Background

1. Isolating brain RNA to determine genes involved in hygienic behavior in the honey bee, Apis mellifera. Laura Chopp (Department of Biological Sciences) Daniel P. Toma, Faculty Mentor, (Department of Biological Sciences) • Background • Honey bees are social insects exhibiting a division of labor throughout their adult life (1,2) (Figure 1). • In addition to being a model research organism for sociality, they are agriculturally valuable, pollinating billions of dollars of crops a year (3, 4). • Problem • Honey bees are under assault from a variety of diseases, including American foul brood, chalk brood, and varroa mite, (Figure 2) devastating colonies and incurring millions of dollars in damage (Reviewed in 5 & 6). • To combat these diseases, honey bees have a socially mediated immune response called hygienic behavior (Figure 3), where bees 14-20 days old uncap infected brood cells and remove larvae (Reviewed in 5 & 6). • This behavior is actively being selected for in colonies to introduce them into commercial bee operations (6, 7). • To aid this selection, genes underlying the behavior could be used as markers in the selection, enhancing its propagation in commercial colonies. • Methods • Bee collection • Day old bees were painted differing colors corresponding to colony they originated from and age. • Set up three observation hives with each consisting of bees from a different hygienic hive. • Place painted bees from a hygienic hive and a non-hygienic hive into each. • Collect bees 14-19 days old performing hygienic behavior from hygienic marked bees and those not performing hygienic behavior from non-hygienic marked bees into liquid N2. Store at -80 C. • Methods – continued • Dissect brains under dry-ice conditions. • Isolate RNA • Send RNA to University of Illinois genomics facility to perform honey bee brain cDNA microarrays to screen for differentially expressed genes between hygienic and non-hygienic bees. • Results • Hygienic and non-hygienic bees were marked and collected as described at the University of Minnesota honey bee research facility. • Bees brains have been successfully dissected on dry-ice and stored at -80 C for RNA isolation (Figure 4). • Conclusions • We have successfully optimized the technique to dissect the brains of bees for the gene expression comparison. • Continuing/Future work • Isolate RNA from 10 sets of hygienic and 10 sets of non-hygienic bee brains. • Send the RNA to the University of Illinois genomics facility at the University of Illinois, Urbana/Champaign to do bee brain microarrays. • Analyze results for differentially expressed genes. A1 A2 A B B1 B2 C USDA.gov Figure 1. Honey bee division of labor. Bees emerge from their cells to perform jobs, such as brood care, inside the colony until 3 weeks old, then perform jobs outside the colony, such as foraging, until 6 weeks old. Figure 3. A. Non-hygienic/B. hygienic behavior. Circular sections of brood are frozen and 24 hrs later the amount of dead brood removed is assayed. If > 95% are removed then repeat. If > 95% are removed again the colony is hygienic. Figure 2. Diseases of the honey bee controlled by hygienic behavior: A. American foulbrood; B. Chalkbrood; C. Varroa mite A B C D E References 1. Wilson, E.O. (1985) The sociogenesis of insect colonies. Science 228: 1489-95. 2. Robinson, G.E. (1992) Regulation of division of labor in insect societies. Annu Rev Entomol 37:637-65. 3. USDA-NASS 2008 reports. 4. Helmholtz Association of German Research Centres (2008, September 15). Economic value of insect pollination worldwide estimated at U.S. $217 billion. 5. Spivak, M., and Gilliam, M. (1998) Hygienic behavior of honey bees and its application for control of brood diseases and varroa, Part I. Hygienic behavior and resistance to American Foulbrood. Bee World 79(3): 124-134. 6. Spivak, M., and Gilliam, M. (1998) Hygienic behavior of honey bees and its application for control of brood diseases and varroa, Part II. Studies on hygienic behavior since the Rothenbuhler era. Bee World 79(4): 169-186. 7. Spivak, M., and Reuter G.S. (2001) Resistance to American foulbrood disease by honey bee colonies Apis mellifera bred for hygienic behavior. Apidologie 32: 555-565. . Figure 4. Stages of dissection of honey bee brains on dry ice: A. Bee head, B. Opened head capsule, C. Dissected brain (anterior), D. Dissected brain (posterior), E. Dissected brain diagram.