(r1) Barrick Lab :: ProtocolsHornwormCare

---++ Tobacco hornworm (<em>Manduca sexta</em>) care

The tobacco hornworm is the caterpillar stage of the five-spotted hawk moth (_Manduca sexta_). They constitute a major pest of nightshade (<em>solanaceae</em>) crop plants, such as tobacco, tomato, and eggplant. Tobacco hornworms are often used in classrooms to demonstrate the process of lepidopteran metamorphosis. However, much tobacco hornworm research focuses on assessing pest control methods. 

   * 5th instar tobacco hornworm caterpillar: <br />
     <img src="%ATTACHURLPATH%/manduca_sexta.jpg" alt="manduca_sexta.jpg" width='521' height='293' />
      * Image via [[https://commons.wikimedia.org/wiki/File:Tomato_Hornworm_in_hand.jpg][Wikimedia Commons]]

---+++ Rearing and Caring for Tobacco Hornworms

---++++ Feed
 
Tobacco hornworms have a flexible diet, with the ability to feed on the following:
   
   * *Artificial chow (pre-prepared):* Tobacco hornworm chow can be obtained from online sellers such as Amazon. Once obtained, refrigerate and handle under sterile conditions. Cubes may be cut out of the pre-prepared chow, or the chow may be re-liquified by adding 15 mL of sterile water to 6+ g of chow and microwaving at 100% power for 40 seconds.
   * *Artificial chow (powder):* Tobacco hornworm chow powder can be obtained from online sellers such as Amazon. It may be stored at room temperature. To prepare, mix 80.6 mL sterile water with 19.4 g of chow powder and heat for 2 - 3 minutes in a microwave on 40% power. Pour into a container and let solidify at room temperature or cooler.
   * *Leaves of natural host plants:* Not all solanaceae plants are edible to tobacco hornworms. See [[https://doi.org/10.1111/j.1570-7458.1985.tb03513.x][Jones et al. 1985 for mortality rates on different plants in the nicotina genus]]. Never provide wilted leaves.
   * *Note:* Caterpillars may only switch between artificial and leaf diets during their early larval stages. 

---++++ Container Construction

When designing tobacco hornworms containers, consider the following: 
   
   * *Air:* _Manduca sexta_ caterpillars require air holes in their containment chambers. Three or four holes poked with thumb-tacks is sufficient. 
   * *Containment:* _Manduca sexta_ caterpillars may escape through small holes if they are not covered by mesh. Additionally, they may chew through plastic films such as parafilm or shrink wrap. They are also excellent climbers and capable of holding onto vertical surfaces or the ceiling of their containers. 
   * *Moisture:* _Manduca sexta_ caterpillars may receive all necessary moisture from their feed if they are fed regularly. Some classroom guides recommend including damp paper towels in containers, but this may lead to mold infection. Dessicated caterpillars take on a brownish and shrunken appearance.
   * *Food:* _Manduca sexta_ caterpillars grow rapidly and require a steady source of food, though the amount will depend on their current size. A cubic centimeter of diet or single leaf each day should be enough to sustain five second-instar larvae. 
   * *Warmth:* _Manduca sexta_ caterpillars can be reared at room temperature, but 30 degrees celsius is ideal. Cooler temperatures will hinder their growth and delay hatching. 
   * *Example Container:* A sterile, medium-sized, clear plastic solo cup, perforated several times with thumb-tacks and covered with mesh. 

---++++ Handling and Transfer

Tobacco hornworms begin life very small and fragile, but grow to the size of a finger before entering the pupal stage. To handle them when small or large, use the following methods:
   
   * *Note:* Caterpillars should always be set down gently. They should be set directly onto food as often as possible. 
   * *Straw:* To transfer young and fragile caterpillars (first and second instar), attach mesh to the end of a hollow plastic tube. Fit a disposable plastic micropipette tip over the end with the mesh. Suck air through the other end, pointing the micropipette tip at a caterpillar's posterior. This will break the caterpillar's grip. Peel the hornworm off of a surface from posterior to anterior and then transport to the desired location. Regularly replace the pipette tip and sterilize the end of the straw coming into contact with human saliva. 
   * *Tweezers:* Tobacco hornworm larvae have sensitive bodies, but they can be picked up by their distinctive tail horns with a pair of tweezers. Limit this to older instars (third and beyond).
   * *Hands:* Larvae may be gently picked up and transported with a gloved hand. However, thus may be too forceful in first or second instar larvae, and the caterpillar's silk may make it difficult to put back down.

---++++ Preventative Healthcare

While tobacco hornworms reared in classrooms are often raised in bulk with very little maintenance, this may lead to disease and higher mortality rates. For optimal caterpillar survival rates:

   * Bleach tobacco hornworm eggs before placing them in their containers. Submerge in 10% bleach for one minute. 
   * Limit the number of caterpillars per container. They cannibalize each other even when plenty of alternative food is available. No more than five caterpillars per containers is prudent, and fewer is preferable. 
   * Transfer caterpillars into new containers daily, or clean containers daily: ethanol is not necessary unless disease is obvious, but hornworm frass (faeces) and old food must be removed.
   * Provide fresh food for caterpillars daily.

---+++ Experimenting on Tobacco Hornworms

---++++ Microbiome Extraction

This procedure is intended to assess the colonization of the tobacco hornworm gut by bacteria species.

   1 Prepare spot plates with antibiotic resistance selective for a particular species, as well as multi-well plates.
   1 Secure one microcentrifuge tube per caterpillar.
   1 Transfer caterpillars into the tubes. Preferrably, place them head-down, horn-up. 
   1 Fill each tube with enough 10% bleach to completely submerge the caterpillars. This will kill all surface bacteria and may kill the caterpillars themselves. 
   1 Let sit for one minute. Watch for caterpillars attempting to climb out of the bleach. If they begin to climb out of the bleach, close the top of the microcentrifuge tub and tap it firmly down on a tabletop to knock the caterpillar back into the liquid.
   1 Use a pipette to empty the tubes of bleach.
   1 Wash the caterpillars with autoclaved water, using the same volume as the 10% bleach. The hornworms, if still alive, must stay in the water to get washed.
   1 Use a pipette to remove the water and fill the tube with the same volume of saline. Do not remove the saline.
   1 Using a clean plastic pestle for each hornworm, grind up the hornworm remains inside the microcentrifuge tube. Twisting in place with the pestle is generally more effective than lifting and driving back down. 
      * Note: some of the hornworms may have survived the previous steps. Squash these hornworms first. They have earned a swift death.
      * Note: there will be some hornworm skin leftover. This is acceptable, though it may interfere with pipetting. As a rule of thumb, continue grinding up the hornworm until you can remove the pestle without any skin clinging to it. 
   1 Use each caterpillar's solution to spot-plate. 

---++++ Droplet Feeding Assays (RNAi)

This protocol is adapted from [[https://doi.org/10.3389/fpls.2019.00313][Burke et al. 2019]], who tested dsRNA as a potential pest-control method. 

   1 Transcribe enough dsRNA to provide 2.5 &#956;g per caterpillar.
   1 Concentrade dsRNA down to 2.5 &#956;g.
   1 Starve second instar (5 - 8 days) larvae for 4 - 6 hours in individual containers.
   1 Use a micropipette to place a 1 &#956;L droplet of 2.5 &#956;g dsRNA solution in front of each caterpillar.
   1 Watch and wait for each caterpillar to consume its dsRNA droplet. Discard any individuals who do not consume the entire droplet.
   1 Transfer caterpillars into appropriate containment chambers and allow to feed _ad libitum_. Observe mortality rates over the course of 7 days. 

-- Main.ElizabethRobinson - 08 Apr 2020
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Barrick Lab > ProtocolList > ProtocolsHornwormCare
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Topic revision: r1 - 2020-04-08 - 23:35:50 - Main.ElizabethRobinson