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.
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 preparation takes approximately 5 minutes to perform.
Before beginning it is important to have ready several fine dissection pins (see below and Part 2) and a dissection needle (see Part 2). Commercially available dissection pins and needles are often too large for this protocol, so we have taken to fabricating our own in lab. To create the dissection needle, tungsten wire (0.005″) is first threaded through the shaft of hypodermic needle (27G, aluminum hub). Thread the wire through the tip of the hypodermic needle so that it emerges from the hub. Crimp the wire protruding from hub; this prevents the wire from being pulled out of the hypodermic needle. Next, cut the wire at the opposite end of the crimp such that several millimeters (~3-4mm) of wire protrude from the tip of the hypodermic needle. Passing the tip of exposed tungsten through a flame will sharpen the wire to a very fine point. A disposable 5ml plastic pipette when wedged into the hub of the hypodermic needle acts as convenient handle for this dissection tool. The fine dissection pins are made in a similar way. After the sharpening step, grasp the very tip of the wire firmly with forceps to prevent it from moving and make a cut approximately 3mm from the tip to release the small dissection pin. Use care to prevent pin from being flung from the grasp of the forceps.
To fillet prepare the adult Drosophila abdomen:
Part 2. Oenocyte dissection
The abdomen fillet preparation exposes the internal surface of the abdominal cuticle and attached tissues including the heart, fat body, tracheae, body wall muscles, epidermis, and the oenocytes. The heart lies along the dorsal midline. Fat body (opaque tissue) covers most of the internal cuticular surface. Tracheae (white tubular structures) extend from the lateral spiracle openings, and make extensive branches which infiltrate these tissues. Lying beneath these tissues, the pigmented oenocytes (amber-colored cells) radiate from the midline to the lateral edge of the cuticle in the posterior region of each segment. The oenocytes lie directly beneath the darkly tanned regions of each tergite (dorsal cuticular plates) and can be difficult to identify without prior knowledge of their location. Another population of ventrally located oenocytes is associated with the sternites (ventral cuticular plates); although it is possible to isolate these cells, this video protocol will only demonstrate the dissection of the dorsal oenocytes. In each abdominal segment, a prominent leaf of fat body tissue lies anterior to the oenocytes; a smaller less obvious leaf of fat body lies posterior to the oenocytes. The prominent leaf of fat body tissue often covers the oenocytes of the segment preceding it. The dorsal vessel and fat body must be removed first in order to expose the oenocytes.
It is important to note that the amber pigmentation of the oenocytes, from which the their name derives (oeno– from Greek oînos, ‘wine’)1, clearly distinguishes these cells from the surrounding tissues; the intensity of the pigmentation increases with the age of the fly. Moreover, a basal lamina ensheathes the oenocytes2,3, providing support to the cell clusters and preventing the cells from becoming dissociated during the dissection. The basal lamina also aids in the removal of extraneously adhered tissues, such as fat body tissue, by creating a weak point where these adhesions can be easily disrupted.
The procedure outlined below provides detailed instructions for the removal of the oenocytes from the abdominal integument. The oenocytes can be easily removed from abdominal segments 2-5 (male), and 2-6 (female). The oenocytes from the first and last abdominal segments (segments 1, and 6 in males or 7 in females) are often disrupted by the dissection pins holding the corners of the cuticle and are therefore avoided. The removal of the oenocytes takes approximately 10 minutes to perform.
To isolate the adult oenocytes:
Figure Legend:
Figure 1. Expression of the desaturase genes, desat1 and desatF, in male and female oenocytes of D. melanogaster. Reverse transcription-PCR amplification of desat1 and desatF from cDNA derived from adult male and female oenocytes. The desat1 gene is expressed in both male and female oenocytes; desatF expression is sexually dimorphic, being expressed exclusively in female oenocytes. Corresponding to these differences in gene expression, desat1 is required for the production of hydrocarbon compounds common to both males and females9, and desatF is involved in the production of female-specific hydrocarbon compounds10,11. desatF has been shown to be specifically expressed in female oenocytes by in situ hybridization12. Note that on average 15-20ng of total RNA is obtained from the oenocytes dissected from an individual fly. RNA was isolated using the RNeasy micro kit (Qiagen); cDNA was produced using the qScript cDND synthesis kit (Quanta BioSciences)
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. A protocol for an oenocyte explant culture would aid investigations aimed at determining the autonomous metabolic capacity of these cells to synthesize and secrete the chemical constituents of cuticular hydrocarbons. These techniques in combination with a recently developed oenocyte-Gal4 line4, which permits the targeted genetic manipulation of the oenocytes, will allow for the detailed molecular genetic analysis of oenocyte function in the adult fruit fly.
Oenocyte cells are found both in the larva and the adult fly. While these cells share many morphological and ultrastructural similarities, it is important to note that the oenocytes of the adult fly are developmentally distinct from those that differentiate during embryonic development and exist during larval stages5. The adult oenocytes have been reported to be derived from the histoblasts6, forming de novo during pupation and continuing to increase in number during the first week of adult life7. While the oenocytes of the adult fruit fly are required for the production of cuticular hydrocarbons4, it remains to be determined if larval oenocytes support a similar function. Likewise, larval oenocytes maintain hepatocyte-like functions with regard to lipid metabolism8; a similar role has not yet been shown for the oenocytes of the adult. The preparation demonstrated here will aid investigations that are aimed at determining the degree to which the larval and adult oenocytes are functionally similar.
The authors have nothing to disclose.
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Sylgard | Dow Corning | |||
Tungsten Wire | Ted Pella, Inc. | 27-11 | 0.005”/20’ | |
Forceps | Dumont | 11252-30 | #5 | |
Shields and Sang M3 Insect Medium | Sigma | S3652 | ||
RNeasy Micro Kit | Qiagen | 74004 | ||
qScript cDNA Synthesis Kit | Quanta Biosciences | 95047-100 | ||
Monoject Hypodermic Needle | Harvard Apparatus | 722289 | 20G1 with aluminum hub |