A histochemical method for identifying sex of Schistosoma mansoni cercariae (under construction)

Author: James Liberatos

Introduction
The sex of Schistosoma mansoni cercariae can be determined rapidly and reliably using a modified C-banding technique, originally developed for use in chromosome banding.

S. mansoni females (♀ZW, ♂ZZ) have a large block of heterochromatin in their W chromosome. The W-heterochromatin can be seen in the tail interphase nuclei of female cercariae, but is not present in male cercariae.

Equipment
Centrifuge
Water bath
Compound microscope

Materials and reagents
15 ml conical centrifuge tubes (siliconized tubes work best)
Coplin jars
Pasteur pipettes
Glass slides
Methanol – acetic acid (3:1) mixed fresh before each use
0.2 N HCl
Saturated Ba(OH)2
2 X SSC (6.3 M NaCl, 0.03 M Na3C6H5O7) at pH 7.0
Giemsa (4% at pH 7.0)

Procedure

  • Collect cercariae in centrifuge tubes, and add methanol-acetic acid.
  • Fix the cercariae for 15 minutes; centrifuge 5 minutes at about 1,300 rpm; discard supernatant.
  • Resuspend the pellet, and fix cercariae again for 15 minutes using fresh methanol-acetic acid; repeat centrifugation.
  • Remove all but a small amount of fixative with a pipette; resuspend the pellet.
  • Drop the cercarial suspension onto clean, wet slides; air dry.
  • Immerse the slides in:  (A) 0.2 N HCl for 15 minutes; (B) Ba(OH)2 for 15 minutes; (C) 2X SSC at 60 – 65ºC for 2 hours.
  • Stain the cercariae in 4% Giemsa for 2 – 4 minutes.
  • Examine the tail nuclei of cercariae with compound microscope for the presence or absence of W-heterochromatin (see pictures below).

 Reference
Liberatos, J.D. and Short, R.B. 1983. Identification of Sex of Schistosome Larval Stages. Journal of Parasitology 69: 1084-1089.

malecellsedited 2 feb.jpg                   femalecells two copy.jpg

A histochemical method for identifying sex of Schistosoma mansoni cercariae image003A histochemical method for identifying sex of Schistosoma mansoni cercariae image004A histochemical method for identifying sex of Schistosoma mansoni cercariae image005A histochemical method for identifying sex of Schistosoma mansoni cercariae image006A histochemical method for identifying sex of Schistosoma mansoni cercariae image007A histochemical method for identifying sex of Schistosoma mansoni cercariae image008  Tail nuclei of female cercariae                                                  Tail nuclei of male cercariae

Arrows indicate W-heterochromatin