This method represents a portion of a larger ex situ conservation breeding protocol used for laboratory mass production of at risk butterflies. It illustrates how basic husbandry methods can be adapted for scientific research to help address key behavioral, life history, or ecological data gaps. The specific methods presented for assessing larval development time and the number of larval stadia have broad applicability to other butterfly conservation breeding or research programs.
This technique is an effective approach for documenting the life history metrics of an endangered butterfly such as number of larval instars, duration of developmental stages, and size of all life stages. We've streamlined our protocol so that productivity and efficiency may be increased in the laboratory which is especially important when collecting data in a short time frame. This method requires dexterity and attention to detail to avoid organism harm, as butterfly larvae are very small especially as neonates.
Demonstrating the procedure will be Jacob Hornfeldt, an undergraduate from our laboratory. To begin, use a small camel hair watercolor paintbrush to carefully move and isolate a single larva and place it under a dissecting microscope in a petri dish. Dip a single hair of the insect pen in nontoxic luminous paint of contrasting color to that of the larva, and carefully put one small drop of paint on the back of the larva.
After 30 seconds, the paint dries. With the help of a small camel hair watercolor paintbrush, place each individual larva in its own two ounce clear plastic portion cup containing approximately one to three small leaves of fresh terminal host material, and write a unique identifier on the cup and lid. Firmly secure the lid.
Carefully check each larva daily. With forceps, remove the leaves and set them onto a white surface. Inspect the cup and the clear lid.
Examine the leaves under a dissecting microscope for the presence of larval exuviae and head capsules. When a larval exuvia is observed, remove it from the cup and place it in a vial filled with 0.2 microliters of glycerin. Label the top of the lid and the side with the larva number, date of molt, and head capsule.
Place the larval exuvia and the associated head capsule in a clear plastic portion cup lid, and put a couple of drops of ethanol in it. Observe the larval exuvia under a dissecting microscope. If the larval head capsule is already separated from the exuvia, place a drop of glycerin on the tip of pointed entymological forceps and gently touch the head capsule to the glycerin.
Place the head capsule in the associated microcentrifuge tube. If the head capsule is still attached to the larval exuvia, use pointed forceps and an insect pin to separate the head capsule from the larval exuvia. Once it is separated use the glycerin technique to pick up the head capsule.
After each molt, repaint the larvae and record the molt dates. Each day, use digital calipers to measure the total body length from the head to the last abdominal segment of each larva. Take three measurements and record the average of the three along with the date and time.
Check the corresponding plastic cup to make sure no mold is in it. Remove all frass and old host debris and add fresh host material as needed. Return the larva to the cup.
Maintain the cups under laboratory temperature between 27 and 32 degrees Celsius for optimal larval activity and development. Maintain the condition until all larvae reach their final instar and begin the prepupal stage. When larvae cease feeding, turn a uniform dull greenish-brown color, lose their chevrons, and often wander off the host, place a small piece of corrugated paper in the cup.
Once each larva has fully pupated measure its total length and record the date of pupation. This is the final molt of each individual. Check on the pupae daily.
When they have grown up into butterflies, use forceps to hold the adult butterfly and gently blow on the wings to reveal the upper wing surface color. Males appear to have blue all over the wings and females have reduced blue and an orange eyespot on the hind wing. Record the eclosion date and sex of each resulting adult butterfly.
To measure the wing chord length of each butterfly gently hold the butterfly with forceps and use digital calipers. In case the butterfly is too active to be measured place it in a refrigerator for 30 seconds or less and try again. This protocol has enabled directed research and extensive data collection on numerous key data gaps important to improve best laboratory breeding and husbandry practices.
Here are the four head capsules collected for one individual. The majority of larvae had four molts with each immature life stage less than five days. Females typically developed quicker in all immature stages compared to males.
Although this was not a significant effect with a p value at 0.625. This step is very meticulous especially when larvae are freshly hatched neonates. It's important to remember to use a microscope during this step to make sure the paint is applied on the appropriate place on the back of the caterpillar.
These methods can be expanded to help evaluate developmental stage survival rates, differential performance on multiple larval hosts, as well as help researchers to better interpret data from the field. This straightforward method illustrates how with a little planning basic organism husbandry practices can be readily adapted for scientific research. The results can then be used to help inform, and potentially adapt, ex situ methods to enhance success.
