A protocol for processing the delicate larval and prepupal salivary glands ofDrosophilafor scanning electron microscopy

Abstract

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton-derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four-step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination.The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate-based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline-based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point-dried, mounted on aluminum stubs, sputter coated with gold-palladium alloy and examined in the SEM. K E Y W O R D S delicate tissue, dirt removal, Drosophila melanogaster, salivary glands, SEM, surface contamination

Drosophila Glue: A Promising Model for Bioadhesion

“…On the substrate near the posterior part of the animal is often found a whitish material that appears to be expelled by the intestine prior to metamorphosis and that mixes partly with the glue that is expectorated from the anterior part of the larva ( Figure 2 B,C) [ 21 , 22 ]. Whether this whitish material contributes to the adhesive properties of the glue is unknown.…”

“…Scanning electron microscopy reveals that the surface of the glue is uniformly smooth and that its internal aspect is complex and structured [ 21 , 22 ]. The glue seems to be organized in thin layers separated by air bubbles [ 21 ] and is made of a multidirectional arrangement of thick fibers of various densities ranging from 30 to 90 nm diameter [ 22 ].…”

“…Scanning electron microscopy reveals that the surface of the glue is uniformly smooth and that its internal aspect is complex and structured [ 21 , 22 ]. The glue seems to be organized in thin layers separated by air bubbles [ 21 ] and is made of a multidirectional arrangement of thick fibers of various densities ranging from 30 to 90 nm diameter [ 22 ]. The variability in inner glue fiber thickness may serve as an elastic buffer that can accommodate mechanical stress exerted onto the animal and may allow its firm attachment to the substrate [ 22 ].…”

“…Unfortunately, there is currently no means to trigger glue expectoration. A recent study [ 22 ] that managed to collect glue monitored the larvae under a stereomicroscope until glue expectoration and required action within a few seconds before the glue solidified completely, which is a time-consuming approach.…”

“…The same suite of behavioral events accompanying glue expectoration was observed in D. virilis [ 41 ], which diverged from D. melanogaster about 45 million years ago [ 42 ]. After expectoration, the glue is liquid and it hardens in a few seconds, depending on air humidity, and becomes completely dry and solid after 3–5 min [ 4 , 22 ]. The movements of the larva during expectoration allow the glue to completely wet the body and increase contact with the animal surface topography by flowing into the folds and crevices of the cuticle, thus maximizing adhesiveness between the animal and the substrate.…”