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.

Measurement of Ca²fluorescence

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 Ca²⁺ signal recordings were acquired and analyzed using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA). Exposure times were 150ms with 2x binning. Maximal Ca²⁺ signal amplitudes (ΔF/F₀%) 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 CaCl₂ bath solution control experiments (Fig. 3A & C) F0 was determined to be the value before the largest rise in Ca²⁺. For the control CaCl₂ 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%.

Regions used for measurements of the Ca²⁺signals and % response area

Ca²⁺signals from each intestine were collected from 50 squares each 50 µm x 50 µm from rectangular 125,000 µm² 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 CaCl₂ APF and followed over 6 hours. A 10 mM CaCl₂ test pulse was added to the chamber as a test of viability of the preparation. An increase >10% in relative Fluo-3 fluorescence to the 10 mM CaCl₂ pulse was taken as an indication of the positive health of the preparation: preparations were rejected if the responses were <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 Ca²⁺ amplitude responses to the anthelmintic stimulus that was smaller than the average amplitude of the spontaneous Ca²⁺ 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.

Statistical Analysis

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 Ca²⁺ 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 t-tests P < 0.05. The t-test (paired or unpaired) used is stated in the figure legends.

Ascaris intestine calcium fluorescence dataset showing synergism of levamisole and Cry5B

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