1. BeeSpace Biology Overview BeeSpace Workshop IGB, UIUC 21 May 2006

2. BeeSpace Goals Analyze the relative contributions of Nature and Nurture in Societal Roles in Honey Bees Experimentally measure brain gene expression for important societal roles during normal behavior varying heredity (nature) and environment (nurture) Interactively annotate gene functions for important gene clusters using concept navigation across biological literature representing community knowledge

3. Overview Understanding Social Behavior Honey bees have only 1 million neurons Yet… Worker bee exhibits social behavior She forages when she is not hungry but the Hive is She fights when she is not threatened but the Hive is

4. Biology: The Model Organism Western Honey Bee, Apis mellifera has become a primary model for social behavior Complex social behavior in controllable “urban” environment Normal Behavior – honey bees live in the wild Controllable Environment – hives can be modified Small size manageable with current genomic technology Capture bees on-the-fly during normal behavior Record gene expression for whole-brain or brain- region

6. Biological Foundations of BeeSpace There is a robust relationship between brain gene expression & social behavior Gene expression is the “first phenotype” and can be used to understand nature/nurture

7. Nature and Nurture both act on the genome HeredityEnvironment

8. (Whitfield et al, 2002)

9. Home Comb build, Remove corpses, Hygienic behavior (remove diseased brood) Offspring Brood care, Attend queen, Personal reproduction (worker) Defense Guard, Soldier Food Forage for nectar, Forage for pollen, Forage for water Forage for resin, Scout, Process food (nectar to honey) Dance communication: sender, Dance communication: receiver Principal Societal Roles in the Honey Bee Colony

10. Nature/Nurture Dissection I Defense Roles: Guard and Soldier (Hunt, Hoffmann, Alaux) Nature: Types of Bees (European, African) Nurture: Level of Threat (Alarm pheromone; Alaux)

11. Nature/Nurture Dissection II Role: Forager (onset age of foraging) Hereditary factors: European and Africanized Bees (Hunt, Hoffmann) ligustica and mellifera (Leconte, ) High/Low Pollen Hoarding Lines (Page, )

12. Nature/Nurture Dissection III Role: forager (onset age of foraging) Social factors Precocious vs Normal Forager (Newman, Zhang, Ament) Normal vs Overage Nurse Typical vs Single-cohort colony Males (?) Queens (Corona, Hughes)

13. Nature/Nurture Dissection IV Role: Forager (onset age of foraging) Physiological Manipulations affecting: cGMP, Manganese, Insulin, Vitellogenin Juvenile Hormone TOFA (Fatty Acid inhibitor) (Maleszka, Ament) Brood Pheromone, Queen Pheromone ( Alaux)

14. Nature/Nurture Dissection V Dissection of Dance Communication Species differences, Pts. 1 & 2 (Sarma) “Distance genes” (Sarma) “Direction genes” (Brockmann) Time training (Moore and Naeger) Scouts (Liang)

15. Experimental Status Genome Complete and Microarray Fabricated (one + year late) Bees collected for Experiments –3/4 last summer; 1/4 this summer Experiments complete with EST array Experiments in progress with full array Initial Use of Interactive Annotation on-going Planning Meta-level Functional Analysis

16. Goals of Functional Analysis Identify genes regulated by heredity and environment… so what? Discover candidate genes (gene clusters, molecular pathways) for behavioral regulation… set up for causal experiments –Kr-h1 (Grozinger lab) –In situ analysis project (Fahrbach lab)

17. Examplar: Regulation of Brain Kr-h1 Expression by Queen pheromone Regulates worker behavior: Retinue response Delay nurse to forager transition** Inhibit queen rearing Inhibit ovary development Christina Grozinger, NC State

18. Worker responses to queen pheromone In young bees: Retinue response (Slessor et al, Science 1988) Alter brain gene expression patterns (Grozinger et al PNAS 2003) In forager bees: No retinue/attraction Still have antennal responses (Pham-Delegue et al 1993) How is response modulated??

19. Do foragers respond to QP at all? Look at brain gene expression –Kr-h1: downregulated by QP in the brains of young bees Place 30 foragers and 30 day-olds in a cage with a queen for 3 days

20. Does JH modulate response? JH levels much higher in foragers than young bees Treat young bees with methoprene (JH analog) Methoprene reduces QP’s effects on gene expression But Met-treated bees still attracted to QP in retinue Kr-h1 relative expression levels Grozinger and Robinson JCPA, 2007 Grozinger et al, Naturwissenschaften, 2007

21. What does Kr-h1 do? Downregulated by QP (2x) (Grozinger et al 2003) High in foragers compared to nurses (4x) (Grozinger and Robinson 2007) QP regulates transition to foraging… so Kr-h1 involved in foraging? What aspect of foraging? (Fussnecker and Grozinger in prep) –Preparation for flight –Neuroanatomical changes –Phototaxis

22. Function of Kr-h1? But what does Kr-h1 actually DO??? Study effects on neuron structure in Drosophila (Tzumin Lee, UMass) Use MARCM to study single neurons WTLOFGOF Shi, Lin, Grinberg, Grozinger, Robinson, Lee. Dev Neurobio. in press

23. Associated with permanent brain changes? MB expand in foragers When foragers revert to nursing behavior, MB stay the same size (Fahrbach et al 2003) Is Kr-h1 associated with flight behavior, or “permanent” brain changes? Kr-h1 expression correlates with permanent change in brain (Fussnecker and Grozinger, in prep)

24. Effect of cGMP on Kr-h1 expression? (Fussnecker and Grozinger, in prep) Background: cGMP treatment causes premature onset of positive phototaxis (Ben-Shahar et al 2003)

25. Regulated directly/indirectly by cGMP? cGMP response element (Hum et al 2004) N = ATCG; R = A, G; K = G/T; Y = T/C (Fussnecker and Grozinger, in prep) Consensus Apis Drosophila Aedes Tribolium AaAtRKaNTTCaAcAKTY AAATAGTCTTCCAAAGTA AAACATTCTTCAAAATTC AAATGTTTTTCCAAATTG AAATATTTTTCTAAATTT

26. Comparative studies… Bumble bees Honey bee Kr-h1 associated with foraging… –QP regulates transition from nursing to foraging –Foragers less responsive to QP Kr-h1 & foraging or Kr-h1 & QP? Bumble bee Nurse vs. forager – determined by size Queen presence regulates dominance status (reproduction) In collaboration with Guy Bloch… Z. Huang

27. Comparative studies… Bumble bees Partially sequenced Bt_Kr-h1 and Bt_PKG –the foraging gene; increased expression in honey bee forager brain (Fan, Patch, Bloch and Grozinger, prelim results)

28. Associated with flight? Drones become competent to take mating flights when they are approx 5-7 days old Compare drones of different ages, with and without flight experience Kr-h1 expression matches likelihood of flight; not affected by experience Z. Huang, MSU (Fussnecker and Grozinger, in prep)

29. Cellular localization of gene expression using in situ hybridization information in the brain flows through chains of connected neurons (circuits) different populations of neurons express distinctive complements of proteins (gene products) in situ hybridization localizes mRNAs encoding specific proteins to individual neurons this allows gene expression to be studied in the context of the neural circuits that produce behavior

30. BeeSpace in situ hybridization projects Fahrbach Lab Goal: create “brain maps” for genes identified in microarray studies as associated with bee behavior using in situ hybridization –leverages more than a century of neuroanatomical studies BUT –requires develop of efficient approaches –current bottlenecks: frozen sections (10 μm), color reaction to reveal digoxigenin-labeled probes –proposed solutions: Vibratome sections (100 μm), fluorescent probes Test set: 36 neuropeptide-encoding genes identified in the bee genome by a UIUC team using bioinformatics and proteomics (Hummon et al. 2006 Science paper) –set is “right size” for a test of new methods –peptides are expressed abundantly in very small populations of neurons (often fewer than 10, out of approximately 1 million total neurons in the bee brain), so identification is unambiguous –data produced will be of interest to insect neurobiologists independent of methods advances

31. Progress report Rodrigo Velarde (Ph.D., Entomology, UIUC, May 2007) has recently joined Fahrbach laboratory at Wake Forest University Velarde’s previous experience in the Robinson laboratory included use of in situ hybridization to map the feeding-related neuropeptide NPF as a postdoctoral researcher, Velarde will coordinate all BeeSpace in situ hybridization projects

32. Goals of Functional Analysis Array studies as papers? Identify genes regulated by heredity and environment… so what? Ghostbusters Paradigm (descriptive, then analytical) MAPK (Whitfield lab, Jason Ebaugh) Discover candidate genes (gene clusters, gene pathways) for behavioral regulation… set up for causal experiments Kr-h1 (Grozinger lab)