#ib427

Introduction:

Manduca sexta is a model organism used in insect physiology, but what goes on inside is still not completely known. There are still so many things to be discovered!

M. sexta is commonly found in the United states, most commonly in the gulf coast states. It has the potential to defoliate many garden plants, so keep an eye on that garden! They feed on Solanaceae plants like tomatoes but will also attack eggplant, peppers and potatoes. At first they can be hard to spot because of their small size, but don’t be fooled-- in just a few weeks they can grow as fat as a grown man's pointer finger (10 grams)-- that’s when the damage comes from these eating machines.

I will be focusing on the life cycle of M. sexta and molting mechanisms. Many of these mechanisms are seen in holometabolous (pupal stage before adult) insects. Some examples are other butterflies and moths like the monarch butterfly.

A little over 3 weeks ago, on January 22nd, I started rearing M. sexta along with my classmates in order to study the life cycle and molting responses. Every week day I would check on my caterpillars and every two days wash and replace their artificial food. Two of my 5 eggs successfully hatched and I’ll be referring to them as ‘Cat 1’ and ‘Cat 2.’ These names correspond to the order they hatched in. Along with rearing M. sexta, I also ligated two Elateridae larvae to prevent molting.

Life Cycle:

Manduca sexta start out as eggs, as all insects do. From there they go through 5 instars before pupation. Their entire life cycle from egg to pupae lasts about 2-3 weeks and Dyar’s rule states that the size increment of each molt stays constant (Grunert, 2016). The complexity of M. Sexta’s life cycle have a lot to do with hormones. Some hormone or hormone-like mechanisms I will mention are: JH (juvenile hormone), PTTH, and ecdysteroid. These are very important in the life cycle as they regulate and control growth and the molting process. Hundreds of scientific papers regarding M. sexta will incorporate JH, PTTH, and ecdysteroid as key mechanisms for growth and development.

The life cycle of M. sexta is known in the world of entomology as a great physiological tool to study. It has become a model organism because of the ease in which it can be kept in laboratories and fed an artificial diet. Many people who study parasitoid wasps also rear Manduca sp. caterpillars to study different mechanisms within that complex parasitism.  

Molting:

Molting is a complicated process that happens after each instar of M. sexta. It is also a very vulnerable time in an insect's life, because after molting an insect must allow the new cuticle, or skin, to harden.

The steps in molting include:

The intermolt stage

Apolysis: The separation of epidermis from cuticle

Secretion of fluid & growth of epidermis

Secretion of new cuticle

Activation of enzymes in molting fluid

shedding of old cuticle

The video provided below, From Larvae to Pupae, is a great way to see what happens when all of these steps line up.

In the figure above it’s easy to see how these mechanisms inhibit or promote growth. When looking closely every molt has large peaks in ecdysteroid and PTTH which are important factors in telling the organism, in this case, M. sexta, to molt. Interestingly it’s only when both of these are available that the caterpillar does molt.

Growth is exponential until adulthood. All of the nutrients are stored for energy to find a mate and pupate. The entire body changes during pupation and ensuring enough energy for that strenuous project is a priority to the caterpillar. Eat, eat, eat!

JH helps determine the pupation stage and in some insects it also fluctuates with molting, though it is not shown in the example above. When the critical weight is attained and at the same time the stretch receptors receive the message it’s time to pupate JH drastically decreases. It’s easy to think of JH like this: The more JH the more likely to pupate. The reason it peaks again in adulthood is for another purpose having to do with egg production.

Ligation (M. sexta and others):

Ligation is the act of cutting off the flow of an insect’s hemolymph (or blood) in order to stop the molting process. This works because the mechanisms needed for molting are found in the hemolymph. We do this by tying a string or dental floss, tightly enough to stop bloodflow, around the insect. In different species the segment of the body where ligation occurs is different. For example, in M. sexta ligations are to be done between the first and second abdominal segments with dental floss. In other insects, like Elateridae (click beetle) larvae and fly larva it is different and it can sometimes be difficult to find the correct time and place to ligate.

Out of 2 Elateridae larvae I successfully ligated one (figures 7-9). The other larvae successfully molted even with the sewing thread tied around the first and second segment. The fly larvae unfortunately perished shortly after ligation occurred and would probably be deemed unsuccessful by just about anyone who sees it (figure 10).

With Cat 1 and Cat 2, the M. sexta caterpillars, I was unable to ligate them because of the inability to tell what instar they were at. When living together in the same cup it was impossible to tell what instar the caterpillars were at: As Cat 1 began to grow, Cat 2 was unable to feed and I moved him to his own cup after reaching about one inch in length. The head capsules were commonly found at the bottom of the cup but because they weren’t separate I again wasn’t able to use the head capsules to identify which instar they were at.

Rearing

Every day I would check on my M. sexta caterpillars. Along with taking pictures of them, I would rinse their cups, wipe them of excess water, and give them fresh food. The diet that was fed to the M. sexta is artificial and made with Wheat Germ and Casein as the main two ingredients. There were multiple times the food was completely consumed. This was especially true when my M. sexta reached the 4th and 5th instar.

When I first got my eggs I was very excited. They were already in a cup with food so all I had to do was wait a few days before they hatched (figure 1).

Once they finally did hatch two days later they were very, very small. In fact, they didn’t even weigh one gram (figure 2).

Washing and drying the cup for the first time was a simple task. Carefully take the caterpillars out with forceps and rinse the cup with water and dry out the cup with a paper towel.

They started to grow at an exponential rate so more and more food was required. I found the frequency at which I checked them was ever growing quicker. The frass (poop) started getting larger and they would trample all over it and their food. For that reason a nice washing was needed every so often.

As of right now my caterpillars are still growing. Cat 1 is in his 5th instar and Cat 2 is still in his third or fourth. I hope to rear them to adulthood. 

The hardest part about rearing is finding time to check on your caterpillars. I did not have them in my room, but in a different building about a 5 minute bike ride away. In the cold that’s a long way. If I were to do this experiment again I would likely take my caterpillars and some food to my room to watch them grow.

I also became quite attached to my caterpillars and was very relieved when the ligation of the Elateridae larvae was successful.

Videos to watch before you start rearing:

Rearing M. Sexta

https://www.youtube.com/watch?v=ZLYRcOLkhGA

Great if you want to get started rearing your very own Manduca. 

From Larvae to pupa

https://www.youtube.com/watch?v=qsRFF8i7Bhw

This video shows the final (5th) instar pupating. It is easy to see the epidermis separate from the cuticle.

Caterpillar Feeding: A matter of taste- Cornell University (Great video!)

http://www.cornell.edu/video/caterpillar-feeding-with-marta-del-campo

Wonderful video about the feeding and different environmental controls the Manduca need to grow properly. 

Cat 1 Eating:

https://www.youtube.com/watch?v=PjZfyBuCCek&feature=youtu.be\

My Caterpillar eating the artificial diet provided.

Blogs on M. sexta:

https://neuroecology.wordpress.com/tag/manduca-sexta/

Why do humans like certain foods?

Primary Literature:

Asuncion-Uchi M, Shawa H, Martin T, Fuse M. Different actions of ecdysis-triggering hormone on the brain and ventral nerve cord of the hornworm, Manduca sexta. General & Comparative Endocrinology [serial online]. March 2010;166(1):54-65. Available from: Academic Search Complete, Ipswich, MA. Accessed February 16, 2016.

Grunert, L. W., Clarke, J. W., Ahuja, C., Eswaran, H., & Nijhout, H. F. (2015). A Quantitative Analysis of Growth and Size Regulation in Manduca sexta: The Physiological Basis of Variation in Size and Age at Metamorphosis. Plos ONE, 10(5), 1-23. doi:10.1371/journal.pone.0127988

Novicki A, Weeks J. The initiation of pre-ecdysis and ecdysis behaviors in larval manduca sexta: The roles of the.. Journal Of Experimental Biology [serial online]. August 1996;199(8):1757. Available from: Academic Search Complete, Ipswich, MA. Accessed February 16, 2016.

Pictures:

Figure 1 (below):

Figure 2:

Figure 3:

Figure 4:

Figure 5:

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Figure 10:

Our experiment, as with many experiments in zoology, has been geared at gaining a better understanding of organic systems. The insect endocrine system, while differing in its effects and capacity for whole-body change, has its parallels with our own. Insects are hardy and able to survive invasive procedures of brain removal and transplanting that would be too dangerous or unethical to be attempted on vertebrates (4). Testing the effects of removing key components may lead to the discovery of alternate sources of chemical signals in the insect body.

4. Wigglesworth, V. B. 1936. The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera). Quart. J. Micr. Sci 79: 91-121.

As for our difficulties, many replicates may be needed in order to eliminate the individual responses of our test organisms. Ideally, we would try to simulate the natural diet and environment of our test species as closely as possible in order to gain a more precise understanding of how these systems work. Unfortunately for us, Manduca aren’t very hygienic organisms in a small environment. Frass, bacteria, and fungi are a constant concern, so that the containers had to be cleaned every two days. As it stands, our experimental design appears to have allowed the Manduca to survive until the point of pupation and possibly beyond. 

I placed one of the Manduca in a dark container over the weekend, as it had exhibited wandering behavior.  Since then, it’s heartbeat has become much more visible on the dorsal integument and its head has become slightly translucent. The stumpy prolegs have become much less firm in shape, a possible sign of internal changes. I assume that this is a precursor to the final molt. 

The other Manduca is still eating, though its pulse is also becoming visible. It may be ready for the wandering phase within the next day or so.

The Manduca appear about the same. Looking at the mealworm that was ligated as a practice exercise, there doesn’t appear to have been any development on either end of the tie. Based on what I have seen of other student’s specimens, I would have expected at least one molt of the forward segment. The rear end looks a little larger, but there is no discarded cuticle in the container.

It should be noted that there have been a few reports of molting hormone being produced in isolated abdomens in this species as well as others (3). It is unlikely that this is a perfect substitute, but it may explain our failure to see any significant differences between the fore and posterior segments after several weeks.

Hsiao, T., Hsiao, C., and J. De Wilde. 1975. Moulting hormone production in the isolated larval abdomen of the Colorado beetle. Nature, 255, 727-728.

They appear to be eating much more than previously noted. Given their size, I assume it is still around the same rate, though I must note the duration between molts.

Both Manduca have been very agitated since the ligation, though they continue to eat. It may be necessary to separate them soon due to wandering-stage aggression.

Both Manduca appear to have molted over the weekend. The ligations don’t appear to have stayed in place. I have replaced them, but it will be anyone’s guess whether or not we will see the predicted partial molt.

Ligating behind the thorax and before the abdomen should allow us to see a clear division in molt activity for each ligated caterpillar. A tight ligation will cut off chemical signals from the brain as well as the prothoracic gland. We would expect to see slower development in the separated portion (2).

Most early insect ligation experiments noted the effects of ligating behind the brain before or after a critical period. Ligating before the critical period would inhibit development, while ligating after this period would have little effect on development. Timing is key in these experiments in order to correctly influence the development of a chosen instar.

Safranek, L., Squire, C., and C. Williams. 1986. Precocious termination of diapause in neck- and abdomen-ligated pupal preparations of the tobacco hornworm, Manduca sexta. Biol. Bull, 171, 126-134.

There hasn’t been much change since the last few inspections. Both of our subjects remain at the fourth instar. The non-ligated Manduca appears non-responsive during care and maintenance. This could be a sign that they will be advancing to the fifth instar over the weekend. Unfortunately, I will not be around.

For a quick look at what the final instar will look like, skip to the end of this video.