Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The salivary glands of insects, are required for transmitting many human diseases. So a better understanding, of salary gland biology should help us, add to existing strategies for disease prevention. This technique will allow us, to quickly ask if mutations in the candidate regulators, affect the morphology, of the mosquito larval salivary glands, and potentially affect the transmission, of malarial parasites.

The first few times you work with the mosquito larvae, they are challenging to grasp as they move quickly. The more you practice, the better you get. Getting started with the procedure, will be Marisol Luchetti, an undergraduate research assistant for my laboratory.

To start the experiment, place a potty plate on the stage of a dissecting microscope, and transfer 10 mosquito L4 larvae onto the potty plate. Then place a drop of the dissection solution, onto the potty plate separate from the larvae. Use forceps to place one L4 larva, into the dissection solution drop of 25%ethanol.

By holding a number five forceps in each hand, grasp the larvae with forceps just below the head, with the dominant hand, and grip the head of the larva with the non-dominant hand, then gently pull the head with minimal constant force, to detach from the rest of the body, while the glands remain attached to the head. After discarding the body portion of the larva carcass, collect the head or salivary glands, into one milliliter of PBS, in a 1.5 milliliter micro centrifuge tube, and wait for the salivary glands, to sink to the bottom of the buffer. On the next day, drain the solution to fixate the tissues, with one milliliter of cold 100%acetone for 90 seconds.

After removing the acetone, gently rinse the tissues three times, with one milliliter of PBS. For immunostaining, add a primary antibody such as Rab 11, at the appropriate dilution, in 200 microliters of the total volume of PBS. Swirl the tube contents gently with a pipette tip.

Incubate the primary antibodies overnight at four degrees, followed by washing three times in one milliliter of PBS. Then add the secondary antibody, Alexa Fluor 488 Goat anti-Rabbit, at the appropriate dilution, in 200 microliters of total volume. After mixing the contents with a pipette tip, incubate the tissues with dyes, such as Nile red, and Hoechst into 200 microliters of PBS, at room temperature for 60 minutes, followed by washing three times with PBS.

Prepare a microscope slide, by adding 200 microliters of 100%glycerol with a pipette, and transferring up to 20 stained heads, with the attached glands and internal structures, per microscope slide using a soft brush. Spread the head samples out, to evenly distribute them along the glass slide. Viewing under a dissecting microscope, separate the larval head from the glands, using two pairs of forceps, by carefully pulling the two tissues in opposite directions.

When all the samples are done, gently place a 1.5 millimeter thick cover slip, on top of the slide, avoiding the formation of air bubbles, and then seal the slide with clear nail Polish. The salivary glands are relatively easy to dissect, from stage four larvae. Male and female larvae can be distinguished, at the late L4 larval stage by a red stripe, along the dorsal thorax of females.

The antennal morphology is also more elaborate, in males than in female L4 larvae as can be seen, with the white arrows pointing out, the female antennae on the left image, and the male antennae on the right. The salivary glands isolated from early L4 stage larvae, stained with Hoechst reveal the proximal and distal lobes, separated by a narrow constriction. The forming lumen was examined, in the immuno stained distal salivary gland, of a mid to late L4 larva.

The apical domains of the secretory cells, surrounding the forming lumen had intense Nile red staining, suggestive of microvilli like structures. The Rab 11 staining was observed close, to the apical surface. There's only one step with the time, and that's the 90 second incubation, of the tissues in cold acetone.

This protocol was only recently developed, but we expect it will be helpful, to anyone working on the mosquito salivary gland. We fully expect to use it often.