Effects of Frass on Tobacco Hornworm Feeding Choices: Implications for Insect Communication

The presence of conspecific insects or evidence of prior feeding may inform larval feeding site choice by indicating the presence of potential competitors on a host plant. Frass contains plant defense and competition indicators that Manduca sexta (Tobacco Hornworm) can detect, which could be used to predict feeding success at that site. This study attempts to determine if Manduca sexta larvae respond to conspecific frass at potential feeding sites to avoid competition. 

We asked if Manduca sexta frass acts as a cue of competition that deters conspecific larval feeding on host plants. 

Frass Production

• Frass used as a conspecific competitor cue was collected from 4th-instar larvae fed on an artificial diet mixed with Solanaceous plant material.

Choice Trials

• Larvae were offered a choice between leaves without evidence of competitors (control leaves) or those with frass or both frass + a conspecific larva (Figure 1).

• A small amount of frass was smashed into filter paper under leaves, and a few frass pellets were placed on top of the leaf as visual cues. 

• 3 different instar stages were tested to determine if age affects choice.

Data Collection

• Choices were collected ~2 hours and 24 hours after set up. 

• Feeding damage was visually assessed to determine larval choice (Figure 2)

• Competitors and trial larvae were assessed for molting. Competitors were used during molting to avoid movement and feeding. Experimental larvae that did not eat during the trial due to molting were excluded (Figures 3 and 4). 

Results

No Feeding Preference

• Across larvae, frass did not affect feeding choice (n = 55), with 58.2% choosing leaves with frass and 41.8% choosing control leaves (binomial test, P = 0.28) (Figure 5).

Larval Age

• Larval age did not affect choice (second Instar: P = 0.07, third Instar: P = 0.31, fourth Instar: P = 0.60) (Figure 5).

Conspecific Larva

• Molting conspecifics did not influence choice among fourth instar larvae (P = 0.60) 

Discussion

Manduca sexta Larvae Ignore Competition Cues

• No evidence that Manduca sexta avoid eating leaves with either direct or indirect competition cues (conspecific larvae (Figure 6) and frass presence (Figure 5) respectively). 

Manduca sexta Adults May Determine Larval Feeding Sites

• Other studies suggest that Manduca sexta may avoid competition by oviposition rather than larval choice7

Lab Conditions May Alter Response

• Chemical composition of frass may have affected the larval choice (frass was from larvae that were reared on a partly artificial diet rather than fully leaves) 

Next Steps

• Next step: test with moth species may display higher conspecific competition (e.g. those with high cannibalism rates)

•  Test with field-collected Manduca sexta, or lab-raised larvae reared entirely on plant diets. 

Larval and plant care and project discussions: Alec Bullard and Laszlo Leedy

All photos by Cole Coomer. 

1. Denno R. F., M.S. McClure, J.R. Ott. 1995. Entomology.

2. Agelopoulos N. G. and M.A. Keller. 1994.  Journal of Chemical Entomology. 

3. Zhang XG, et al. 2019. Neotropical Entomology. 

4. Self L. S., F.E. Guthrie, E. Hodgson. 1964. Journal of Insect Physiology.

5. Blakstad J.I. et al. 2023. Biocatalysis and Agricultural Biotechnology.

6. Mutyambai D. M. et al. 2005. Scientific Reports. 

7. Zhang, J. et al. 2019. PNAS.

1. Teamwork/Communication: We coordinated every aspect of this project through lab notebooks and task lists, ensuring that each task got done efficiently

2. Critical Thinking: Since Manduca sexta showed no preference, we developed alternative hypotheses based on literature reviews and personal experience in the lab. 

3. Technology: Creative used for Google Suite products enabled constant, active team communication during both experimental work and colony care

4. Career & Self-Development: Living Manduca sexta provides opportunities for development across numerous fields, including animal welfare, experimental design, and field collection processes.