Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

Abstract

Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of drugs that target AChRs in filarial worms.

Keywords: Acetylcholine receptors; Anthelmintic; Brugia malayi; Filarioideae; In situ hybridization; Reproduction.

Graphical abstract

Fig. 1

In situ hybridization patterns for Bm1_20330 in adult B. malayi adult worms. The sense RNA probe (negative control) did not label tissues in female (A) or male worms (F). In contrast, the antisense probe produced strong signals in female (B–E) and male worms (G–I). Oocytes in ovary (B), morulae stage embryos (C) and the uterine wall (arrow in E) adjacent to pretzel or stretched microfilariae (Mf) were intensely labeled and stretched Mf was partially labeled (E). The antisense probe also strongly labeled spermatogonia in the male testis (G, H) and the wall of the Vas deferens (arrow) (I), the lateral cord was weakly labeled (G, H). Weak to moderate labeling was also observed in both male and female body muscle. Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 2

In situ hybridization patterns for Bm1_38195 in adult B. malayi adult worms. The sense RNA probe (negative control) did not label tissues in female (A) or male worms (F). In contrast, the antisense probe produced moderate to strong signals in female (B–E) and male worms (G–I). Oocytes in ovary (B), morulae stage embryos (C), and the uterine wall (E, arrow) adjacent to pretzel or stretched Mf were intensely labeled; and pretzel stage and stretched Mf were partially labeled (D, E). The antisense probe also strongly labeled spermatogonia in the male testis (G) and the wall of the Vas deferens (arrow) (I), the lateral cord was weakly labeled in female (B, C) and male (H, I), respectively. Weak to moderate labeling was also observed in both female and male body muscle. Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 3

In situ hybridization patterns for Bm1_35890 in adult B. malayi adult worms. The sense RNA probe (negative control) did not label tissues in female (A) or male worms (F). In contrast, the antisense probe produced strong signals in female (B–E) and male worms (G–I). Oocytes in ovary (B), morulae stage embryos (B and C), pretzel stage (D), and the uterine wall (arrow) adjacent to pretzel or stretched Mf (E, arrow) were intensely labeled; stretched Mf was partially labeled (E). The antisense probe also strongly labeled spermatogonia in the male testis (G) and the wall of the Vas deferens (arrow) (H, I), and sperm was not labeled (I). The lateral cord was moderately labeled in male and female. Weak to moderate labeling was also observed in both male and female body muscle. Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 4

In situ hybridization patterns for Bm1_48815 in adult B. malayi adult worms. The sense RNA probe (negative control) did not label tissues in female (A) or male worms (F). In contrast, the antisense probe produced strong signals in female (B–E) and male worms (G–I). Oocytes in ovary (B), morulae stage embryos (C) and the uterine wall adjacent to pretzel or stretched Mf (E, arrow) were intensely labeled; pretzel stage (D) and stretched Mf (E) were partially labeled. The antisense probe also strongly labeled spermatogonia in the male testis (G, H) and the wall of the Vas deferens (arrow) (I); the lateral cord was strongly labeled in male (G, H). Weak to moderate labeling was also observed in male body muscle. Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 5

In situ hybridization patterns for Bm1_40515 in adult B. malayi adult worms. The sense RNA probe (negative control) did not label tissues in female (A) or male worms (F). In contrast, the antisense probe produced strong signals in female (B–E) and male worms (G–I). Oocytes in ovary (B), morulae stage embryos (C) and the uterine wall (arrow) adjacent to pretzel or stretched Mf (E) were intensely labeled and stretched Mf was partially labeled (D). The antisense probe also strongly labeled spermatogonia in the male testis (G) and the wall of the Vas deferens (arrow) (I); the lateral cord was strongly labeled (G, H). Weak to moderate labeling was also observed in both male and female body muscle. Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 6

Localization of the predicted peptide from Bm1_48815 in adult B. malayi adult by immunohistochemical labeling. Negative control did not label any tissue except areas where the internal enzymes are high such as intestine in female (A) or male (F). Polyclonal antibodies against a predicted peptide from Bm_48815 labeled developing embryos, lateral cord, the wall of uteri with stretched Mf (arrow) in female (B–D); the antibody also labeled spermatogonia (G) and the body muscle and lateral cord (G–I), and the wall of the Vas deference with mature sperms (arrow, I). Abbreviations: Ov, ovary; I, intestine; U, uterus; M, muscle; Lc, lateral cord, Vd, Vas deferens; T, testis. Scale bar 10 μm in panel A–E and 5 μm in panel F–I.

Fig. 7

Higher magnification of the Localization of the predicted peptide from Bm1_48815 in B. malayi female by immunohistochemical labeling (100×). Negative control did not label any tissue except areas where the internal enzymes are high such as intestine in female (A and C) while the uterine walls (arrows in B and D corresponding to Fig. 6D and E) were moderately labeled. Scale bar 10 um.