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                                                                                                                                               

 

  Tail nuclei of female cercariae                                                  Tail nuclei of male cercariae

                Arrows indicate W-heterochromatin