The overall goal of this procedure is to assess physiological parameters of the feeding in fluid-feeding insects using a sucrose solution mixed with fluorescent magnetic nanoparticles. This method can help answer key questions in the entomology field such as structure function relationships of insect mouth parts and insect feeding mechanisms. The main advantage of this technique is that it allows us to determine if insects are able to ingest fluids that are present either as pools or as liquid films.
This demonstration utilizes blue bottle flies because their fluid uptake abilities and mouth part morphology are well documented. House flies, cabbage butterflies, and other fluid feeding insects could be used. Obtain fly pupae and place them in an environmental chamber with 18 hours of light per day and at 23 degrees Celsius.
After adults emerge from their pupae do not feed them before the experiment. For the experimental food prepare 20 percent sucrose solution with 0.5 milligrams nanoparticles per milliliter. Then, prepare a feeding station consisting of a manipulator, a clamp and a feeding stage.
On the feeding stage, hold the food with a concave slide. Also, prepare various nylon net filters and membrane filters of different pore sizes to cover the concave slide. Begin with wrapping the insect's bodies, legs and wings into a folded tissue paper.
Leave only the head and mouth parts exposed. While doing this, keep the insect secured by pinching it's wings together. Now, secure the wings wrapped in tissue paper between two microscope slides.
Then clamp the two slides and secure the clamp to a manipulator. Then, secure the insect above the feeding stage using the manipulator. Next, deposit 30 microliters of feeding solution into the middle of the concave slide and cover the solution with a porous piece of filter paper.
The droplet will spread along the filter paper filling the pores. Next, position the insect so the distal regions of the mouth parts can make contact with the wetted filter paper such that only the flat part of the slide can be reached and the insect cannot feed from the concave region. Now, use an insect pin to extend the mouth parts onto the filter paper and allow the insect to feed for 45 seconds.
If the insect does not express an interest in feeding hold the mouthparts to the filter paper for the fill 45 seconds using the pin. This completes the feeding protocol. For the dissection, load a 50 milliliter watch glass with enough PBS to cover the insect's body.
Set this up along with all the dissecting tools at the microscope. Now, remove the insect from the tissue paper keeping its wings closed. Then, remove the head, legs and wings using spring micro-dissecting scissors and place the thorax and abdomen into the PBS.
To dissect the gut, first take hold of the distal abdomen cuticle and cut the cuticle in an anterior direction along the lateral side from the posterior end to the thorax. Take special care to ensure that only the cuticle is cut and that the alimentary canal is not damaged. Next, remove the abdominal cuticle, fat body, and other structures with the assistance of insect pins until only the thorax and alimentary canal remain in the watch glass.
Now, identify the crop. It's a sack-like structure, located near the juncture of the thorax and abdomen. If needed, make additional incisions into the thorax to reveal the crop.
Then, cut away the remaining thorax leaving only the alimentary canal and the crop. To test for the presence of ingested nanoparticles begin by using a fine-point dissecting forceps to transfer the crop to a cover slip for imaging. Use care to prevent rupture of the crop.
Immediately image the crop on an inverted confocal microscope at 20 times magnification using the CY3 channel or phase contrast. To identify ingested nanoparticles, wave a magnetic stir bar about 10 millimeters from the crop. Make each back and forth motion take approximately one second.
While waving the magnet, slowly move the operating stage to scan for nanoparticle movement inside the nearly transparent crop. If the nanoparticles are present in the crop they will wave in unison with the magnetic stir bar. An insect feeding on solution from a porous surface must create a stable liquid bridge via plateau instability to overcome the capillary pressure that keeps the liquid in the pores.
It is predicted that the size of an insect's feeding conduit will determine the smallest pore size from which they can feed. After insects were dissected to assess if nanoparticles were ingested, their mouth parts were imaged to measure the size of the food canal conduit. For flies, the diameter of the pseudotrachea and the oral opening were measured.
Analysis of the ingested nanoparticles suggests a close relationship between the distal mouth part conduit size, and the smallest pore size from which an insect can feed. This relationship is not seen with the proximal mouth parts. Consequently, fluid uptake for dipterans would first occur in the pseudotrachea rather than the oral opening and capillary action must have an important role in feeding.
After watching this video, you should have a good understanding of how to feed insects a nanoparticle solution, dissect them to isolate their crop, and then inspect the crop for the nanoparticles. While attempting this procedure, it is important to be careful during the dissections. If the crop is ruptured, it can spill the liquid contents and nanoparticles thus ruining your sample.
