1. The Remarkable Natural Defenses of Honey Bees
Marla Spivak
University of Minnesota
Penn State 2015

2. How do insects fight off disease?
Innate Immune System:
Non-specific defenses against foreign microbes
Exoskeleton barrier
Blood cells encapsulate
Gliński & Jarosz 1995,

3. How do insects fight off disease?
Antimicrobial peptides (AMPs):
Proteins with broad spectrum antibiotic, antiviral and antifungal activity
Gliński & Jarosz 1995

4. Insects do not produce antigen-specific antibodies with “memory”

5. Social Immunity
(Cremer et al., 2007)
Collective defenses arising from behavioral cooperation among individuals

6. Hygienic behavior Resin collection Individual Immunity Social Immunity
Behavioral defenses
Grooming
Hygienic behavior
Antimicrobial defenses
Resin collection
Mechanical barriers
Antimicrobial peptides

7. What is Hygienic Behavior?
Ability of bees to detect and remove diseased brood, BEFORE pathogen forms infectious spores
Park et al. 1938
Woodrow 1942
Rothenbuhler 1964

8. Hygienic Behavior also is a defense against Varroa
Bees detect and remove mite infested pupae AFTER mite has started egg-laying
Mite offspring killed during removal
Mite reproductive cycle interrupted

9. Plant Resins
Resin is a sticky exudite produced by some plants as a defense mechanism against herbivores, pathogens and UV
Resins contain complex and diverse mixtures of antimicrobial compounds (monoterpenes, flavanoids)
1. Resin physically exclude pests, makes plants unpalatable, and chemically inhibits the growth of
microorganisms
2. Resin can be secreted by both internal structures (cysts and ducts) or external structures (glandular tricombs). In rare cases it is a reward for pollination.
Plants that secrete resin can vary greatly by region and are especially abundant in warmer climates
Langenheim, 2003

10. Bees scrape resins off plant with mandibles, then move to hind legs
Resin foraging is energetically demanding and provides no direct, individual reward

11. Bees need help removing sticky resin loads!
PHOTOS:
Christine Kurtz
Sonoma, CA

12. Resin to Propolis

13. Propolis deposition in commercial beekeeping equipment vs tree cavity
But this is addition of a propolis trap is for commercial purposes by beekeepers that wish to sell the propolis and not intended for bee health necessarilly.

14. Resin and Propolis Questions
What is the benefit of a propolis envelope?
Do honey bee colonies with a propolis “envelope” inside nest have less disease?
What are plant sources of resins and are some more antimicrobial than others?
Do bees choose resins? Do they self-medicate?
Mike Simone-Finstrom
Renata Borba
Michael Wilson

15. What is benefit of propolis envelope?
Simone-Finstrom painted a propolis extract (propolis in 70% Ethanol) inside boxes
Borba stapled propolis traps (with 7mm gaps) inside boxes for bees to deposit a natural envelope.
Controls: Boxes with no added propolis

16. Propolis envelope provides constitutive benefit to colony immunity
Healthy bees in propolis-envelope colonies had significantly lower immune gene expression throughout the summer
bees did not have to invest as much energy in baseline immune function
Simone M, Evans J, Spivak M Evolution
Borba R, et al J. Exp. Biology

17. Immune system of bees with propolis envelope
without
propolis envelope

18. Does propolis envelope have an inducible benefit to colony health?
Does it provide a therapeutic defense against pathogens?
Borba and Spivak, Submitted

19. Propolis-extract envelope
After challenge with fungal pathogen, Ascosphaera apis, colonies with propolis-extract envelope had fewer clinical signs of chalkbrood disease
Treatment
Chalkbrood mummies
Propolis-extract envelope
No envelope
In a previous work, it was found that a propolis–extract envelope had a therapeutic benefit to the colony when the colony was challenged with fungal pathogen, Ascosphaera apis, that causes chalkbrood
This is a brood pathogen, so it only kills the brood. And they found that propolis-rich colonies had significantly less infected larvae compared to colonies without the envelope.
6 colonies each treatment
Simone-Finstrom and Spivak (2012)

20. What about the bacterial pathogen, Paenibacillus larvae, that causes American Foulbrood (AFB)?
- Brood disease
Only 1-2 d larvae susceptible
Larvae contract AFB via oral intake of brood food contaminated with P. larvae spores
One mechanism of resistance to AFB is associated with larval food antimicrobial activity
American foulbrood is also a brood disease. 1-2 day old larvae are the only susceptible stage of this disease, older larvae and adult bees that ingest P. larvae spores do not show any signs of AFB.
Rose and Briggs, 1969

21. Experimental Design P. larvae Challenge No P. larvae Challenge
Propolis Envelope
Sucrose solution with 107 P. larvae spores, 5ml / comb
I had 10 colonies with a propolis envelope and half were challenged with a P. larvae spore solution. And 10 colonies without a propolis envelope and again half were challenged with a P. larvae spore solution
No Propolis Envelope
5 colonies each treatment

22. Hypotheses
Colonies with both propolis envelope and P. larvae challenge would have:
Higher level of immune gene expression in nurse age bees
Greater antimicrobial activity of larval food
Reduced level of American foulbrood (AFB) clinical signs

23. P. larvae challenge and propolis envelope: 7d nurse bees had higher immune gene expression levels
This is interesting, but when I was writing my hypothesis for this research I kept thinking that adult bees had a reason to induce an immune response, that was not just related to the presence of the P. Larvae – because P. Larvae spores do not germinate in adult bees.
Gene expression analysis of antimicrobial peptides was conducted using the ΔΔCt method to correct for temporal variation in gene expression between the two collection periods [35]. Ct values for all four treatment groups were first normalized to two reference genes (actin and RPS-5) and second, normalized Ct values of bees from challenged colonies were normalized to their respective controls. Unchallenged colonies with and without a propolis envelope served as controls for the respective challenged colonies with and without a propolis envelope (e.g., propolis + no P. larvae treatment colonies served as controls for propolis + P. larvae treatment colonies). Finally, the normalized expression between the propolis + P. larvae and no propolis + P. larvae treatments was compared using two tailed t-test, using R version 2.15, with colony of origin as a random factor and treatment group as a fixed effect.
Antimicrobial Peptides

24. Larval food antimicrobial activity
larval food from each cell was individually homogenized in 30 µl of phosphate buffer by repeated pipetting and then transferred to a 1.5 ml eppendorf tube. Larval food from 32 cells, located in the same frame, was collected from each colony and stored individually.
Phosphate buffer was removed from each sample by freeze-drying the larval food and controls (30 µl of phosphate buffer from each sample).
P. larvae (from stock strains obtained from the USDA Agricultural Research Service culture collection; NRRL# B-2605) were cultured in brain/heart infusion broth and the bacterial growth assay was conducted following Wilson et al. [18]. Dried larval food samples were resolubilized in 100 µL of brain/heart infusion broth, transferred to 96 well plates and placed in a plate shaker for 30 min at 400 rpm to improve homogenization. P. larvae liquid culture was transferred into the well plates (creating a 1:100 dilution of P. larvae in each well), and the well plates were incubated at 37°C at 400 rpm for six hours. Bacterial growth inhibition was evaluated in 96-well plates by measuring turbidity (optical density at time 0h subtracted from time 6h, OD600) of treated cultures (containing larval food) relative to untreated controls (phosphate buffer only) using a microplate spectrophotometer.
Larval food
Plates containing P. larvae + larval food, or phosphate buffer

25. % P. larvae growth relative to untreated controls
Larval food from challenged colonies with a propolis envelope had the highest inhibitory activity against P. larvae
% P. larvae growth relative to untreated controls

26. Larval food antimicrobial activity
Nurse bees synthesize more antimicrobial peptides and incorporate them into larval food?
Volatile antimicrobial compounds from the propolis envelope in larval food?

27. Challenged colonies with a propolis envelope had less AFB
In sum, the propolis envelope served as an external antimicrobial layer around the colony, constitutively protecting the brood and supporting bees’ ability to induce a strong and effective immune response against infection after challenge with the result of a lower infection load after two months following the challenge.
the reduced level of AFB clinical symptoms in early October in colonies with a propolis envelope compared to colonies without a propolis envelope is likely a result of a combination of the effects of propolis on both the collective and individual behavioral responses (larval food bioactivity and individual bee immune response).

28. A propolis envelope:
Serves as an external antimicrobial layer around the colony
Supports nurse bees’ ability to induce a strong and effective immune response
Protects brood from pathogen infection
Lowers colony-level infection load

29. Do bees increase resin foraging after colony is challenged with fungal or bacterial pathogen?

30. To measure the resin foraging activity
To measure the resin foraging activity. I close the colonies for 15 minutes and quantified the number returning foragers with resin loads. I did 12 observations before and 12 observations after pathogen challenge
After 3 minutes the colonies had been closed, a picture was taken from the front of the colony to quantify the number of Pollen foragers were used as a proxy for total foraging activity.

31. Bees significantly increase the number of resin foragers after colony is challenged with fungal A. apis but not bacterial P. larvae
Simone and Spivak, 2012
R Borba, PhD Thesis

32. Botanical source of resin analyzed using reverse-phase LC-MS
Do bees alter their selection of resin?
Botanical source of resin analyzed using reverse-phase LC-MS
Leaf
Bee
Resin removed from the hind leg of captured bees
Wilson et al., 2013; 2015

33. Colonies increased resin collection from the plants they were already visiting
Populus deltoides
Eastern cottonwood
Populus hybrid
Unknown 1
Unknown 2

34. vs
Given all the evidence provided here, it is important to recognize the significance of the propolis envelope as a crucial component of bees natural defense mechanism.
The process of domestication of Apis mellifera by keeping them in man-made hives has interfered with a critical, natural defense mechanism of the honey bee colony: the bees do not construct a natural propolis envelope inside the hive as they do in natural tree cavities.

35. How does agricultural land use affect survival of honey bee colonies?
Dr. Matthew Smart

36. North Dakota hosts over 500,000 bee colonies every summer and produces over 42 million pounds of honey annually

37. Regional land use change
USDA-NASS crop production summaries,

38. North Dakota flowers for bees
Canola
First 3 weeks of June
CRP grassland, mixed grass/forbs
Last week of June – Sept.
Flowering trees and shrubs
2nd half of May
Mixed grass/flowering plants
Throughout the summer
2nd crop alfalfa
Mid-July – Sept.
Can say some consider them “weedy”. Say a lot is sweet clover**
Oilseed sunflower
3rd week of July – Aug.

39. Study colony locations
144 colonies in 6 apiaries
Sampled every 6 weeks in ND and CA for 3 years
Annual survival May-April
Month
Location
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
CA holding yards
CA almonds
ID holding yards
ND apiaries
ND: highest honey yields of any state in US; summering pasture for CA almond pollinating bees

40. Land use: 6 apiaries from Arc-GIS quantified land use (m2) within 2-mile (3.2-km) radius around each site
Uncultivated forage:
CRP
Fallow
Flowering trees
Grassland
Hayland
Pasture
Ditch
Cultivated forage:
Alfalfa
Canola
Sunflower
Wetlands:
Wetlands
(wet & dry)
Cattails
Non-forage:
Corn
Oats
Soybean
Wheat
How far away from each other??

41. Individual bee “blood tests”
Colony measures
Frames of bees
Frames of brood
Pollen collection/storage
Honey production
Parasites/pathogens
Pesticide exposure
Queen events
Individual bee “blood tests”
Nutrition – Vg, lipids
Immune system status
Colony measures are taken every six weeks May-Sep in ND, and in Nov, Jan, and Mar in CA. The question is whether location (and the forage in that particular area) in ND has a measurable affect on the factors listed above while the bees are in ND and later when they are moved to CA for eventual Almond pollination.

42. Statistical modeling
Apiary survival modeled against land, colony, and individual bee measures using linear mixed effect modeling (R and lme4 package)
Backward selection until all predictors were significant (α<0.05).
Akaike Information Criterion (AIC) for best fit
Land use
Colony
Individual bee
Site and year were analyzed as random effects.
All measures/predictors were fixed effects.
Response (proportion of colonies surviving in each apiary and year) was sin-1 √ transformed.
Relationships between measures and response were examined for normality, homoscedasticity, and linearity.
Backward selection was used to pare down large models by removing least significant predictors one at a time until all predictors left in the model were significant (α<0.05).
Akaike Information Criterion (AIC) was then used to select the best fit of the available models.

43. Colony survival lowest in apiary surrounded by least uncultivated forage
F5,12=6.6, p=0.003
Uncultivated forage, flowers
Uncultivated forage, no flowers
Cultivated forage, flowers
Wetlands
Non-forage

44. Mean honey production ± s.e 2010-2012ab bc c b a
Mean honey production ± s.e
Site

45. Varroa controlled twice/ year with Amitraz by beekeeper Levels never got above 3 mites/ 100 bees over the three years

46. Pollen identification and pesticide residue analyses
Pollen pesticide residues
3-g samples (3-6 per year)
USDA-AMS National Science Laboratory in Gastonia, NC
GC-MS analysis: residues of 174 common insecticides, fungicides, herbicides, and metabolites (ppb)
Pollen identification
3-g samples sorted by color
Identified to lowest taxonomic level via light microscopy
betterbee.com
humblebeehoneycompany.com

47. Colony Pesticide exposure
Pollen hazard quotient (PHQ):
Mean ppb of each pesticide/ honey bee contact LD50
Bee Toxicity
High, ≤2 μg/bee
Moderate, μg/bee
Low, >11 μg/ bee
Make bottom line bigger and emphasize!!!! No neonics. Also bigger font on the left. Bring pie charts in again? Interactions/synergies with fung and herb. Unknown. These are big crop pesticides that must have drifted onto other flowers in the area. Gross.
No neonics detected

48. Losses Not Correlated with Total Pollen Hazard Quotient
Total PHQ
Pollen hazard quotient (PHQ):
Mean ppb of each pesticide/ honey bee contact LD50

49. Colony Level Indicators of Bee Health and Survival
Colony adult pop. size
Brood area (pupae)
in September
Pollen stores
Pollen collection
mean grams/day
Queen status
Pesticide exposure
Parasites/diseases (Varroa)
Increase in colony measure
Apiary Survival
brood
pollen
Varroa
Just use pictures instead of graphs.

50. Individual Bee Blood Test Indicators of Health and Survival
Increase in individual bee measure
Apiary Survival
Lipids Vg
Lys-2
High Lipid stores
in August - good
High Vg expression
in Sept - good
High Lys-2 expression
in Sept – not good
Muscle bee (healthy) has high nutrition, low immune

51. Specifically, amount of pollen coming in to the colony over the summer
When parasites, diseases and pesticides ruled out, floral resources, within 2 mi of apiary significantly increased health and survival of colonies
Specifically, amount of pollen coming in to the colony over the summer

52. Nutrition Honey Bee Problems Environmental Problems Varroa Parasite
Insecticides
Nutrition
Viruses
Herbicides
Fungicides
Flowerless landscape
Pathogens

53. Continuing Work Regional, multi-state study began this summer
Many more apiaries across a more diverse spectrum of land use profiles
Test robustness, discern finer details of model

54. Big Thanks to Graduate Students!
Mike Wilson Elaine Evans Joel Gardner Ian Lane Renata Borba Mike Goblirsch
Katie Lee Judy Wu- Smart Matthew Smart Morgan Carr-Markell

55. Thank you Bees, Beekeepers & Supporters
National Science Foundation
USDA-NIFA
NRCS
National Honey Board
Almond Board
General Mills
MN Environmental and Natural Resources Trust Fund
Minnesota and North Dakota Beekeeping Associations
Individual donors