12 October 2011

Dissecting Tribolium Genitalia

For the past couple of days I have been working on figuring out how to dissect the testes from Triboloium castaneum.  I need the testes for a FISH project for my advisor and also for some future karyotype projects of my own.  It took a quite a bit of trial and error so I wanted to give everybody a run down on how I did it just in case someone else out there is struggling through the same difficulties.


Equipment and Solutions

1.Fine tip forceps (2)
2.Needle probe (2)
3.Deep well slides (2)
4.Farmer’s fixative
  75 ml ethanol
  25 ml glacial acetic acid
5.Insect Saline
  100 ml distilled H2O
  0.9 g NaCl
6.Ethanol

Protocol
1.Beetles should be held on ice for 10 minutes prior to dissection.
2.Place the beetle in a drop of insect saline solution on a depression slid.
3.Orient the beetle so that the ventral side is facing up.
4.Using broad forceps apply increasing pressure to the metasternum.
  • This pressure will cause the abdomen to begin distending and the 8th sternite as well as part of genitalia will evert.
5.Grasp the everted genitalia and gently pull away from the beetle.
  • When this is done you will normally get the majority of the reproductive system separated from the beetles abdomen. Sometimes you also get a portion of the digestive system attached to the genitalia. If the gut pulls away with the reproductive system it is easy to recognize by the attachment of the darkly colored Malpighian tubes as well as the wider and rougher midgut. If the gut is present, it can be gently pulled away from the reproductive organs. In males it is common for one or both of the testes to remain in the abdomen when the rest of the reproductive system is pulled away. If this occurs, you will have to dissect the abdomen to remove them. The testes are approximately .6mm in diameter and are usually lightly attached to the ventral side of the 7th abdominal segment and are easily teased away. Each testis is made up of 6 globular testicular follicles which are tightly attached to the vas deferens.

6.Use the needle probes to remove excess tissues that are stuck to the genitalia by gently agitating the tissue or the solution around it.
7.Once the structures of interest have been isolated they can be moved into the fixative.
8.Tissues should be soaked in the fixative for 5 minutes.
9.Move tissues into ethanol and refrigerate.

  • Once in ethanol the tissue should remain viable for a number of months or even years if refrigerated.
Here are three pictures of what you will actually see when you start pulling the genitalia out.  The pictures are my own while the line drawing is from the Biology of Tribolium Volume I by A. Sokoloff.

1 comment:

  1. Hello! I just found your blog and I want to say that it's an amazing resource, not only for beetles, but for the ways you learn about them. I'm currently an undergrad at a jc, taking my general education courses, but I hope to transfer to UC Riverside and get a masters. :)

    I love beetles too, and I want to study them exclusively someday! Thank you so much for posting all the cool and curious content on your blog.

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