Tobacco Hornworm: Memory Ability With A Food Source

By Abbey Audesse and Maiah Jones

Background and Significance: 

It’s well known that diet has an overall influence on the fitness of an organism. Humans have the ability to localize specific, nutrient rich foods and retain for later use, but do other organisms do the same thing? Male moths, and other species, have the ability to utilize the pheromones emitted by females to find a mate (Grunbaum and Willis). Throughout the lifetime of a moth, they carry this cognitive ability to recognize the pheromones emitted and determine what they are. At the School of Biological and Molecular Sciences, they explored the possible role of cell survival through metamorphosis as a mechanism of the persistence of memory (Ray 1). We would like to dive deeper into the aspect of memory within our study.

Insects undergo a metamorphosis experience, changing their complete morphology, actions, and lifestyle (Blackiston and Casey). During pupation, extreme lysis, neurogenesis, and reorganization of tissues all occur (Ray 1). We want to test the ability of memory and how it is stored throughout a species developmental periods. Moths and butterflies transform from crawling little creatures to fully winged adults. A phenomenon called ‘chemical legacy’ is the connection between the larval stage and adult stage in which exposures can be carried to (Blackiston and Casey). If aspects such as odour and host plant can be recognized from prior experience, food should be memory as well. In our study, we hope to identify if food preference can be a chemical legacy. If associated behavior from the larval stage, through the pupal stage, and into the adult is possible, then food preference should remain the same. 


Objective, Hypothesis and Prediction

The objective of this experiment is to test whether or not the Tobacco Hornworm, over the course of pupating, is capable of recognizing from past experience. We would like to test the ability to acknowledge previously exposed food sources. 

The specific hypothesis to be tested is:

If the Tobacco Hornworm is presented with two food sources, it will choose the one that it has had prior experience with. 

Our prediction includes: 

The Tobacco Hornworm will choose a food source it has had prior experience with.

Methods:
Studied Species
The Tobacco Hornworm or the Manduca Sexta is a commonly known pest in various parts of the world. This species of caterpillar is referred to as a “hornworm” because of its green worm-like body and the horn on its posterior. The tobacco hornworm is known as a pest for its diet consists of plants from the Solanaceae family. This family includes crops such as tobacco, tomatoes, peppers and eggplants (M. Byron et al). This diet of plants from this family plays a significant role in the biology of the tobacco hornworm. Plants in the Solanaceae family contain steroidal and triterpenoid glycosides, which are chemical compounds that can have an influence on food preference (M. Byron et al). When in the larval stage, these caterpillars can cause major damage to plants by eating all of the leaves off or the actual vegetable. We will be taking the full vegetables out of the picture and utilizing only the leaves. 

Housing/Diet
The tobacco hornworms lifespan lasts from 30 days to 50 days. Since we are managing food, we want to minimize the variety of foods it comes in contact with. We will start with hornworm eggs and feed them the ‘ready to use hornworm diet’ food. The larvae will receive constant light to increase the time of the stage. It will take around three days of the eggs to hatch and we will then start testing and observations. Once the larvae are 2 cm long they will be moved into separate plastic containers with a hole and wire covering for ventilation. The hornworms will have access to leaves and small branches to climb on. These branches will be in a small amount of water so that they don’t dry up. The hornworms will have access to food, and it will be different for each enclosure. Each enclosure will be exactly the same in regards to size and layout. The amount of food, temperature, and humidity will remain the same throughout.

Experimental Design:
In order to test if the tobacco hornworms will choose a food it has had prior experience with, we will have to create replicates. There will be a total of three enclosures held within the science center. In one enclosure, 5 tobacco hornworms will only be consuming a diet of a tomato plant, or Lycopersicon esculentum. In another enclosure, 5 tobacco hornworms will only be consuming a diet of a potato plant, or Solanum tuberosum. The third will be our control, contain 5 tobacco hornworms that have access to both a tomato and a potato plant. We will let the tobacco hornworms become accustomed to their particular diets. After pupating, we will be presented with moths. Due to moths laying eggs around 3-4 days after hatching, we will have to observe quickly. During the pupation stage we will transfer a different food source into the enclosures, keeping out control the same. For example, the hornworms with the tomato plant will be introduced to potato plants and vise versa. This way we will be able to observe whether or not the moths will prefer a food that it has prior experience with or if it will go for the newly introduced food. We will observe them eating for all three days to collect data on how many of the moths continued eating their pre pupating diet. We do not want to start the cycle over again unless necessary. Therefore, when we have completed the observations we will freeze the moths for 48 hours and dispose of them. 

Example of enclosure for eggs/larvae stage: 

Image from Carolina Biological Supply Co. 

Data Analysis
Within our study we will run data analysis on the number of moths that either ate or did not eat the pre recognized food. We will use a two tailed t-test to do this. We will also create a bar graph to visually display the number that used memory to choose a food source. 

References Cited
Blackiston DJ, Silva Casey E, Weiss MR (2008) Retention of Memory through Metamorphosis: Can a Moth Remember What It Learned As a Caterpillar? PLoS ONE 3(3): e1736. https://doi.org/10.1371/journal.pone.0001736
Byron, Morgan A, and Jennifer L Gillett-Kaufman. “Featured Creatures.” Tobacco Hornworm, Manduca Sexta (Linnaeus); Tomato Hornworm, Manduca
Quinquemaculata (Haworth), 2017, entnemdept.ufl.edu/creatures/field/tobacco_hornworm.htm.
Grünbaum, Daniel, and Mark A Willis. “Spatial Memory-Based Behaviors for Public Library of Science. “Can Moths Or Butterflies Remember What They Learned As Caterpillars?.” ScienceDaily. ScienceDaily, 8 March 2008. <www.sciencedaily.com/releases/2008/03/080304200858.htm>.
“Living Organism Care Guide: Hornworms.” Carolina Biological Supply, https://www.carolina.com/teacher-resources/Interactive/living-organism-care-guide-hornworms/tr10510.tr.
Locating Sources of Odor Plumes.” Movement Ecology, vol. 3, no. 1, Apr. 2015, doi:10.1186/s40462-015-0037-6.

Resources Required
3 Plastic storage container
About 20 Manduca s. Eggs
Water
Potato Plant
Ready ro use hornworm diet
Tomato Plant
Heated light source
Wire (to cover hole for vent)
Branches



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