Ultrastructural alterations in Schistosoma mansoni juvenile and adult male worms after in vitro incubation with primaquine

Abstract

Background: Praziquantel has been cited as the only drug for treating schistosomiasis. However, concerns over drug resistance have encouraged the search for novel drug leads. The antimalarial drug primaquine possesses interesting anti-schistosmal properties.

Objectives: This study is the first to document the potential role of primaquine as a schistosomicide and the ultrastructural changes induced by primaquine on juvenile or adult male worms of Schistosoma mansoni.

Methods: Ultrastructural alterations in the tegumental surface of 21-day-old juvenile and adult male worms of S. mansoni were demonstrated following primaquine treatment at different concentrations (2, 5, 10, 15, and 20 µg/mL) and incubation periods (1, 3, 6, 24, and 48 h) in vitro, using both scanning and transmission electron microscopy.

Findings: At low concentrations (2, 5, and 10 µg/mL) both juvenile and adult male worms were alive after 24 h of incubation, whereas contraction, paralysis, and death of all worms were observed after 24 h of drug exposure at 20 µg/mL. The tegument of juvenile and adult male worms treated with primaquine exhibited erosion, peeling, and sloughing. Furthermore, extensive damage of both tegumental and subtegumental layers included embedded spines, and shrinkage of muscles with vacuoles. The in vitro results confirmed that primaquine has dose-dependent effects with 20 µg/mL as the most effective concentration in a short incubation period.

Main conclusions: The schistosomicidal activity of primaquine indicates that this drug possesses moderate in vitro activity against juvenile and adult male worms, since it caused high mortality and tegumental alterations. This study confirmed that the antimalarial drug primaquine possesses anti-schistosomal activity. Further investigation is needed to elucidate its mechanism of action.

Fig. 1. : mortality rate (%) of Schistosoma mansoni juvenile worms treated in vitro with different concentrations of primaquine for different incubation times. The mortality rate of both the negative control group and the group treated with 2 µg/mL of primaquine was 0%. Error bars represent the ± SD from three independent experiments.

Fig. 2. : mortality rate (%) of Schistosoma mansoni adult male worms treated in vitro with different concentrations of primaquine for different incubation times. The mortality rate of both the negative control group and the group treated with 2 µg/mL of primaquine was 0%. Error bars represent the ± SD from three independent experiments.

Fig. 3. : scanning electron microscopy (SEM) of Schistosoma mansoni juvenile (A-B) and adult male worms (C-D) incubated in pure medium (negative control) for 24 h showing: (A) juvenile worm with an oral sucker (OS), ventral sucker (VS) and a bore (BO) marking the beginning of the gynaecophoric groove (×85); (B) the dorsal surface of the juvenile worm showing rows of tegumental folds (TF) (×1600); (C) adult male worm showing the oral sucker (OS), ventral sucker (VS), and gynaecophoric groove (GG) (×100); (D) adult male worm with well-developed tubercles (T) and tegumental ridges (TR) on the dorsal surface (D) (×2.047).

Fig. 4. : scanning electron microscopy (SEM) of Schistosoma mansoni juvenile (A-B) and adult male worms (C-D) exposed to 5 µg/mL of Praziquantel (PZQ) (positive control) for 24 h showing: (A) juvenile worm contraction with vesicles (VE) and focal lesions (FL) in the middle part of body (×130); (B) magnification of the anterior region of a juvenile worm showing tegumental wrinkles (W) and blisters (BL) (×400); (C) adult male worm bending in the ventral (V) direction with oral sucker (OS), ventral sucker (VS), and gynecophric groove (GG) (×70); (D) ventral surface (V) of an adult male worm showing peeling (P) and erosion (E) of the tegument with blebs (B) (×270).

Fig. 5. : scanning electron microscopy (SEM) of Schistosoma mansoni juvenile worms exposed to different concentrations of primaquine (A-F) for 24 h showing: (A) juvenile worm bending dorsally (D) with tegumental peeling (P) on the ventral surface (V) of the worm at concentration 10 µg/mL (×140); (B) magnification of the anterior region of a juvenile worm showing erosion (E), peeling (P) and blisters (BL) with tegumental wrinkles (W) at the concentration 10 µg/mL (×400); (C) juvenile worm with curving body in the ventral direction (V) showing oral sucker (OS) at the concentration 15 µg/mL (×75); (D) magnification of the anterior region of a juvenile worm showing focal lesions (FL) on the tegument with large vesicles (VE) at the concentration 15 µg/mL (×750); (E) juvenile worm with ventrally (V) bending body showing oral sucker (OS) and ventral sucker (VS) at the concentration 20 µg/mL (×60); (F) magnification of the anterior region of a juvenile worm showing erosion (E), peeling (P), and vesicles (VE) at the concentration 20 µg/mL (×330).

Fig. 6. : scanning electron microscopy (SEM) of Schistosoma mansoni adult male worms exposed to different concentrations of primaquine (A-G) for 24 h showing: (A) convoluting (CV) of the adult male worm body with destruction of tubercles (T) at the concentration 10 µg/mL (×50); (B) magnification of the anterior region of an adult male worm showing pitted (PI) tegumental layer (TL), destruction of tubercles (T) and changes in the oral sucker (OS) at the concentration 10 µg/mL (×400); (C) twisting (TW) of the adult male worm at the middle region of the body at the concentration 15 µg/mL (×65); (D) magnification of the anterior region of adult male worm showing destruction of the oral sucker (OS) and sloughing (S) of the tegument with peeling (P) at the concentration 15 µg/mL (×400); (E) adult male worm showing coiling body (CO) at the concentration 20 µg/mL (×90); (F) magnification of the middle region of an adult male worm showing extensive peeling (P) and erosion (E) of the tegumental layer (TL) at the concentration 20 µg/mL (×1300); (G) adult male worm with destruction of the tegumental layer (TL) at the concentration 20 µg/mL (×430).

Fig. 7. : ultrastructural alterations in adult male Schistosoma mansoni worms: (A) Adult male worm incubated for 24 h in pure medium (negative control) showing normal pointed spines (S), tegumental layer (TL), subtegumental layer (STL), and normal muscles (M) (×4400); (B) adult male worm maintained in a medium with Praziquantel (PZQ) (positive control) for 24 h showing embedded spines (ES) and shrinkage in muscles (M) with formation of vacuoles (VC) (×4400); (C) adult male worm exposed to primaquine for 24 h showing embedded spines (ES) and pointed spines (S) with small vacuoles (VC) between the normal muscles (M) at the concentration 10 µg/mL (×4400); (D) more embedded spines (ES) and a large vacuole (VC) in adult male worm at the concentration 15 µg/mL (×4400); (E) adult male worm with degeneration in both the tegumental (TL) and subtegumental layers (STL), embedded spines (ES), large vacuoles (VC), and shrinkage in the muscles (M) at the concentration 20 µg/mL (×4400).