DOI: 10.5061/dryad.b5mkkwhkk
title: Ascaris intestine calcium fluorescence dataset showing synergism of levamisole and Cry5B

description: A novel class of biocidal compounds are the Crystal 3D (Cry) and Cytolytic
 (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry
 proteins have a selective nematocidal activity, with Cry5B being the most
 studied. Cry5B kills nematode parasites by binding selectively to membrane
 glycosphingolipids, then forming pores in the cell membranes of the
 intestine leading to damage. Cry5B selectively targets multiple species of
 nematodes from different clades and has no effect against mammalian hosts.
 Levamisole is a cholinomimetic anthelmintic that acts by selectively
 opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs)
 that have been found on muscles of nematodes.  A synergistic
 nematocidal interaction between levamisole and Cry5B has been described
 previously on whole worms, but the location, mechanism and time-course of
 this synergism is not known. In this study we follow the timeline of the
 effects of levamisole and Cry5B on the Ca2+ levels of enterocyte cells of
 the intestine of Ascaris suum using fluorescence imaging.
  The peak Ca2+ responses to levamisole were observed
 after approximately 10 minutes and the peak responses to activated Cry5B
 were seen after approximately 80 minutes.  When levamisole and
 Cry5B were applied simultaneously, we observed that the responses to Cry5B
 were bigger and occurred sooner than when it was applied by itself. It is
 proposed that there is an irreversible cytoplasmic Ca2+ overload that
 leads to cell-death in the enterocyte that is induced by levamisole
 opening Ca2+ permeable L-subtype nAChRs and the development of Ca2+
 permeable Cry5B toxin pores in enterocyte plasma membranes. The effects of
 levamisole potentiate and speed the actions of Cry5B.  
description: <em>Measurement of Ca<sup>2</sup><sub>­ </sub>fluorescence </em> All recordings were performed with a Nikon Eclipse TE300 microscope (20X/0.45 Nikon PlanFluor objective), fitted with a Photometrics Retiga R1 camera (Photometrics, Surrey, BC, Canada). Light control was achieved using a Lambda 10-2 two filter wheel system with a shutter controller (Lambda Instruments, Switzerland). Filter wheel one was set on a green filter (510-560 nM bandpass, Nikon USA) between the microscope and camera. Filter wheel two set on the blue filter (460-500 nM, bandpass, Nikon, USA) between a Lambda LS Xenon bulb light box which delivered light via a fiber optic cable to the microscope (Lambda Instruments, Switzerland). The blue light emission was controlled using a shutter. All Ca<sup>2+</sup> signal recordings were acquired and analyzed using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA). Exposure times were 150ms with 2x binning.  Maximal Ca<sup>2+</sup> signal amplitudes (ΔF/F<sub>0 </sub>%) for all stimuli applied were calculated using the equation F-F0/F0 where F is the fluorescent value and F0 is the baseline fluorescent value, which was determined as the lowest value before the largest rise in fluorescence for all recordings analyzed. Representative traces were generated using the same formula, with F0 being the value before a detectable increase in the fluorescence. For the 1 mM CaCl<sub>2</sub> bath solution control experiments (Fig. 3A & C) F0 was determined to be the value before the largest rise in Ca<sup>2+</sup>. For the control CaCl<sub>2</sub> responses (Fig 3) F0 was determined as the value before stimulus application. Rise times were calculated by normalizing the trace during stimulus exposure, with the lowest fluorescence value being represented by 0% and the highest being 100%. The peak time was calculated by subtracting the time when the stimulus was applied from the time the signal reached 100%.  <em>Regions used for measurements of the </em>Ca<sup>2+ </sup><em>signals and % response area</em> Ca<sup>2+ </sup>signals from each intestine were collected from 50 squares each 50 µm x 50 µm from rectangular 125,000 µm<sup>2</sup> areas of the intestine that included 800-1000 enterocytes (Fig. 1B & C). The relative fluorescence amplitude was determined and followed over time for each of the individual 50 square regions. Long-term control preparations were not exposed to any test agents and were incubated in 1 mm CaCl<sub>2</sub> APF and followed over 6 hours. A 10 mM CaCl<sub>2</sub> test pulse was added to the chamber as a test of viability of the preparation. An increase &gt;10% in relative Fluo-3 fluorescence to the 10 mM CaCl<sub>2</sub> pulse was taken as an indication of the positive health of the preparation: preparations were rejected if the responses were &lt;10% as not being viable.  For the 6-hour incubations, intestines were exposed to either 10 µg/ml Cry5B, 100 µg/ml Cry5B, or 100 µg/ml Cry5B and 100 mM galactose. For the 2-hour recordings preparations were exposed to either 10 µg/ml Cry5B, 30 µM levamisole as the sole active agent or a combination of 10 µg/ml Cry5B and 30 µM levamisole. Any of the 50 µm x 50 µm regions whose Ca<sup>2+</sup> amplitude responses to the anthelmintic stimulus that was smaller than the average amplitude of the spontaneous Ca<sup>2+</sup> signals (2.4% ±0.1% Fig. 2C) were discarded. The reason for their rejection was that we could not rule out the possibility that these signals were, themselves, spontaneous rather than produced by the anthelmintic stimulus. <em>Statistical Analysis</em> Statistical analysis of all data was made using GraphPad Prism 9.0 (Graphpad Software, Inc., La Jolla, CA, USA). We repeated our experiments to ensure reproducibility. The total number of female worm intestinal preparations, the total number of regions showing responses, the concentrations, and durations of applications are provided in the figure legends of the figures.    Analysis of Ca<sup>2+</sup> amplitudes and time to peak were made using either unpaired for separate preparations or paired when the responses in the same preparation were being followed using student <em>t</em>-tests <em>P &lt; </em>0.05. The <em>t</em>-test (paired or unpaired) used is stated in the figure legends.   
description: # Williams et al: 2024 Ascaris suum intestine data sets for figures
 [https://doi.org/10.5061/dryad.b5mkkwhkk](https://doi.org/10.5061/dryad.b5mkkwhkk) We list the the raw observations in .xlsx format (Excel Workbook) that was entered into Prizm 5 files for the construction of Figures, 2C, 3B, 4D, 4E, 4F, 8D, 8E, 8F, 9B, 9C and 9D for the Figs 2C, 3B, 4D, 4E, 4F, 8D, 8E, 8F, 9B, 9C and 9D of the Plos Pathogens manuscript. ## Description of the data and file structure The data files refer to each of the Figures for the manuscript prepared for Plos Pathogens. The data are listed in columns for each of the different treatments. **Cry5B message targets are present in *A. suum* intestine** Fig 2C:  Cry5B message targets are present in *A. suum* intestine. Datasets of the differential expression of Cry5B targets of Asu-bre-5 and *Asu-cdh-8)* between intestine and body wall. Bar chart (expressed as mean ±SEM) demonstrating transcript level analysis from intestinal samples for *bre-5* (grey bar) (5.24 ± 0.68) and *cdh-8* (white bar) (6.86 ± 2.32) when compared to paired body wall samples. n = 15 individual worms from 3 separate groups of A. suum, collected at different times.   **Long-term Ca2+ imaging** Fig 3B: Dataset for the Amplitudes of spontaneous Ca2+ signals (white bar) and control 10 mM CaCl2 amplitudes (red bar). **** Significantly different to spontaneous. Spontaneous vs 10 mM CaCl2 P &lt;0.0001 t = 41.76, *df* = 249, paired *t*-test, 5 individual female intestines.  **Cry5B stimulates concentration dependent Ca2+ peak and time-to-peak responses on A. suum  intestines that are inhibited by galactose** Fig4D: Dataset  for the maximal amplitudes of Ca2+ fluorescence over 6 hours in response to 100 µg/ml Cry5B treated, 10 µg/ml Cry5B treated and 100 µg/ml Cry5B + 100 mM galactose treated. 11 female intestines; 100 µg/ml Cry5B vs 100 µg/ml Cry5B + 100 mM galactose P = &lt;0.0001 t = 14.55, *df* = 485, unpaired *t*-test; 10 female intestines). † Significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 100 µg/ml Cry5B + 100 mM galactose P = &lt;0.0001 t = 6.437, *df* = 531, unpaired *t*-test, 11 female intestines. Fig 4E: Dataset  for the time to maximal peak amplitude over 6 hours in response to 100 µg/ml Cry5B treated, 10 µg/ml Cry5B treated and 100 µg/ml Cry5B + 100 mM galactose treated. Significantly different to 100 µg/ml Cry5B. (100 µg/ml Cry5B vs 10 µg/ml Cry5B, P= &lt;0.0007, t = 3.412, *df* = 518, unpaired *t*-test, 11 female intestines. Significantly different to 100 µg/ml Cry5B. 100 µg/ml Cry5B vs 100 µg/ml Cry5B + 100 mM galactose P = &lt;0.0001 t = 14.55, *df* = 485, unpaired *t*-test, 10 female intestines). † Significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 100 µg/ml Cry5B + 100 mM galactose P = &lt;0.0001 t= 6.437, *df* = 531, unpaired *t*-test, 11 female intestines. Fig 4F: Dataset % Region Responses: % of regions showing Ca2+ responses bigger than the average spontaneous amplitude in untreated intestines in:  100 µg/ml Cry5B treated; 10 µg/ml Cry5B treated; 100 µg/ml Cry5B + 100 mM galactose treated. N.S. not significant compared to 100 µg/ml Cry5B (100 µg/ml Cry5B vs 10 µg/ml Cry5B, P = &lt;0.9264 t = 0.095, *df* = 9, unpaired *t*-test, 11 female intestines; 100 µg/ml Cry5B vs 100 µg/ml Cry5B + 100 mM galactose P = &lt;0.271 t = 1.183, *df* = 8, unpaired *t*-test, 10 female intestines. 100 µg/ml Cry5B n = 5 female intestines with 237 of 250 regions providing viable responses which were used to generate the mean and SEM for D & E; 13 of the 250 regions showed no response. *10 µg/ml Cry5B *n = 6 female intestines with 283 of 300 regions produced responses which were used to generate the mean and SEM for D & E; 17 of 300 regions showed no response.  100 µg/ml Cry5B + 100 mM galactose n = 5 female intestines with 195 of 250 regions produced viable responses which were used to generate the mean and SEM for D & E, 55 of their 250 regions showed no response.  **Levamisole potentiates Cry5B signal in *A. suum intestines*** Fig 8D: Dataset showing the maximal amplitudes of Ca2+ fluorescence over 2 hours in 10 µg/ml Cry5B treated. The 30 µM levamisole responses and the 10 µg/ml Cry5B + 30 µM levamisole responses. N.S. not significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 30 µM levamisole, P = &lt; 0.3117 t = 1.013, *df* = 393, unpaired *t*-test, 11 female intestines). Significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 10 µg/ml Cry5B + 30 µM levamisole, P = &lt; 0.0001 t = 13.58, *df* = 482, unpaired *t*-test, 12 female intestines). † Significantly different to 30 µM levamisole (30 µM levamisole vs 10 µg/ml Cry5B + 30 µM levamisole P = &lt; 0.0001 t = 13.41, *df* = 465, unpaired *t*-test, 12 female intestines. Fig 8E: Datasets of the times-to-peak amplitude over 2 hours in 10 µg/ml Cry5B treated, 30 µM levamisole and 10 µg/ml Cry5B + 30 µM levamisole . Significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 30 µM levamisole, P = &lt; 0.0001 t = 22.11, *df* = 363, unpaired *t*-test 11 female intestines; 10 µg/ml Cry5B vs 10 µg/ml Cry5B + 30 µM levamisole, P *= &lt; 0.0001 t*= 23, *df* = 482 unpaired *t*-test, 12 female worms). † Significantly different to 30 µM levamisole (30 µM levamisole vs 10 µg/ml Cry5B + 30 µM levamisole P = &lt; 0.0001 t= 12.37, *df* = 465, unpaired *t*-test, 12 female intestines. Fig 8F: Dataset % regions showing a Ca2+ response larger than the average spontaneous Ca2+ amplitude in untreated intestines in 10 µg/ml Cry5B treated, 30 µM levamisole and 10 µg/ml Cry5B + 30 µM levamisole. Not significant compared to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 30 µM levamisole, P = &lt; 0.102 t = 1.821, *df* = 9, unpaired *t*-test, 11 female intestines).  Significantly different to 10 µg/ml Cry5B (10 µg/ml Cry5B vs 10 µg/ml Cry5B + 30 µM levamisole P = &lt; 0.0357 t = 2.468, *df* = 9, unpaired *t*-test, 11 female intestines).  Significantly different to 30 µM levamisole (30 µM levamisole vs 10 µg/ml Cry5B + 30 µM levamisole, P = &lt; 0.0004 t = 5.266, *df* = 10, unpaired *t*-test, 12 female intestines. 10 µg/ml Cry5B was tested on n = 5 female intestines showed 191 out of 250 regions that produced viable responses that were used to generate the means and SEM for D & E;   59 of 250 regions showed no response. 30 µM levamisole was tested on n = 6 individual female intestines and 174 of 300 regions produced viable responses which were used to generate the mean and SEM for D & E, 126 of 300 regions showed no response. 10 µg/ml Cry5B + 30 µM levamisole was tested on *n* = 6 individual female worms that had 293 of 300 regions showing a viable response that were used to generate the mean and SEM for D & E,  7 of the 300 regions did not respond. **Mecamylamine inhibits levamisole mediated potentiation Cry5B** Fig 9B: Dataset of the maximal amplitudes of Ca2+ fluorescence over 2 hours in 30 µM levamisole + 10 µg/ml Cry5B treated (green bar) and 10 µM mecamylamine + 30 µM levamisole + 10 µg/ml Cry5B (blue bar) treated intestines. Significantly different: 30 µM levamisole+10 µg/ml Cry5B *vs* 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B (P = &lt; 0.0001 t = 12.92, *df* = 494, unpaired *t*-test, 11 female intestines). Fig 9C: Dataset of times to maximal peak amplitude over 2 hours in 30 µM levamisole+10 µg/ml Cry5B treated (green bar) and 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B (blue bar) treated intestines. Significantly different: 30 µM levamisole+10 µg/ml Cry5B vs 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B (P*= &lt; 0.0001 t = 72.6, *df* = 494, unpaired *t*-test, 11 female intestines). Fig 9D: Dataset  of the percent of regions showing a Ca2+ response larger than the average spontaneous Ca2+ amplitude in untreated intestines. 30 µM levamisole+10 µg/ml Cry5B treated and 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B .  Significantly different: 30 µM levamisole+10 µg/ml Cry5B vs 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B (*P *= &lt; 0.0176, t*= 2.899, *df* = 9, unpaired *t*-test, 11 female intestines). 10 µg/ml Cry5B+30 µM levamisole *n* = 6 individual female worms with a total of 293 of 300 regions providing a viable response that were used to generate the mean and SEM for B & C. Note: 7 of 300 regions showed no response. 10 µM mecamylamine+30 µM levamisole+10 µg/ml Cry5B n = 5 individual female worms with a total of 203 of 250 providing a viable response to generate the mean and SEM for B & C. Note that 47 of  250 showed no response. The means, +/- Standard errors may be constructed from the data to see if they show statistically different values and therefore the effects of our test procedures. We have measured qPCR values for Fig 2C. The values for Figures 4F, 8F and 9D show % regions that respond to the treatments. The remaining observations are measurements of (F-F0)/F0 % relative fluorescence. ## Sharing/Access information The sequence datasets analyzed during the current study are available in Wormbase Parasite, The European Nucleotide Archive (ENA) and UNIPROT repositories, [https://parasite.wormbase.org/index.html](https://parasite.wormbase.org/index.html), [www.elixir-europe.org/platforms/data/core-data-resources](http://www.elixir-europe.org/platforms/data/core-data-resources), [www.uniprot.org](http://www.uniprot.org). Links to the datasets are presented in supplementary Table 3. ## Code/Software Statistical Analysis was conducted using Prizm 5 Software. Calcium fluorescence was measured using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA). Exposure times were 150ms with 2x binning.