Tobacco Hornworm (Manduca sexta) Behavior




Animal behavior can broadly be defined as the responses of an organism to stimuli in its environment.  Early ethologists (or scientists studying animal behavior) laid the groundwork for current behavioral biologists by studying and characterizing relatively simple and repeatable behaviors seen in animals.  Many of these behaviors were instinctive or innate, i.e., learning was not involved.  For such behaviors, a given stimulus would always produce the same, stereotyped behavior.  A signal in the environment that elicits a given response by the organism is known as a sign stimulus, or a key stimulus.  The stereotyped or fixed behavior that is performed as the result of the stimulus is known as a fixed action pattern.


Simple, stereotyped behaviors such as fixed action patterns, are much more common among animals with relatively simple neural circuitry, such as insects, than among animals with more complex brains, such as mammals.  One of the questions we’re going to ask in this lab is whether insects, in this case, Tobacco Hornworms, are capable of even a simple kind of learning known as nonassociative learning.  Using nonassociative learning, an animal does not need to make a connection between two stimuli or between a stimulus and a response.  One type of nonassociative learning is called habituation, which involves a decrease in the response by an animal to a repeated stimulus that provides no information to the animal.  That is, there is neither positive nor negative feedback as the result of the stimulus.  If an animal is exposed to such a stimulus over and over again, it might become habituated to the stimulus, and it responds less and less.


Simple kinds of movements exhibited by animals are known as kineses (sg. = kinesis) and taxes (sg. = taxis).  A kinesis involves an increase in activity rate or movement in response to a stimulus.  A taxis involves the movement toward or away from a specific stimulus such as light (phototaxis) or gravity (geotaxis) or temperature (thermotaxis).  In this lab exercise, we will attempt to find out if Tobacco Hornworms exhibit any taxes.


Study Organism—Tobacco Hornworm (Manduca sexta)


The Tobacco Hornworm (Manduca sexta) is the developmental stage (larva) of a moth that is commonly known as the Tobacco Hawkmoth (or Tobacco Hornworm Moth).  The Tobacco Hawkmoth is very closely related to the Five-spotted Hawkmoth (or Tomato Hornworm Moth, Manduca quinquemaculata), and both are members of the family of moths known as Sphingidae, which include Hummingbird Moths.  Moths and butterflies collectively make up the Order Lepidoptera (literally “scaled wing”), one of approximately 30 Orders within the Class Insecta [see classification]. 





            Domain:  Eukarya

                        Kingdom:  Animalia

                                    Phylum:  Arthropoda

                                                Class:  Insecta

                                                            Order:  Lepidoptera

                                                                        Family:  Sphingidae

                                                                                    Genus:  Manduca

                                                                                                Species:  sexta


Fifty percent of all named organisms on the earth are insects and 75% of all known animals on the planet are insects. In fact, just three Orders of insects—Coleoptera (beetles), Lepidoptera (butterflies and moths), and Hymenoptera (ants, bees, and wasps)—constitute one-half of all animal species.  Insects are characterized by the four following characteristics:

1.     three body segments:  head, thorax, abdomen

2.     three pairs of legs

3.     two antennae

4.     an exoskeleton

Insects are thought to be so successful because of their external protective exoskeleton, their small size, and their ability to fly.  They inhabit virtually every environment on the earth, they are critical components of the community of organisms that is involved in the decomposition of dead organic matter, they eat more plants than any other group of animals, and they, in turn, provide a food source for many other animals.


Two Tobacco Hornworms can readily defoliate an entire tobacco plant and thus a population of these insects can cause considerable damage to a tobacco crop.  Besides the use of pesticides, Tobacco Hornworms can sometimes be controlled by natural enemies.  Larvae are preyed upon by wasps in the family Vespidae (Polistes spp.), and the Braconid wasp Apanteles congregatus, as well as several Tachinid flies, parasitize the larvae.


As many of you probably know, Tomato Hornworms, the larvae of Five-spotted Hawkmoths, commonly ravage tomato plants in Indiana and elsewhere in the United States, denuding tomato plants and reducing their productivity. Tobacco and Tomato Hornworms are very closely related and the same kinds of parasites that infect Tobacco Hornworms also parasitize Tomato Hornworms.


Tobacco Hornworms range from southern Canada to Argentina, while Tomato Hornworms extend only from southern Canada through the southern U.S.  As implied by the names, tobacco and tomato plants are the principle host plants for Tobacco and Tomato Hornworms respectively.  Tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants belong to the family Solanaceae, or Nightshade family.  Other members of the Nightshade family, such as Horse Nettle (Solanum carolinense), Eggplant (Solanum melongena), Potato (Solanum tuberosum), various peppers (Capsicum spp.), and others are sometimes used as host plants by these hornworms.  Hornworms are easily reared in the laboratory on artificial diets.


Life History:

Hornworms exhibit a life cycle characterized by several distinct stages:  egg, larva, pupa, and adult.


Š      Egg – Hornworm eggs are smooth, spherical, and about 1.0 mm in diameter.  Initially, they are light green but they turn white before hatching.


Š      Larva – After hatching, hornworm larvae go through five stages, or instars.  In between each instar, the larva molts, or sheds, its exoskeleton and grows a new, larger one.  Fifth instar, or mature hornworm larvae are 75-85 mm long (how many inches is that?), usually have green bodies with fine, white hairs, and seven diagonal white stripes (M. sexta) or 8 “V”-shaped marks (M. quinquemaculata) on each side.  The “horn” on the posterior end is either red (M. sexta) or blue-black (M. quinquemaculata).  Spiracles (breathing holes), which look like portholes, occur in pairs on the sides of all but the two anterior-most segments.  Upon maturation of the larva, the dorsal aorta becomes visible along the dorsal midline.  This dark, pulsating line is first visible just anterior to the horn.


Š      Pupa – Pupae are brown, hard, and spindle-shaped, and are about 50 mm in length.  They have a curved, pitcher-handle-like tongue case, which is long and has a slight curve.


Š      Adult – Adult hornworm moths, i.e., hawkmoths, have a wingspan of 105-130 mm, and are brownish-gray with black, brown, and whitish lines.  The abdomen is gray and black with six large orange spots on each side.  Very wavy (sexta) or only slightly wavy (quinquemaculata) lines on the hind wings are fairly distinct.



    First -nstar larva                 Second-instar larva                Third-instar larva



     Fourth-instar larva     Adult Tobacco Hawkmoth


Hornworms overwinter in the soil as pupae.  Moths of the overwintering generation (there can be 2-3 generations per year depending on the locality) begin to emerge in early June and may continue to emerge as late as August.  Nocturnal in habit, hawkmoths can be seen hovering over plants at dusk.  At night, females lay eggs on the undersides of the leaves of host plants.  Each moth deposits one to five eggs per plant visit and may lay up to 2000 eggs.  Larvae hatch about four days later, depending on the temperature.  After feeding for approximately three weeks, hornworms burrow into the soil and spend three weeks there, after which a new generation of moths emerges.  Female moths with mature eggs produce a pheromone that attracts males.  The pheromone is produced primarily by unmated females, so females typically mate with a single male, while males might mate with several females.


Manduca experiments—Week 1


1.     Righting experiment


-Get a hornworm from the front of the lab and put it into a plastic Petri dish.


a.  Turn the hornworm upside down.  Describe the response of the hornworm.





b.  Waiting 30 seconds in-between trials, repeat the procedure 4 times (total of 5 times).  Does the response of the hornworm change from trial 1 to trial 5?  Explain.






2.    Stimulus-response experiment (smaller larvae might be more responsive than larger larvae); if you get no response, try cooling or warming (DON’T OVERHEAT) the tip of the probe:


     a.  With a probe, prod the right side of the hornworm close to the front, or head, end and note the response.  Pay close attention to which direction the hornworm turns and to what degree it turns.  Describe the response of the hornworm.


-Wait 30 seconds and then prod the hornworm on the right side about mid-way    between the head and rear end.  Again, describe the response.





-Wait another 30 seconds and prod the hornworm on the right side near the posterior end (i.e., the end where the horn is located).  Describe the response.





b.     Repeat the same series of stimuli on the left side of the hornworm and describe the responses.







     c.  Repeat the same series of stimuli on the dorsal midline of the hornworm and describe the responses.







3.  Habituation experiment:


     -After completing #2, wait 5 minutes before resuming.


a.     Using the body location that generated the most extreme response on the part of the hornworm, prod it in that location and note the response.





b.     Waiting 10 seconds in-between trials, repeat the prod nine more times in the same location (total of 10 times).  Does the response of the hornworm change from the first to the final trial?  If so, how?  Has habituation occurred?






4.  Taxes:


-After completing #3, wait 5 minutes before resuming.


a.     Does the hornworm show a preference for a relatively lighted area (positive phototaxis) or for a relatively dark area (negative phototaxis)?  [Use aluminum foil to cover ½ of the plastic Petri dish.]











b.  Does the hornworm show a preference for an area that is warmer than room temperature (positive thermotaxis) or for an area that is cooler than room termperature (negative thermotaxis)?  [Orient the Petri dish such that ½ of it is on the black bench and ½ of it is on the white bench paper; then shine a lamp from above onto the Petri dish—the side of the dish above the black surface should warm up faster than the side above the white bench paper.]
























Manduca Experiments—Week 2—Designing your own experiment:




1.  Food choice experiments—we do not have tobacco plants at our disposal but we do have access to tomato plants as well as (in the greenhouse) several other members of the Solanaceae, namely Brugmansia insignis (Angel’s Trumpet), Brunfelsia jamaicensis (Lady of the Night), Brunfelsia nitida (Lady of the Night), Cestrum nocturnum (Night Jasmine), Cestrum purpureum (Purple Jasmine), Solandra nitida (Chalice Vine), Solanum melongena (Eggplant), and Solanum pseudocapsicum (Jerusalem Cherry).  The greenhouse obviously has lots of other plants from many different plant families.


a.     Do Tobacco Hornworms prefer tomato leaves to leaves of other plants in the Solanaceae?


b.     Do Tobacco Hornworms prefer tomato leaves to leaves of other plants that are not in the Solanaceae?


c.     Do Tobacco Hornworms prefer non-tomato Solanaceous leaves to leaves of other plants that are not in the Solanaceae?


d.     Do Tobacco Hornworms avoid nicotine?


e.     Do Tobacco Hornworms avoid caffeine?




2.  Temperature-dependent growth rate—you will need to set this up during week 1 so that you can measure differences during week 2:


a.  Do Tobacco Hornworms grow faster at 20° C or at 25° C?


b.  Do Tobacco Hornworms grow faster at 25° C or at 30° C?




3.  Photoperiod-dependent growth rate—you will need to set this up during week 1 so that you can measure differences during week 2:


a.  Do Tobacco Hornworms grow faster when exposed to a 12:12 L:D photoperiod (12 hours of light and 12 hours of dark) or a 6:18 L:D photoperiod?


b. Do Tobacco Hornworms grow faster when exposed to a 12:12 L:D photoperiod or a 18:6 L:D photoperiod?



4.  Metabolic rate experiment:


How does the metabolic rate of earlier instars of Tobacco Hornworms compare to the metabolic rate of later instars?  This question can be approached in a couple of different ways:


  1. Use a respirometer to see if later hornworm instars consume more O2 than earlier instars.


b.   Using dyed artificial food, determine how long it takes for food to pass through the gut of earlier vs. later instars.  [Note:  you might need to return later in the day or the following day to check on the presence of colored feces in the experimental chamber.]




5.  Response to flooding:


Can a Tobacco Hornworm survive submersion under water for 1 minute?  …….for 10 minutes?  ……..for one hour?



6.  Response to desiccation (?)