Observation of metaphase by DAPI staining

In butterflies and moths (order Lepidoptera), males have ZZ sex chromosomes but females have either ZW or ZO sex chromosomes depending on species or populations. Here I describe a simple method to observe metaphase plate of females using Conger-Fairchild quick-freeze method and DAPI staining.

Photos: metaphase of Ostrinia scapulalis females (n=31)

Preparation

• Fixative (Carnoy's fluid) [ethanol: chloroform: acetic acid = 6:3:1]
• PBS buffer
• 60% acetic acid
• dry ice
• 100% ethanol
• DAPI concentrate [200 ug/ml in distilled water]
• DAPI solution for staining [20 ug/ul in 2xSSC] (10 ul of DAPI concentrate in 100 ml of 2xSSC)
• antifade
• nail varnish

Procedure

1. Dissection of insects
   Open a matured female adult in PBS using fine forceps. In the PBS, remove fat bodies sticking to ovarioles. Pick out the ovarioles and remove extra PBS.
2. Fixation
   Put the ovarioles in the fixative (Carnoy's fluid). After 15 min, place the fixed ovarioles on a glass slide. 
3. Squash
   When most of the extra fixative evaporate (but should not be dried up), add 3 or 4 drops of 60% acetic acid. Place a cover slip over the material and squash by fingers using paper towel. [The purpose of the squash is to flatten the material into a single focal plane.] Put this glass side on dry ice. After 5 min, flip off the cover slip using a scalpel. Immediately put the glass slide into 100% ethanol. Gently move the slide for 10 sec. Stand the slide and air dry.
4. Staining
   When excessive ethanol evaporates, put the slide into DAPI solution. After 10 min, put 40 ul antifade onto the material and place a cover slip. Seal the edge of the cover slip with nail varnish and preserve in the dark (e.g., refrigerator) until observation.

Observation

• Observe under a fluorescence microscope. A single set of chromosomes (in the metaphase) should be found in a single egg.

Reference

• Conger AD & Fairchild LM (1953) A quick-freeze method for making smear slides permanent. Stain Technol. 28: 281-283.
• Kageyama D & Traut W (2004) Opposite sex-specific effects of Wolbachia and interference with sex determination of its host Ostrinia scapulalis. Proceedings of the Royal Society of London, Series B  271: 251-258.

Protocols

Here I post some protocols which might be useful to someone.

1. Observation of female metaphase by DAPI staining
2. Observation of male metaphase by orcein staining (coming soon)
3. Sex diagnosis using W chromatin
4. Giemsa staining of bacteria (coming soon)
5. PCR primers for endosymbiotic bacteria
6. PCR primers for insects

Sex diagnosis using W chromatin

What is W chromatin?
In many species of butterflies and moths (Order: Lepidoptera), sex chromosome constitution is ZW in females and ZZ in males. Intriguingly, W chromosome is condensed and conspicuous in each cell nucleus during interphase. This is called W chromatin or sex chromatin. Hence, sex diagnosis is available by a relatively simple method (compared to conventional karyotyping) even in the very young stages when it is almost impossible to distinguish male and female morphologically. For details, please refer to Traut and Marec (1996).

[Photo] A Malpighian tubule of a female moth, Ostrinia scapulalis. Octopus-like structure surrounded by a broken line is a single nucleus in highly polyploid state. Arrows indicate W chromatins.

Preparation

• Insects
  
• PBS buffer (or Ringer solution)
  
• Fixative (methanol: acetic acid = 3:1) [the fixative MUST be prepared every time]
  
• Stain (lactic acetic orcein solution) [should be prepared beforehand*]

*To make lactic acetic orcein: Condensed solution: 5 g of Orcein + 100 ml of lactic acid + 100 ml of acetic acid -> Keep it for more than 2 weeks (mix occasionally). -> Add equal volume of 50% acetic acid to the condensed solution.

Procedures

1. Dissection of insects
Open fresh insects in PBS using fine forceps. Pick out Malpighian tubules and remove extra PBS.
2. Fixation
Put the Malpighian tubules in the fixative (10-30 sec) and then on a glass slide.
3. Staining
When most of the extra fixative evaporate (but should not be dried up), add 2 or 3 drops of lactic acetic orcein solution (use supernatant).
4. Observation
After 3 min, place a cover slip over the material and observe under a light microscope.

Reference

• Traut W and Marec F (1996) Sex chromatin in Lepidoptera. The Quarterly Review of Biology 71: 239-256. 

A list of papers that examined W chromatin of lepidopteran insects