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Induction Of Preference Or Oligophagy In Manduca Sexta Oluwaseye Ayoade, Howard Community College Mentored by: William C. Gretes, Ph.D. |
Abstract
The tobacco hornworm, larval Manduca sexta, is an oligophagous insect that primarily feeds on plants in the family Solanaceae. In a laboratory setting, however, M. sexta larvae can be reared on select non-solanaceous plants. Our research focuses on the relationship between the rearing plant and later feeding decisions of the larvae. This study attempts to provide evidence for an induction of preference (species-specific effect) or an induction of oligophagy (family-specific effect) within non-solanaceous plant families by observing the rate of consumption of non-solanaceous plants from larvae. The two plant families tested were Fabaceae and Brassicaceae.
Our data may suggest a family-specific effect within larvae reared on plants from the Fabaceae family. If such an effect existed, it would be expected that larvae raised on non-fabaceous plants would mostly reject those plants, but larvae reared on plants from the Fabaceae family would have a greatly increased acceptance of those plants. The data presented here seems to support this. Our data does not suggest a similar effect on larvae reared on plants from the Brassicaceae family, as these plants were generally accepted to larvae from all rearing meaning that prior experience had little effect on the palatability of these plants. Our findings suggest that the mechanisms that control feeding inductions in non-solanaceous plants are more complex than previous papers have suggested and certainly, future studies will be needed to better understand this.
Introduction
Manduca sexta, commonly known as the tobacco hornworm, is an herbivorous insect with an oligophagous feeding pattern. Larvae primarily consume plants within the solanaceous family, more commonly known as the nightshade family, such as tobacco, tomato, and eggplant. These insects are destructive as larvae due to their feeding habits, resulting in agricultural losses and necessitating increased insecticide use [1]. In the past, researchers established that a preference for a single species of plant can be induced within M. sexta larvae. However, there has yet to be substantive evidence pointing towards the potential for inducing a preference for an entire family of plants, rather than just a single species [2],[3],[5],[6]. The induction of preference to a plant family–referred to as induction of oligophagy–was first proposed by Yamamoto [2] in his experiments on tomato-reared larvae. Further research by del Campo & Renwick [3] added to the body of literature supporting an induction of oligophagy in M. sexta larvae raised on solanaceous plants [5]. Additionally, del Campo & Renwick later suggested a mechanism by which M. sexta that fed and were exposed to a plant compound unique to solanaceous plants became chemically tuned to that compound and would later reject all non-solanaceous plants [4].
Following this, Gretes et al. [5] sought to determine whether diet experience on solanaceous plants in early development would increase the palatability of all solanaceous plants or simply the rearing plant, effectively asking if oligophagy was induced or inherited. The experiments that Gretes et al. performed were a series of no-choice tests, wherein larvae that had been reared up to the fourth instar–a stage in the M. sexta larval cycle–on a rearing plant species were then evaluated on leaf discs from a test plant. The authors concluded that the universal acceptance of solanaceous plants in M. sexta larvae is innate and was present regardless of which plant the larvae were reared on [5].
Our research aims to expand on the results from previous researchers by rearing larvae on select non-solanaceous plants from two different plant families and testing how this modified the palatability of those same non-solanaceous plants. Our larvae were reared on a type of plant from one of two families, Brassicaceae (rapeseed, cabbage, turnip) or Fabaceae (cowpea, soybean, pea), and were given a no-choice test with a leaf from one experimental or control plant. By testing the larvae on these groups of non-solanaceous plants, we were able to determine a pattern of feeding behavior and whether larvae reared on plants from one family preferred other plants of the same family.
Methods
Study Site: This study was conducted in the greenhouse and research room at Howard Community College. The plants used for this study were grown at temperatures ranging from 21 to 26°C from Aug 29, 2025, to Oct 3, 2025. Manduca sexta eggs were obtained from Carolina Biological. Larvae were reared on either tobacco, cowpea, cabbage, soybean, pea, turnip, or rapeseed in an incubator kept at 27°C with 18 hours of light and six hours of darkness. Larvae were reared from Oct 9, 2025, to Oct 23, 2025.
Plant Preparation: Plant pots were filled about ¾ of the way with soil and Osmocote fertilizer and divided into trays. Each tray had 6 pots and was designated for one of the seven experimental plants. Three seeds from each plant were placed in their respective pots, and each tray was labelled with the plant and date of planting. All trays were left in the greenhouse with a water source and monitored daily. This process was repeated weekly until the M. sexta eggs were received, at which point leaves from each plant were cut off and placed into separate containers with six to seven M. sexta eggs.
M. sexta Rearing: Larvae were reared in containers inside an incubator located in the HCC greenhouse. The containers used were rinsed with water daily, and new leaves were added as needed. Observations on the growth of the larvae were recorded during this process. As larvae approached the fifth instar, they were isolated for data collection.
Data Collection: In the research room, each post-molt fifth instar larvae were placed into a Petri dish containing a single leaf from a plant of the 6 plant species. Each Petri dish was labelled with the date, rearing plant, and experimental plant. A GoPro camera was mounted overhead and took photos of the dish in 30-second intervals for 12 hours. After 12 hours, the hornworm was euthanized in a freezer at -80°C, and the images from the camera were extracted and stored on a USB stick. Each batch of photos was labelled with the date, rearing plant, and experimental plant. Once extracted, the batches were examined, and any sets of photos with any consumption were put aside for data analysis.
Data Analysis. The photos analyzed were from experiments done between Oct. 23, 2025, and Oct. 27, 2025, and from prior research groups, ranging from 2018 to 2024. Each batch of photos that showed consumption was analyzed using the software SketchAndCalc. An image from every hour was used to calculate the consumption rate by hour. The size in cm², measured by SketchAndCalc, of the leaves before consumption and after each hour was recorded in Google Sheets. The difference between initial leaf size and hourly leaf size was used to calculate the rate of consumption over time in Google Sheets. Once all the selected photos had been analyzed, the averages of all of the data were calculated and graphed. A graph was created for each plant that the larvae were tested on, depicting the averages of the data relating to it, and once all six graphs were created, the rate of consumption of each plant was compared through visual inspection of the generated graphs.
Results
Consumption of Plants from the Brassicaceae Family (Cabbage,Turnip,Rapeseed)
Figure 1: Average area of cabbage leaf consumed over 12 hours by 5th instar larvae reared on one of six experimental rearing plants, excluding soybean
Figure 1 depicts the average area of cabbage leaves in cm2 consumed over time by larvae for a 12-hour span, with data collected over 260 hours total using 21 larvae total. Rapeseed-reared larvae, from the Brassicaceae family, consumed the greatest area of cabbage leaves over 12 hours. Pea-reared larvae, from the Fabaceae family, consumed the lowest area of cabbage leaves over 12 hours. Turnip-reared larvae, from the Brassicaceae family, consumed a similar area of cabbage leaves as the pea-reared larvae over 12 hours. Cabbage-reared larvae, from the Brassicaceae family, consumed a similar area of cabbage leaves as the cowpea-reared larvae from the Fabaceae family over 12 hours.
Figure 2: Average area of turnip leaf consumed over 12 hours by the 5th instar larvae reared on one of the six experimental rearing plants, excluding soybean, pea, and cabbage
Figure 2 depicts the average area of turnip leaves in cm2 consumed over time by larvae for a 12-hour span, with data collected over 160 hours total using 13 larvae total. Turnip-reared larvae, from the Brassicaceae family, consumed the greatest amount of turnip leaves over 12 hours. Cowpea-reared larvae, from the Fabaceae family, and rapeseed-reared larvae, from the Brassicaceae family, consumed a similar area of turnip leaves over 12 hours.
Figure 3: Average area of rapeseed leaf consumed over 12 hours by the 5th instar larvae reared on one of the six experimental rearing plants, excluding cabbage, pea, and soybean
Figure 3 depicts the average area of rapeseed leaf in cm2 consumed over time by larvae for a 12-hour span. The data was collected over a period of 144 hours using a total of 12 larvae. Turnip-reared larvae, from the Brassicaceae family, consumed the greatest area of rapeseed leaf over 12 hours. Cowpea-reared larvae, from the Fabaceae family, consumed the lowest area of rapeseed leaf over 12 hours. Rapeseed-reared larvae, from the Brassicae family, consumed a lower area of rapeseed leaves than turnip-reared larvae but a higher area than cowpea-reared larvae over 12 hours.
Consumption of Plants from the Fabaceae Family (Cowpea, Soybean, Pea)
Figure 4: Average area of cowpea leaf consumed over 12 hours by the 5th instar larvae reared on one of the six experimental rearing plants, excluding soybean and cabbage
Figure 4 depicts the average area of cowpea leaf in cm2 consumed by larvae over a 12-hour span. The data was collected over a period of 240 hours using a total of 20 larvae. Pea-reared larvae, from the Fabaceae family, consumed the greatest area of cowpea leaves over 12 hours. Rapeseed-reared larvae, from the Brassicaceae family, consumed the lowest area of cowpea leaves over 12 hours. Cowpea-reared larvae, from the Fabaceae family, consumed the second greatest area of cowpea leaves over 12 hours. Turnip reared larvae, from the Brassicaceae family, consumed a lower area of cowpea leaves than cowpea-reared larvae and a higher area of cowpea leaves than rapeseed-reared larvae.
Figure 5: Average area of soybean leaf consumed over 12 hours by the 5th instar larvae reared on one of the six experimental rearing plants, excluding rapeseed, pea, and cabbage
Figure 5 depicts the average area of soybean leaves in cm2 consumed by larvae over a 12-hour span. The data was collected over 84 hours total using 7 larvae total. Soybean-reared larvae, from the Brassicaceae family, consumed the greatest area of soybean leaves over 12 hours. Turnip-reared larvae, from the Brassicaceae family, consumed the lowest area of soybean leaves over 12 hours. Cowpea-reared larvae consumed a lower area of soybean leaves than the soybean-reared larvae and a greater area of soybean leaves than the turnip-reared larvae.
Figure 6: Average area of pea leaf consumed over 12 hours by the 5th instar larvae reared on one of the six experimental rearing plants, excluding soybean, pea, rapeseed, and cabbage
Figure 6 depicts the average area of pea leaves in cm2 consumed by larvae over 12 hours, with data collected over 36 hours total using 3 larvae total. Cowpea-reared larvae, from the Fabaceae family, consumed the greatest area of pea leaves in 12 hours. Turnip-reared larvae, from the Brassicaceae family, consumed the lowest area of pea leaves in 12 hours.
Larvae Diet
Figure 7: Rearing plant larvae and the diet they were tested on. X in each position represents the presence of data representing a combination of host plant and reared larvae. Empty cells reflect a lack of trials due to insufficient plant supply or unviability of larvae to test
Conclusion
Plants from the Brassicaceae family appear to be broadly palatable to all non-solanaceous reared larvae tested. As seen in Figures 1-3, larvae reared on plants from both tested non-solanaceous families consumed plants from the Brassicaceae family, with little to no difference between the two groups. This may suggest that these plants are generally acceptable, which implies they contain relatively few chemical deterrents, meaning that feeding experience on these plants has little effect on their palatability.
Plants from the Fabaceae family were not broadly palatable to all non-solanaceous reared larvae tested. Only larvae reared on Fabaceous plants, as seen in Figures 4-6, consumed plants from the Fabaceae family, while larvae reared on plants from the Brassicae family near-universally rejected Fabaceous plants. This may suggest that plants from the Fabaceae family possess chemical deterrents that make them generally unpalatable, and that feeding experience on these plants leads to a desensitization to those deterrents.
If these inductions are present, this indicates we should be more cautious when making broad conclusions about an induction of preference or oligophagy in M. sexta when testing only a small selection of non-solanaceous plants. It also implies that caution should be taken when testing non-solanaceous plants from different families alongside each other in a broad ‘non-solanaceous’ category. Because quantity of plant ingestion may differ by plant family, results may be skewed by this partiality if measures are not taken to statistically normalize data.
Discussion
More experiments with plants from the Fabaceae family should be performed due to a lack of data from soybean and pea-reared larvae. M. sexta is known to be difficult to raise on non-solanaceous plants, leading to a limited number of viable 5th instar larvae for testing. The vast majority of larvae reared on soybean, pea, or cabbage plants died before reaching the fourth instar, and a larger sample size would have allowed a higher chance of certain larvae surviving to the fifth instar when reared on some of the less-preferred plants. The insignificant sample sizes present in Figures 1-4 and 6 and the lack of data for pea-reared larvae observed in Figures 2, 3, 5, and 6 are due to these difficulties. Cabbage showed the same difficulties with rearing, leading to the lack of data for cabbage-reared larvae seen in Figures 2-6.
Previous studies have tested feeding inductions in M. sexta larvae using multi-choice feeding assays [2],[3],[6]. No-choice tests can be advantageous here because they subject only one variable to testing at a time, allowing the experiment to examine the underlying reasons why an induction of preference may occur. Our research was centered around these no-choice tests because they allowed us to compare the individual results for each larva reared on each plant against each other in the absence of any form of selection bias.
Further experiments would be required to determine the range of feeding inductions in M. sexta for all non-solanaceous plant families, but this study suggests it is possible to test the degree to which these inductions extend to closely related plants. A wider range of data to compare could be achieved by rearing more larvae per experimental plant. Other plants in the Fabaceae and Brassicaceae families should be considered as well to determine the robustness of these conclusions. Using two families of non-solanaceous plants and through no choice tests, it has been demonstrated that larvae feeding patterns have some degree of adjustability through rearing.
Contact: wgretes@howardcc.edu, ugonna.onukaogu@howardcc.edu, katherine.mena@howardcc.edu, oluwaseye.ayoade127@gmail.com, oluwaseye.ayoade@howardcc.edu, gburke15@terpmail.umd.edu
References
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