Anthelminthic Activity of Assassin Bug Venom against the Blood Fluke Schistosoma mansoni

Abstract

Helminths such as the blood fluke Schistosoma mansoni represent a major global health challenge due to limited availability of drugs. Most anthelminthic drug candidates are derived from plants, whereas insect-derived compounds have received little attention. This includes venom from assassin bugs, which contains numerous bioactive compounds. Here, we investigated whether venom from the European predatory assassin bug Rhynocoris iracundus has antischistosomal activity. Venom concentrations of 10-50 µg/mL inhibited the motility and pairing of S. mansoni adult worms in vitro and their capacity to produce eggs. We used EdU-proliferation assays to measure the effect of venom against parasite stem cells, which are essential for survival and reproduction. We found that venom depleted proliferating stem cells in different tissues of the male parasite, including neoblasts in the parenchyma and gonadal stem cells. Certain insect venoms are known to lyse eukaryotic cells, thus limiting their therapeutic potential. We therefore carried out hemolytic activity assays using porcine red blood cells, revealing that the venom had no significant effect at a concentration of 43 µg/mL. The observed anthelminthic activity and absence of hemolytic side effects suggest that the components of R. iracundus venom should be investigated in more detail as potential antischistosomal leads.

Keywords: Rhynocoris iracundus; Schistosoma mansoni; assassin bug; cell proliferation; in vitro culture; natural compound; stem cells; venom.

Figure 1

The European predatory assassin bug Rhynocoris iracundus. Stimulation of R. iracundus on the hind legs using entomological forceps (white arrow heads) encourages the insect to use its proboscis (black arrow head) to inject venom through laboratory film (Parafilm) stretched over a collection tube containing phosphate-buffered saline (PBS).

Figure 2

Rhynocoris iracundus venom affects the vitality of Schistosoma mansoni. Worm pairs were treated with different concentrations of venom (25 or 50 µg/mL). Representative images show worms after 72 h. (A) Untreated control worms remained paired and attached via their suckers to the base of the culture plate. The addition of venom at (B) 25 µg/mL or (C) 50 µg/mL induced the separation of pairs and detachment from the plate. Scale bars = 250 µm.

Figure 3

Effect of Rhynocoris iracundus venom on Schistosoma mansoni motility, pairing and egg production. Worm pairs were treated with different concentrations of venom (10–50 µg/mL) for a period of 72 h. We measured (A) motility, (B) the percentage of worms attached to the base of the plate, and (C) pairing stability every 24 h. (D) The number of eggs produced within 72 h relative to the untreated control. The shape of the eggs appeared normal after venom treatment (inserts). Graphs show a summary of two experiments with 5–8 worm pairs (mean ± SEM). Significant differences vs. the control are indicated (* p < 0.05, Wilcoxon rank sum test). (E,F) Representative images showing the number of eggs produced by untreated control worms and venom-treated worms (50 µg/mL). Scale bars = 250 µm, for inserts = 60 µm.

Figure 4

Effect of Rhynocoris iracundus venom on the proliferation of Schistosoma mansoni stem cells. (A) Overview of the location of parenchymal stem cells (neoblasts) and gonadal stem cells (spermatogonia and oogonia) in male and female worms. Stem cells are labeled with EdU (green), and nuclei are counterstained with Hoechst 33342 (blue). Scale bars = 100 µm. (B) Worm pairs were treated for 72 h with 25 or 50 µg/mL of venom or cultured without venom as a control. EdU was added during the final 24-h period. The abundance of EdU-positive proliferating stem cells was comparable in worms of the control group (a–d) and those treated with 25 µg/mL of venom (e–h) whereas 50 µg/mL of venom reduced the number of proliferating stem cells in males (i, j) but not in females (k, l). Scale bars = 50 µm. Representative images of four worms per treatment group are shown.

Figure 5

Reduction in stem cell frequency and density in male Schistosoma mansoni treated with Rhynocoris iracundus venom. Worm pairs were treated for 72 h with 25 or 50 µg/mL of venom or cultured without venom as a control. Proliferating stem cells were labeled with EdU and nuclei of all cells with Hoechst 33342. Cell numbers were quantified in z-stacks using the software package “IMARIS for cell biologists” (Bitplane). The percentage of EdU-positive cells related to the total cell number (C, G) and the number of EdU-positive cells per 1e5 µm³ tissue were calculated (D, H). (A) Representative images of testes from one worm which was digitally separated from the surrounding tissue using IMARIS. Nuclei are depicted in blue, stem cells in green. Scale bar = 40 µm. (B) All EdU-positive stem cells (spermatogonia) from the testes shown in (A) were aligned and quantified. Scale bar = 25 µm. The frequency (C) and density (D) of spermatogonial stem cells in testes were calculated. (E) Representative images of parenchyma from one worm after processing with IMARIS. Nuclei are depicted in blue, stem cells in green. Scale bar = 30 µm. (F) All EdU-positive stem cells (neoblasts) from the parenchymatic area shown in (E) were aligned and quantified. Scale bar = 15 µm. The frequency (G) and density (H) of neoblasts were calculated. Four worms per treatment group were analyzed. Statistical differences compared to the untreated group are indicated with * p < 0.05 (Wilcoxon rank sum test).

Figure 6

Rhynocoris iracundus venom reduces the number of spermatogonia in the testes of Schistosoma mansoni. Pairs of worms were treated with 50 µg/mL of venom for 72 h and males were stained with carmine red to reveal morphological details. (A) Control males feature typically pronounced testicular lobes filled with large spermatogonia (red arrows show examples) and different stages of maturing cells. Mature spermatozoa appear as small white comma-shaped cells (yellow arrows). (B) Testicular lobes in venom-treated males appear shrunken, include atypical cell-free areas (marked with *), and lack most of the spermatogonia, whereas mature spermatozoa are still present. The remaining spermatogonia show evidence of intracellular degradation. Scale bars = 100 µm (left), 50 µm (center), 20 µm (right).

Figure 7

Hemolytic activity of Rhynocoris iracundus venom against porcine red blood cells. Relative proportion of cells lysed by R. iracundus venom (43 µg/mL) compared to 10% Triton X- 100 as a positive (+) control (100% lysis) and PBS as a negative (–) control.