on a gum flo NBG, ACT Effect of on bee March 24, 2009 Outline Physiology and behavior • What is Biology of the pathogen • division of labor Biology of the honey bee • The Nosema-JH connection Extended phenotype? • The test of hypothesis

Effect of on Zachary Huang •

Michigan State University 1 2

The Nosema parasite Life cycle of Nosema

• Nosema apis Zander 1909 • A spore forming microsporadian • Transmitted by spores • Classification: • Spore ingested by bees Protista (now a !): : Microsporea: Long, coiled, polar filament everts Microsporida • • Obligatory parasite of epithelial cells of midgut • Sporoplasm injected into cell of honey bee adults (all castes) • Multiply through vegetative stages • A new species (N. ceranae) was described in • Spores released when host cells burst 1996, initially found in . • Spores voided and re-infect other bees or re-infect other midgut cells

3 Photo: http://www.biol.lu.se/cellorgbiol/microsporidia/proj_descr.html4

1 Life Cycle

http://www.ojhas.org/issue19/micro2.gif

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Nosema spores (x400) Dead Colonies

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2 Regression of hypopharyngeal gland Effect of Nosema on workers

Protein deficiency due to indigestion lower protein, lipid and sugar levels in blood

• Earlier regression of food glands: poor nursing • Earlier onset of foraging and guarding • Shorter life span (22-44% reduction)

Wang & Moeller, 1970; 1971

Healthy nurse Infected nurse 9 10

Worker bees: behavioral development JH affects onset of worker foraging and and changes with juvenile hormone hypopharyngeal gland development Days since emergence 2 - 10 11 - 20 21 - 35 JH treatment induces • Earlier regression of hypopharyngeal glands • Earlier onset of foraging and guarding • Shortening of life (due to limited foraging life)

Jaycox, 1976, Jaycox et al 1974, Robinson 1987, Sasagawa, 1989

Low JH titer High JH titer

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3 Effect of Nosema on workers Effect of JH on workers

Protein deficiency due to indigestion Protein deficiency due to indigestion lower protein, lipid and sugar levels in blood lower protein, lipid and sugar levels in blood

• Earlier regression of food glands: poor nursing • Earlier regression of food glands: poor nursing • Earlier onset of foraging and guarding • Earlier onset of foraging and guarding • Shorter life span (22-44% reduction) • Shorter life span

Wang & Moeller, 1970, 1971. Jaycox 1976, Jaycox et al. 1974, Robinson 1986.

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R. Dawkins (1982): “The Extended Phenotype.” All phenotype is a means for replicating genes. Host manipulation by a parasite (to increase the fitness of the parasite): Nosema JH Foraging? * Caterpillars climb high on grass when dying from virus * Slugs change color when parasitized * Honey bees forage early when infected by Nosema -- cites a Science paper suggesting Nosema produces JH -- infected hemipterans have supernumerary instars -- allatectomized animals with Nosema show JH activity

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4 Bleeding Radioimmunoassay for Radiochemical Assay for JH titers Dissection JH biosynthesis Extraction

Developed by Hunnicutt et al. (1989) Developed by Pratt & T obe (1974), Tobe and Pratt (1974) Validated for honey bees by Huang et al. Incubation Incubation (1994) Validated for honey bees by Huang et al. (1991) Allows JH measurement on individual bees Allows JH level measurement on individual bees Results of this RIA agree with two other RIAs Separation (Strambi, Goodman), both of which validated Extraction with GC / MS (Goodman et al. 1993). Rates are significantly correlated with JH titers as measured by Strambi's RIA (Huang et al., 1991) Quantification Quantification

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Nosema changes honey bee behavior Materials and Methods but how?

Newly emerged workers obtained in incubator We asked: • • Bees paint-marked or individually tagged 1. Do nosema infected workers have higher JH titers? • Bees individually fed with Nosema spores 2. Do they have higher rates of JH production? 3. Do they have lower rates of JH degradation? • Bees isolated for 30 min, introduced into colony 4. Does nosema produce JH directly? • Foraging observation of marked/tagged bees

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5 Obtaining Newly Emerged Bees Tagging bees

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Inoculation Inoculation

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6 Observation Sampling Bees

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Armed with bees that respond to Nosema infection, now we can ask the 1st question: Collecting Hemolymph

1. Are the earlier foraging in these bees mediated through JH (but not a nosema specific foraging-inducer) if so, we expect higher JH titers in bees just before they embark on foraging (we did not know if foraging performance itself would elevate JH titers).

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7 JH titers are significantly higher in Nosema infected bees, even before foraging started (age=12 days) Are the higher JH titers due to higher

Control Nosema-fed JH production by infected bees?

150

t = 2.37 JH biosynthesis: P<0.01 100 remove CA, incubate in vitro t = 2.07 with radioactive precursor P<0.02 and measure JH production

JH Titers (ng/ml) JH Titers 50

Colony 20 Colony 8 Corpora allata (CA)

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Rates of JH biosynthesis are also higher in young, Nosema infected bees Do bees with corpora allata removed 8 (allatectomized) still forage early? Control Nosema Fed Nosema 6 Allatectomized bees P<0.01 in each colony Control Colony 4

2 JH Biosynthesis ( pmol/h/CA ) JH Biosynthesis ( pmol/h/CA

10 9 9 9 10 9 9 10 Foraging 31830 45 observation Colony number 31 32

8 The majority of infected bees did not forage earlier after CA removal

Allatectomzied control 60 Allatectomized bees infected with Nosema Evidence that Nosema does not produce JH directly:

50 1. Allatectomized bees showed no behavioral changes 40 2. Allatectomized bees contained no JH in their blood 30

G=1.1 G=1.2 G=4.6 3. No radioactive JH was produced when midgut was 20 ns ns * incubated with radio-labelled methionine. 10 64 30 39 32 50 37

31845

Colony number 33 34

Nosema apis affected food glands and blood protein Conclusion: Control bees 125 20 Nosema-infected  Nosema does not produce JH directly. 100 15 75 l)

µ 10 50 Control bees g/  Nosema-infected workers forage earlier: due to higher µ 25 Nosema-infected 5 JH titers, higher rates of JH biosynthesis, and despite Colony 26 Colony 26 25 of higher rates of degradation. 150 125 20  Is this host manipulation? Probably not: 100 15 1. There is genetic variation (some bees do not respond) 75 10 50 5 2. Rates of degradation also high (no redundancy) 25 Colony 27 Colony 27

 Alternative explanations? 25 125 20 3. Possibly simply higher metabolism due to “stress?” 100 Hemolymph protein concentration ( 15 4. “Malnutritioned” bees forage earlier because they are 75 10 not good nurses? (µm) acinus gland of hypopharyngeal Diameter 50 25 5 Colony 28 Colony 28

4 8 12 16 20 24 28 4 8 12 16 20 24 28

35 Age of workers (days) Age of workers (days) 36

9 New twist on Nosema

1. The original species was Nosema apis. 2. A new species was discovered in 1996 by Ingma Fries, in Apis cerana, named Nosema ceranae. 3. In 2005 it was reported in our species (Apis mellifera) in Taiwan and Europe. 4. Now it seems all the nosema we can found in US is also Nosema ceranae. 5. Recent studies in Spain attribute 50% of colony loss to N. ceranae.

Nosema apis 37 38

No nutrition x Nosema ceranae interaction, but pollen shows a huge impact on worker longevity.

No pollen ->  pollen provided Number of surviging bees surviging of Number No nosema

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10 Co-infected bees die much earlier than single-species-infected-bees Co-infected bees died earlier in nucs

# Treatment 1 0 spores, control group 100 90 2 30,000 N. ceranae spores 80 3 30,000 N. apis spores 70 Control (50% Sugar

4 25k N. c + 5k N.a spores 60 Solution) 50 5 20k N. c + 10k N.a spores N. apis (50,000 40 spores) 30 N. ceranae (50,000 6 15k N. c + 15k N.a spores 20

Survial rates (%) rates Survial spores) 10 7 10k N. c + 20k N.a spores 0 Mixed (25,000 Apis + 25,000 Ceranae)

85k N. c + 25k N.a spores

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Method of infection makes a difference Co-infected bees foraged faster than others

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50 Control (50% Sugar 40 Solution) N. apis (50,000 spores) 30

20 N. ceranae (50,000 10 spores)

0 Mixed (25,000 Apis + Cumulative % foragers % Cumulative 25,000 Ceranae)

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11 Thank you for your attention.

Ron Lin Joerg Schmidt-Bailey

Laurent Clauda Jayne Michalek Rebecca Michalek

Funding Provided by: USDA (NRI) Bees on Protea, Mt Tomah BG,

45 April 7, 2009 www.beetography.com 46

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