NIAID Schistosomiasis Resource Center

A histochemical method for identifying sex of Schistosoma mansoni cercariae

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

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		*A histochemical method for identifying sex of Schistosoma mansoni* cercariae 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. Tail nuclei of female cercariae Tail nuclei of male cercariae Arrows indicate W-heterochromatin