Going to Mexico?
Protein analysis of the monarch butterfly (Danaus plexippus)
P. Vargas, A. Salcido, S. Perez
Department of Biology, University of Texas at El Paso, El Paso, TX 79968
Eastern monarch butterflies (Danaus plexippus) migrate in the fall from the northeastern United States and southern Canada to the state of Michoacan in central Mexico. There are multiple generations each year, not every generation of monarchs migrates. Migrating monarchs are in reproductive diapause, and have significantly longer life spans (up to 6 months) while non-migratory monarchs are reproductively active, and have life spans of about two months. This project is guided toward identifying molecular differences between migrating and non-migrating generations. We are interested in identifying the presence of Apolipophorins (ApoLp) proteins that associate with lipids and serve as a vehicle for energy transfer. This is especially important during an extensive migration like that of the monarch butterfly. We hypothesized that a significant difference in proteins would be found between migratory and non-migratory monarchs. Preliminary analysis shows an expected difference in proteins when comparing migratory and non-migratory monarchs.
Monarch butterflies (Danaus plexippus) are known for their extensive overwintering migration (Fig.1) from the northeastern U.S. and southern Canada to roosting sites in central Mexico (Herman, 1989). Every year monarchs aggregate at the same sites, although they are separated by several generations from those that over-wintered the year before (Wassenaar, 1998). The significance of this migration would be to help distinguish protein profiles between generations of monarchs.
During the fall migration large amounts of energy must be used. Lipid stores in the abdomen are used to provide energy for the flight muscles in the thorax (Chino, 1997). To enable the transfer of energy, a lipid shuttle system serves as a system in converting High Density lipids (HDLp) to Low Density lipids (LDLp) in association with Apolipophorins (ApoLp), particularly the protein Apolipophorin III (Haunerland). This system is especially important during migration when most flight occurs (Kanost, 1994). In this study we tested the hypothesis that a distinction in proteins will be found between migratory and non-migratory monarchs. This information will enable us to determine molecular differences between migrating and non-migrating monarch butterflies.
Thirty-six randomly selected male and female monarch butterflies were used. Nine male and nine female migratory monarchs were collected October 21 - 22, 2001. Migrants were frozen for storage in a -20C freezer. Another sixteen non-migratory monarchs (seven male, nine female) were collected at the Animal Behavior Laboratory, University of Texas El Paso, El Paso, TX. Two non-migratory males were raised under fall conditions, i.e. decreasing daylight, which can be a cue signaling migration (Dingle, 1972). The contents of the abdomen were extracted from each butterfly. A Protein Extraction Protocol (Das, 1996) was used to isolate proteins in the abdomen, where Apolipophorin III is located. Each sample was seperated into twelve tubes each containing 100ul and quantified using a spectrophotometer and a Bradford Reagent for equal concentrations. Following the protocol, 15ul of equally concentrated sample and 15ul of stain were loaded into a 10% acrylamide electrophoresis gel (a standard was used for comparison). The gel was run for one hour at 100 volts. Coomose Blue stain was used on the gel for 24 hours to show appearance of banding. A series of protein gels was run between 14 June and 19 July 2002. Protein bandings were compared with particular attention to proteins of the approximate sizes of ApoLp II and ApoLp III.
Migratory monarchs exhibited clear dark banding in the area of 85-90kDaltons where ApoLpII is believed to be located (Fig. 2). The non-migratory group showed light banding in the area of protein ApoLpII (Fig.3). When compared to results in migratory monarchs a significant difference could be seen in the presence of these proteins. Non-migratory monarchs raised under “autumn” conditions exhibited an interesting representation of mostly migratory characteristics with some characteristics of non-migratory monarchs (Fig.4). Dark banding was displayed in the areas of 85-90kDaltons, similar to migratory monarchs. Observations were consistent with expectations of findings.
Preliminary findings show that there is a difference in protein profiles of migratory and non-migratory monarchs. As seen in Fig.2 and Fig.3 significant differences can be observed in the area of 85-90kDaltons where ApoLpII is found. Furthermore, monarchs raised in the lab under “autumn” conditions appear to have characteristics of both migratory and non-migratory protein profiles. The above mentioned differences can be seen in Fig.5, “autumn” raised monarchs not only show banding in the 85-90kDalton range, they also show the light banding characteristic of non-migratory monarchs at about 20-25kDaltons. Previous studies (Kanost, 1995) have shown that Apolipophorin III is important component of the lipid shuttle system; however, little reference was made to Apolipophorin II. In this study results indicate that Apolipophorin II may also be responsible for energy storage and usage in migrating insects. Although these results are significant the function of apolipophorins in the monarch butterfly remains unknown.
We would like to thank Cynthia Batkin, Dr. S. Das, Cristina De La Mora, Miguel Garcia, and Jose Pacheco. Support for this study was provided by NIH Grant 2R25GM49011-04 presented to P. Vargas, MIE/REU Grant presented to A. Salcido, and the College of Science of the University of Texas at El Paso.
Chino, H., 1997, Physiological significance of lipid transport by lipophorin for long distance flight insects, Comparative Biochemistry and Physiology PartB: Biochemistry and Molecular Biology, 117(4):455-461
Das, S. and Gillin, F., Giardia lamblia: Increased UDP-N-acetyl-D-glucosamine and N-Acetyl-D-galactosamine tranferase activities during encystations, Experimental Parasitology, 83:19-29
Dingle, H., 1972, Migration strategies of insects, Science, 175(4028): 1327-1335
Haunerland, N., BISC 429: Experimental techniques II separation methods, http://www.sfu.ca/bisc/bis429/lipoprotein.html 15 June 2002.
Herman, W. S., Brower, L.P., and Calvert, W., 1989, Reproductive tract development in monarch butterflies overwintering in California and Mexico. Journal of Lepidopterists Society: 43(1): 50-58
Kanost, M., Sparks, K., and Wells, M., 1995, Isolation and characterization of Apolipophorin III from the giant water bug (Lethorus medius), Insect BioChem Molec. Biol 25(7): 759-764.
Taylor, O., 2002, http://www.monarchwatch.org, 15 June 2002
Wassenaar, L., and Hobson, K., 1998, Natal origins of migratory monarch butterflies at wintering colonies in Mexico: New isotopic evidence, Proceedings of the National Academy of Science, 95:15436-15439.
Fig.1 Fall Migration-Migration occurs from northeastern United States and southern Canada to Michoacan in central Mexico.
Fig.2 (Migratory Gel) Dark banding can be seen consistently in the area of 85-90kDaltons in the migrating monarchs.
Fig.3 Non-Migratory Gel-Light banding can be seen in all but one of the non-migrating group; in comparison with Fig. 3 a significant difference can be noted. Note: subject in 5th column was raised under “autumn” conditions.
Fig. 4 Combination Gel-Subjects in columns 2 and 3 were raised under “autumn” conditions (decreasing photoperiod) exhibit hybrid banding prominently with migratory monarch characteristics. Subjects in columns 4 and 5 show banding in the 85-90kDalton range, where Apolipophorin II is presumably found. Subjects in columns 6 and 7 (non-migratory) show slight banding at the 85-90kDalton mark as compared to migratory monarchs.?