Wonders of Wisconsin: Insect Macrophotography

Here are my ligation experiment results! Since I performed the ligation after the head critical period, molting hormones were already circulating through the larva, and indeed he molted on both the front and half end. However, the problems with ligating include the struggle to actually get out of the old cuticle. This is a problem because the cuticle also gets shed from the tracheal system and the fore and hind-guts. It has been three days since he tried to molt with the ligation, and has not escaped. This means that he has not been able to feed or excrete wastes. I feel bad watching him struggle. These issues I believe have arisen from the ligation and not the hormones.

Last night I helped my larva get out of part of his old cuticle. It took a while, and he was not happy that I was poking him, but I managed to free his pro-legs (the five pairs of leg-like structures on the abdomen) and clear out the spiracles. In the image you can see the head capsule (yellow/tan color over the head) and part of the new cuticle. The old cuticle on the front half has come off the first segment of the body, but has yet to get past the front pair of legs.

Manduca sexta is a pest species, and there are many ways to control them in the wild. One way would be to use pesticides to kill the caterpillars, but this is not the only way. Nature has fostered interactions between these larvae and a parasitoid wasp. The adult female wasp lays an egg inside the caterpillar. This one egg will then split to form multiple clones of itself. This particular wasp (Trichogramma nubilale) is an extoparasite, meaning it develops on the outside of the caterpillar. They look like small white cocoons on the back of the caterpillar! Below is a link to a paper discussing the rearing process of these wasps on M. sexta for biological control.

The reason people experiment on insects is multifaceted. Insects are generally easy to rear and can tell us a lot about many physiological functions that can even be related back to human disorders (Savva et al. 2016). Figuring out how different physiological pathways work under varying conditions in insects can translate into understanding those same pathways in humans, and can lead to treatments and cures. Check out the other work being done using Drosophila as the study organism at Brown University! https://vivo.brown.edu/display/rreenan.

As a class, we practiced ligating beetle larvae in preparation for ligating our Manduca sexta. Ligation was first performed by Stefan Kopec in his papers published in 1917 and 1922. By restricting the flow of hormones from the upper half of the larva (the brain) and the lower half (the abdomen), Kopec found that the brain actually controlled the initiation of molding. Our class will be conducting similar experiments by tying dental floss around the larva’s abdomen between the first and second segments at different larval developmental stages. I will be performing the ligation procedure two days after the mold between the third and fourth instars.

These little guys are now in the 3rd instar stage (since 2/1/16). I have been watching them both closely for a few days now. Once they molt into the larger 4th instar, I will wait two days to perform ligation. This molt should have occurred on February 3nd (2-3 days after the second molt into the 3rd instar stage), but they have yet to do so. It could be that the diet cube was very dry and not providing the proper nutrients or hydration they needed. After I changed it, they grew in size a little, so the molt into the 4th instar stage should be soon!

The tobacco hornworm (Manduca sexta Linnaeus)(Lepidoptera: Sphingidae) is a sphinx moth that, as a caterpillar, is a major crop pest. The adult moth is not a pests; it is actually a pollinator of many plants. This organism has been used for laboratory experiments because they are large and are easy to rear and manipulate to observe the life cycle. For the insect physiology course at the University of Illinois at Urbana-Champaign, we are rearing these moths to test the effects that hormones have on the process of molting and pupation. The next few posts will be about the rearing process.

Here is the first image of the M. sexta eggs we received on January 22, 2016. They were placed on an artificial diet cub in plastic lidded cups for when they hatch. The artificial diet is a mixture of many ingredients, including wheat germ, yeast, cholesterol, acids and sulfates, and sugars. This is different from what the caterpillars would normally feed on in the wild.  The larvae prefer plants from the family Solanaceae, the nightshade plants which include tobacco and tomato. With both natural and artificial diets, M. sexta spend much of their time (up to 80%) in feeding bouts, and it has been observed that this behavior occurs with both natural and artificial diets (Reynolds et. al. 1986).

 I will be posting more images in information about the M. sexta during the next few weeks, stay tuned to see some cute caterpillars and learn about the laboratory technique of ligation!