Ilić and Von Elert, 2021)). We used
 the CYA43 strain of <em>Microcystis</em> because it strongly
 inhibits chymotrypsin activity in <em>D. magna</em> (von
 Elert, Zitt and Schwarzenberger, 2012), but does not produce microcystins.
 We used the CYA160 strain because a prior study found it protected
 <em>Daphnia dentifera</em> against infection by
 <em>Metschnikowia</em>, yielding much lower levels of
 infection than <em>Scenedesmus</em> (Sánchez <em>et
 al.</em>, 2019). We used the PCC7806 and PCC7806-mycB strains to
 allow us to explore variation among <em>Microcystis
 </em>strains in their effects on the infection process and to
 isolate whether there is an effect of microcystin, since PCC7806-mycB is a
 microcystin deficient mutant of the wild type strain (Dittmann
 <em>et al.</em>, 1997); hereafter, we refer to these strains
 as PCC7806-WT (for wild type) and PCC7806-MT (for the
 microcystin-deficient mutant type). We grew all
 cultures in chemostats with 24-hour light.
 <em>Scenedesmus</em> was grown in standard COMBO media,
 <em>Microcystis</em> CYA43 and CYA160 were grown in Z8 media,
 <em>Microcystis</em> PCC7806-WT in BG11 media, and
 <em>Microcystis</em> PCC7806-MT was grown in BG11 media with
 5µg/mL chloramphenicol (which is necessary to keep microcystin genes
 inactivated; (Dittmann <em>et al.</em>, 1997)). Food for
 experiments was prepared weekly. We harvested cells in 50 mL Falcon tubes
 and centrifuged (Sorvall St 16, ThermoScientific) for 10 min at 3000 rpm.
 After, we decanted media from the tubes and resuspended cells in ADaM
 (Klüttgen <em>et al.</em>, 1994). The volume was adjusted to a
 concentration of 100 mg C/L and this solution was used to feed animals a
 final concentration of 2 mg C/L. <em>Microcystis</em>
 PCC7806-MT was rinsed and spun down twice with milliQ water before
 resuspending in ADaM to rinse out any chloramphenicol that may have been
 left from growth media. <em>Experiment 1.
 Dehiscence assay: isolating the impact of gut extracts and chymotrypsin on
 spore dehiscence.</em> To test whether diet
 impacted dehiscence of <em>Metschnikowia</em> spores, we
 exposed them to extracts from the guts of animals that were fed
 <em>Scenedesmus</em> or from the guts of animals that were fed
 <em>Microcystis </em>diets that are known to inhibit
 chymotrypsin activity in the gut. If chymotrypsin promotes spore
 dehiscence, we expected higher levels of dehiscence for spores exposed to
 gut extracts from <em>Scenedesmus </em>(which should have
 normal chymotrypsin activity) than from <em>Microcystis</em>.
 To further test the impact of chymotrypsin, we also added pure
 chymotrypsin (CAS 9004-07-3, Sigma-Aldrich) to the gut extracts of animals
 fed <em>Microcystis</em>, to see if we could recover the
 levels of dehiscence seen for spores exposed to gut extracts from animals
 fed green algae. This assay was done using extracts from the guts of the
 ‘May 20’ <em>Daphnia magna </em>clone, because prior work
 indicated the susceptibility of this clone differed greatly between
 <em>Scenedesmus</em> and <em>Microcystis</em>
 diets.   We reared 5 individuals of clone ‘May 20’ per
 100 mL beaker filled with 50 mL of filtered lake water, feeding them 2mg
 C/L <em>A</em><em>nkistrodesmus falcatus </em>AJT
 strain (Tessier <em>et al.</em>, 1983; Schomaker and Dudycha,
 2021)<em> </em>until they were 5-6 days old. At this time, we
 moved the animals to clean filtered lake water, keeping 5 individuals per
 beaker, and fed them 2 mg C/L of either <em>Scenedesmus
 </em>or <em>Microcystis aeruginosa </em>CYA43; we had 20
 replicate beakers for the <em>Scenedesmus </em>treatment and
 40 replicate beakers for the <em>Microcystis </em>treatment.
 We had twice the number of <em>Microcystis </em>treatment
 replicates because we needed extra animals to make gut extracts – 100 of
 these animals would be used for the regular gut extract treatment, and 100
 would be used for the gut extract + chymotrypsin treatment, as described
 below. On the next day, when animals were 6-7 days old, we again fed them
 2 mg C/L of their treatment diet. On the following day we placed animals
 in 2mL Eppendorf tubes, placing 20 animals per tube (combining four of the
 beakers into a single tube) for a total of 5 tubes with 100 animals total
 for the <em>Scenedesmus </em>diet treatment, and 10 tubes with
 200 animals total for the <em>Microcystis </em>treatment, and
 stored the tubes with animals at -20 °C. We later dissected the entire
 guts of animals and placed 20 guts in a tube, again for a total of 5 tubes
 with 20 guts each for the <em>Scenedesmus</em> diet treatment
 and 10 tubes with 20 guts each for the <em>Microcystis
 </em>diet treatment. We added 100 µL of 0.1 M potassium-phosphate
 (P-P) buffer pH 7.5 to each of the tubes and homogenized them using a
 pestle. We centrifuged the tubes at 14,000 g for 3 minutes, then
 transferred the supernatant into a 2mL Eppendorf tube. We used bovine
 chymotrypsin (Sigma Aldrich) as a positive control, resuspending it in P-P
 buffer to a concentration of 10% w/v. Finally, we used P-P buffer as our
 negative control. We tested the impact of these gut
 extracts (or controls) on parasite transmission spores. Specifically, we
 measured how many spores lost their ascus over time. The extracts and the
 concentrations we tested are the following: 5% w/v <em>Scenedesmus
 </em>gut extract, 5 % w/v <em>Microcystis </em>gut
 extract, 5% w/v <em>Microcystis </em>gut extract with 1% w/v
 bovine chymotrypsin, 1% w/v bovine chymotrypsin, and finally, P-P buffer
 as a negative control. We predicted that, if chymotrypsin is important for
 dehiscence, the highest level of dehiscence (that is, most spores without
 asci) would occur in treatments with high levels of chymotrypsin activity,
 and the lowest levels would be in treatments with low activity levels of
 chymotrypsin (inhibited due to diet). More specifically, we predicted that
 spores incubated in gut extracts of animals fed
 <em>Microcystis</em> would have lower levels of dehiscence
 than those incubated in gut extracts of animals fed
 <em>Scenedesmus</em>, and that there would be high levels of
 dehiscence of spores incubated in pure chymotrypsin or in those where
 chymotrypsin was added to gut extracts of animals fed
 <em>Microcystis.</em> To test this, we
 placed 250 µL of spore slurry containing ~ 100,000 spores of the fungal
 parasite <em>Metschnikowia bicuspidata </em>in wells of a 96
 well plate<em>. </em>We did this for 20 wells placed randomly
 throughout the plate. One person would place a treatment picked
 haphazardly to one of the wells in the plate and note the time at which
 the treatment was placed. After 30 and 210 minutes another person would
 count the number of spores with and without asci within a given well
 without knowing which treatment was placed in that well. The counts were
 done under a compound microscope at 400X with a Neubauer counting chamber.
 This was repeated for every treatment for a total of 4 replicates per
 treatment. <em>Experiment 2. Quantification of
 effects of diet treatments on gut digestive enzymes and infection
 levels</em> Because susceptibility and tolerance
 to protease inhibitors are <em>Daphnia</em> clone and diet
 dependent, we wanted to measure variation in protease activity on
 different diets and test how these gut enzyme levels corresponded with
 host susceptibility and parasite fitness. To quantify the impact of diet
 on gut proteases, we measured trypsin and chymotrypsin activity of gut
 extracts from animals fed different diets. We also measured variation in
 infection outcomes for animals fed on these different diets. We were
 interested in both the variation in gut protease activity and in whether
 reduced protease activity correlated with decreased infection.
 We ran a factorial experiment with 11
 <em>Daphnia</em> clones and 5 diets. We ran this experiment in
 7 blocks aiming for 5 replicates per clone x diet treatment per block. We
 interspersed blocks that were used to measure enzyme activity and those
 used for infection assays. Blocks 1-3 were used entirely for infection
 assays, blocks 4 and 6 entirely for enzyme assays, and blocks 5 and 7 were
 used for both infection assays and enzyme activity; for blocks 5 and 7 we
 aimed for 10 replicates per treatment and half of those replicates were
 used for the enzyme activity assay. While we aimed for 5 replicates per
 block, in some cases, we lost animals, especially due to cyanobacteria
 toxicity; in these cases, we tried to replace the replicate in a future
 block. This was particularly problematic in blocks 1 and 2. For the two
 PCC7806 diet treatments, very high mortality means that the dataset does
 not include any animals from those two blocks for these two diet
 treatments in our analyses. We first reared individuals
 of each clone under standardized lab conditions for multiple generations
 prior to the experiment. For all blocks, neonates (0-1 day old) were
 harvested from mothers and placed 5 each in 150 mL beaker with 100 mL of
 filtered lake water. Neonates were fed <em>Ankistrodesmus
 falcatus</em> daily for 5 days. When juveniles were 5-6 days old,
 each juvenile was placed individually in a 50 mL beaker with 30 mL of
 filtered lake water. Juveniles were fed their corresponding treatment diet
 as follows: 2 mg/L C of <em>Scenedesmus, </em>2 mg/L C of
 <em>Microcystis </em>CYA160<em>, </em>2 mg/L C of
 <em>Microcystis </em>CYA43, 1 mg/L C <em>Microcystis
 </em>PCC7806-WT with 1 mg/L C <em>Scenedesmus, </em>and
 1 mg/L C <em>Microcystis </em>PCC7806-MT with 1 mg/L C
 <em>Scenedesmus</em>. For the two PCC7806 treatments, we
 needed to use a 50:50 mix of the treatment
 diet:<em>Scenedesmus</em> to promote survival. The first two
 blocks of the experiment used 100% of the <em>Microcystis
 </em>strains, but the very high mortality meant we could not use
 those animals in the experiment. Therefore, for blocks 3-7, animals in
 these two treatments were fed a 50:50 mix of the
 <em>Microcystis</em> strain and <em>Scenedesmus.
 </em> For animals in blocks 4 and 6 and for the
 half of the block 5 & 7 animals that were used to measure protease
 activity, on the next day (that is, when animals were 6-7 days old), we
 transferred animals to clean beakers filled with 30mL of fresh filtered
 lake water<em> </em>and fed them half the amount from the
 previous day of their corresponding treatment food; this was done to be
 consistent with the treatment of animals for the infection assays (see
 below). After 24 hours, we sacrificed these animals and preserved them in
 Eppendorf tubes with no water and stored them at -20 °C. At a later date,
 animals were grouped to have 20 animals per clone x diet treatment (for a
 given clone ⅹ diet treatment combination, animals from blocks 4-7 were
 combined into one tube) in a given tube and used to measure proteolytic
 activity of trypsin and chymotrypsin. Trypsin activity was assayed using
 the substrate N-benzoyl- DL-arginine p-nitroanilide (BapNA), while
 chymotrypsin activity was assayed using
 N-succinyl-L-alanyl-L-alanyl-L-propyl-L-phenylalanine 4-nitroanilide
 (SucpNA). Detailed methods used to quantify potential enzyme activity (as
 ΔmAU/min/µg protein content) are given in the supplement.
 For the five infection assay blocks, when animals were 6-7 days
 old, we transferred them individually to clean beakers filled with 30mL of
 fresh filtered lake water. On this day all animals were exposed to 500
 spores/mL of <em>Metschnikowia </em>and fed half the amount
 from the previous day of their corresponding treatment food; prior work
 has shown that reducing phytoplankton during exposure promotes spore
 uptake by hosts. After 24 hours, we placed each animal singly in a 150 mL
 beaker with 100 mL of filtered lake water and fed them their corresponding
 treatment diet with the original amount of their treatment diet food. We
 checked for mortality daily and counted offspring twice a week during
 water changes, removing offspring from the experiment. Animals were fed
 their treatment diets daily. Animals that died throughout the experiment
 were preserved in 100 µL of MilliQ water and stored at 4° C for later
 spore counts. At the end of 20 days, any remaining animals were preserved
 in 100 µL of MilliQ water and stored at 4° C for later spore counts. We
 determined the final abundance of transmission spores in hosts by counting
 spores under a compound microscope at 400X using a Neubauer counting
 chamber. Animals were diagnosed as infected if they contained transmission
 stages (equivalent to “terminal infection” in Stewart Merrill <em>et
 al.</em>, 2019).
description: # Inhibition of gut digestive proteases by cyanobacterial diets decreases
 infection in a Daphnia host-parasite system
 [https://doi.org/10.5061/dryad.nzs7h44xq](https://doi.org/10.5061/dryad.nzs7h44xq) There are three datasets for this project. * The first dataset focuses on Experiment 1. Dehiscence assay: isolating the impact of gut extracts and chymotrypsin on spore dehiscence. * The second dataset is for Experiment 2. Quantification of effects of diet treatments on gut digestive enzymes and infection levels, and focuses on *Daphnia's* life parameters such as offspring produced, infection status and infection load. * The third dataset is the rest of the data for Experiment 2. Quantification of effects of diet treatments on gut digestive enzymes and infection levels, which contains data on *Daphnia* clone enzyme activity. ## Description of the data and file structure The following explains all of the variables in the dataset dehisence_Jan2023.csv which are part of Sanchez KF, von Elert E, Monell K, Calhoun S, Maisha A, McCreadie P, Duffy MA 2023 Inhibition of gut digestive proteases by cyanobacterial diets decreases infection in a Daphnia host-parasite system. #### datasheet: dehisence\_Jan2023.csv * Well: Position on a 96 well plate where a treatment replicate was performed. * Treat_ID: Hapharzadly assigned number given to a given treatment replicate in order to keep the counts blind. * Treat: Treatment to which spores were exposed to. Abbreviatons as follows: cya43 = gut extract from animals fed Microcystis aeruginosa CYA43; cya43+ = gut extract from animals fed Microcystis aeruginosa CYA43 and spiked with chymotrypsin; sce = gut extract from animals fed Scenedesmus obliquus; chym = chymotrypsin; ppB = potassium-phosphate buffer (negative control). * Rep: Replication ID number. * min: Time in minutes at which spores with and without ascus were counted after being treated with their corresponding extract or treatment. * NA1: Hemocytometer count for one chamber of spores without an ascus * NA2: Hemocytometer count for another chamber of spores without an ascus * NA3: Hemocytometer count for another chamber of spores without an ascus * NA4: Hemocytometer count for another chamber of spores without an ascus * NA_average: Average number of spores counted (mean number of cells NA1 through NA4) * NA_spore_mL: Calculated density of spores in water (average spore count * 10000) * Total_NA: Calculated total number of spores produced in a the well (NA_spore_mL (spores/mL) * 0.25 volume of sample (mL)) * WA1: Hemocytometer count for one chamber of spores with asci * WA2: Hemocytometer count for another chamber of spores with asci * WA3: Hemocytometer count for another chamber of spores with asci * WA4: Hemocytometer count for another chamber of spores with asci * WA_average: Average number of spores counted (mean number of cells WA1 through WA4) * WA_spore_mL: Calculated density of spores in water (WA average spore count * 10000) * Total_WA: Calculated total number of spores produced in a the well (WA_spore_mL (spores/mL) * 0.25 volume of sample (mL)) The following explains all of the variables in the dataset Magna_spores_offspring.csv which are part of Sanchez KF, von Elert E, Monell K, Calhoun S, Maisha A, McCreadie P, Duffy MA 2023 Inhibition of gut digestive proteases by cyanobacterial diets decreases infection in a Daphnia host-parasite system. #### datasheet: Magna\_spores\_offspring.csv * Clone: Daphnia magna genotype * Diet: Treatment diet given to a given clone. Please see methods for more information about how diet were given since in some instances it was a mixture of control algae and assigned treatment algae. Abreviations for the diets are as follows: CYA160 = Microcytis aeruginosa strain CYA160; CYA43 = Microcystis aeruginosa strain CYA43; mycB = Microcytis aeruginosa knock out strain PCC7806 -mycB; PCC7806 = Microcystis aeruginosa strain PCC7806; Sce = Scenedesmus obliquus strain UTEX 3155. * Rep: Replication ID number. * Block: Block in which experimental unit was run. * Infected: Whether the animal in the experimental unite became infected or not with the fungal parasite Metschnikowia bicuspidata. * week1.1: Number of offsprings produced by adult Daphnia during the second half of the week after parasite exposure (or Daphnia the same age for negative controls). * week2.1: Number of offspring produced by a given individual during the end of the first week after parasite exposure (or Daphnia the same age for negative controls).Empty cells mean the individual was already deceased so there is no offspring data available. * week2.2: Number of offspring produced by a given individual during the beginning of the second week after parasite exposure. Empty cells mean the individual was already deceased so there is no offspring data available. * week3.1: Number of offspring produced by a given individual during the end of the second week after parasite exposure.Empty cells mean the individual was already deceased so there is no offspring data available. * week3.2: Number of offspring produced by a given individual during the beginning of the third week after parasite exposure.Empty cells mean the individual was already deceased so there is no offspring data available. * week3.3: Number of offspring produced by a given individual during the end of the third week after parasite exposure.Empty cells mean the individual was already deceased so there is no offspring data available. * week 4.1: Number of offspring produced by a given individual before the last day of the experiment.Empty cells mean the individual was already deceased so there is no offspring data available live animal was not found with offspring. * week 4.2: Number of offspring produced by a given individual on the last day of the experiment.Empty cells mean the individual was already deceased so there is no offspring data available or live animal was not found with offspring. * Offspring: Number of offspring produced in the lifetime of the host (sum of rows week1.1 to wk4.2). * c1: hemocytometer count for one chamber * c2: hemocytometer count for another chamber * c3: hemocytometer count for another chamber * c4: hemocytometer count for another chamber. If cell is empty, it means first three counts were sufficient for precision. * c5: hemocytometer count for another chamber. If cell is empty, it means first four counts were sufficient for precision. * c6: hemocytometer count for another chamber. If cell is empty, it means first five counts were sufficient for precision. * vol_uL: volume of nano pure water in microliters that individuals were resuspended for spore count. If cell empty or NA it means data does not exist. Please see notes for further explanations. * vol_mL: column "vol_uL" converted into milliliters (mL). If cell empty or NA it means data does not exist. Please see notes for further explanations. * Average_count: Average spore count from chambers (average of c1-c6). If cell empty or NA it means data does not exist. Please see notes for further explanations. * cells_mL: Calculated density of spores in water (average spore count * 10000). If cell empty or NA it means data does not exist. Please see notes for further explanations. * spores_daphnia: Calculated total number of spores produced in a single individual Daphnia (average density of spores (spores/mL) * volume of sample (mL)). If cell empty or NA it means data does not exist. Please see notes for further explanations. * notes: contains information for ways in which certain data points were treated. Note: there are two columns because they were transcribed from 2 different "written" data sheets. The following explains all of the variables in the dataset All_clones_assay_data.csv which are part of Sanchez KF, von Elert E, Monell K, Calhoun S, Maisha A, McCreadie P, Duffy MA 2023 Inhibition of gut digestive proteases by cyanobacterial diets decreases infection in a Daphnia host-parasite system. #### datasheet: All\_clones\_assay\_data.csv * Clone: Daphnia magna genotype * Diet: Treatment diet given to a given clone. Please see methods for more information about how diet were given since in some instances it was a mixture of control algae and assigned treatment algae. * Substrate: substrate for trypsin (BApNA) or chymotrypsin (SucpNA) enzyme activity analysis. * DH_vol: volume in uL of *Daphnia* homogeneate used for the enzymatic assay. * T0: Absorbance at 390nm at time 0, time at which substrate and extracts/treatments where mixed. * T1: Absorbance at 390nm ~15 minutes after starting the reaction * T2: Absorbance at 390nm ~30 minutes after starting the reaction * AU_T0-T2: Change in absorbance (390 nm abs at time 2 - 390 abs at time 0) * rate_mAU: Column AU_T0_T2 converted into milli absrobance units (mAU) AU_T0-T2 * 1000 * T2_min: Time elapse for a given sample/well from T0 to T2 in minutes * Rate_mAU_min: Rate at which absorbance change per minute of time (column rate_mAU / column T2_min) * Protein_clone: Total protein content for a given sample as measured with Bradford protein assay * mAU_min_protein: Rate of absorbance change per minute per ug of protein