Method Article

Dissection of Oenocytes from Adult Drosophila melanogaster

DOI:

10.3791/2242

July 18th, 2010

In This Article

Summary

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In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Abstract

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In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Protocol

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Part 1. Fillet preparation of the adult Drosophila abdomen.

The procedure outlined immediate below prepares the abdomen for the subsequent removal of the oenocytes (see Part2 of protocol). The same procedure can also be used for the preparation of the other tissues attached to the inner surface of the cuticle, including the dorsal vessel (i.e. heart) and fat body.

Note that in the video article the oenocyte dissection technique is demonstrated on an adult wild-type (Canton-S) male fly, six days of age. Identical techniques can be used to remove the oenocytes from female flies. The abdomen fillet preparat....

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Discussion

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In this video article we present a detailed dissection protocol for the preparation of the oenocytes from adult D. melanogaster in a manner suitable for molecular analysis. An abbreviated text-based account of the dissection method has been described elsewhere2, and the resulting oenocyte preparation has been demonstrated to be appropriate for the extraction of both RNA and protein2. Using this preparation it may also be possible to develop methods for the culturing of explanted oenocytes........

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Disclosures

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No conflicts of interest declared.

Acknowledgements

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We would like to thank Amsale Belay for her assistance in filming of this video article. This work was funded by CIHR, NSERC and CRC grants to JDL.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
SylgardDow Corning
Tungsten WireTed Pella, Inc.27-110.005”/20’
ForcepsDumont11252-30#5
Shields and Sang M3 Insect MediumSigma-AldrichS3652
RNeasy Micro KitQiagen74004
qScript cDNA Synthesis KitQuanta Biosciences95047-100
Monoject Hypodermic NeedleHarvard Apparatus72228920G1 with aluminum hub

References

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  1. Wielowiejski, H. Ueber das blutgewebe der insekten. Zeit. Wiss. Zool. 43, 512-536 Forthcoming.
  2. Krupp, J. J., Kent, C., Billeter, J. C., Azanchi, R., So, A. K., Schonfeld, J. A., Smith, B. P., Lucas, C., Levine, J. D. Social experience modifies pheromone expression and mating behavio....

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Tags

Oenocyte DissectionCuticular HydrocarbonsAbdominal IntegumentFillet PreparationTungsten NeedleFat Body RemovalBody Wall MusclesBasal LaminaCell Lysis Buffer

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