Bee on a gum flo on honey bee Outline - Puget Sound …...Nosema apis affected food glands and blood...

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Effect of Nosema on honey bee Physiology and behavior

Zachary HuangMichigan State University

Bee on a gum floNBG, ACTMarch 24, 2009

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Outline• What is Nosema apis

Biology of the pathogen• Honey bee division of labor

Biology of the honey bee• The Nosema-JH connection

Extended phenotype?• The test of hypothesis

• Effect of Nosema ceranae on bees

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The Nosema parasite

• Nosema apis Zander 1909• A spore forming microsporadian• Classification:

Protista (now a fungus!): Microsporidia: Microsporea: Microsporida

• Obligatory parasite of epithelial cells of midgut of honey bee adults (all castes)

• A new species (N. ceranae) was described in 1996, initially found in Apis cerana.

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Life cycle of Nosema

• Transmitted by spores• Spore ingested by bees• Long, coiled, polar filament everts• Sporoplasm injected into host cell• Multiply through vegetative stages• Spores released when host cells burst• Spores voided and re-infect other bees or

re-infect other midgut cells

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

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Life Cycle

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

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Dysentery

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DeadColonies

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Nosema spores (x400)

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Effect of Nosema on workers

Protein deficiency due to indigestionlower 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

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Regression of hypopharyngeal gland

Healthy nurse Infected nurse

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Worker bees: behavioral developmentand changes with juvenile hormone

Days since emergence2 - 10 11 - 20 21 - 35

Low JH titer High JH titer

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JH affects onset of worker foraging and hypopharyngeal gland development

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

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Effect of Nosema on workers


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

Wang & Moeller, 1970, 1971.

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Effect of JH on workers


• Earlier regression of food glands: poor nursing• Earlier onset of foraging and guarding• Shorter life span

Jaycox 1976, Jaycox et al. 1974, Robinson 1986.

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Nosema JH Foraging?

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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):

* 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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Incubation

Quantification

Extraction

Radioimmunoassay for JH titers

Developed by Hunnicutt et al. (1989)

Validated for honey bees by Huang et al. (1994)

Allows JH measurement on individual bees

Results of this RIA agree with two other RIAs (Strambi, Goodman), both of which validated with GC / MS (Goodman et al. 1993).

Separation

Bleeding

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Dissection

Incubation

Quantification

Extraction

Radiochemical Assay for JH biosynthesis

Developed by Pratt & T obe (1974), Tobe and Pratt (1974)

Validated for honey bees by Huang et al. (1991)

Allows JH level measurement on individual bees

Rates are significantly correlated with JH titers as measured by Strambi's RIA (Huang et al., 1991)

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Nosema changes honey bee behavior

but how?

We asked:

1. Do nosema infected workers have higher JH titers?2. Do they have higher rates of JH production?3. Do they have lower rates of JH degradation?4. Does nosema produce JH directly?

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Materials and Methods

• Newly emerged workers obtained in incubator• Bees paint-marked or individually tagged• Bees individually fed with Nosema spores• Bees isolated for 30 min, introduced into colony • Foraging observation of marked/tagged bees

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Obtaining Newly Emerged Bees

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Tagging bees

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Inoculation

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Inoculation

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Observation

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Sampling Bees

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

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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Collecting Hemolymph

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

JH T

iters

(ng/

ml)

Colony 20 Colony 8

Control Nosema-fed

50

100

150

t = 2.37P<0.01

t = 2.07P<0.02

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Are the higher JH titers due to higher JH production by infected bees?

Corpora allata (CA)

JH biosynthesis:

remove CA, incubate in vitro with radioactive precursorand measure JH production

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Rates of JH biosynthesis are also higher in young, Nosema infected bees

JH B

iosy

nthe

sis

( pm

ol/h

/CA

) ControlNosema Fed

Colony number

2

4

6

8

3 18 45

9 109 109 9 910

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P<0.01 in each colony

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Allatectomized bees

Do bees with corpora allata removed (allatectomized) still forage early?

Nosema

Control

Foragingobservation

Colony

9

33

10

20

30

40

50

60

3 18 45

Allatectomzied controlAllatectomized bees infected with Nosema

The majority of infected bees did not forage earlierafter CA removal

Colony number

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G=4.6

*G=1.2

nsG=1.1

ns

5039 3264 30

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Evidence that Nosema does not produce JH directly:

1. Allatectomized bees showed no behavioral changes

2. Allatectomized bees contained no JH in their blood

3. No radioactive JH was produced when midgut was incubated with radio-labelled methionine.

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Conclusion:

Nosema does not produce JH directly.

Nosema-infected workers forage earlier: due to higher JH titers, higher rates of JH biosynthesis, and despite of higher rates of degradation.

Is this host manipulation? Probably not:1. There is genetic variation (some bees do not respond)2. Rates of degradation also high (no redundancy)

Alternative explanations?3. Possibly simply higher metabolism due to “stress?”4. “Malnutritioned” bees forage earlier because they are

not good nurses?

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Hem

olym

ph p

rote

in c

once

ntra

tion

(µg

/µl)

Nosema-infectedControl bees

5

10

15

20

282420161284

5

10

15

20

25

5

10

15

20

25

Colony 28

Colony 26

Colony 27

Age of workers (days)

25

50

75

100

125

25

50

75

100

125

150

282420161284

25

50

75

100

125

Colony 27D

iam

eter

of h

ypop

hary

ngea

l gla

nd a

cinu

s (µ

m)

Colony 28

Nosema-infectedControl bees

Colony 26

Age of workers (days)

Nosema apis affected food glands and blood protein

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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.

38Nosema apis

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

No pollen -> pollen provided

No nosema

Num

ber o

f sur

vigi

ng b

ees

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# Treatment

1 0 spores, control group

2 30,000 N. ceranae spores

3 30,000 N. apis spores

4 25k N. c + 5k N.a spores





Co-infected bees die much earlier than single-species-infected-bees

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Co-infected bees died earlier in nucs

0102030405060708090

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Surv

ial r

ates

(%)

Control (50% SugarSolution)

N. apis (50,000spores)

N. ceranae (50,000spores)

Mixed (25,000 Apis +25,000 Ceranae)

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Co-infected bees foraged faster than others

0

10

20

30

40

50

60

70

Control (50% SugarSolution)

N. apis (50,000 spores)

N. ceranae (50,000spores)

Mixed (25,000 Apis +25,000 Ceranae)

Cum

ulat

ive

% fo

rage

rs

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Method of infection makes a difference

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Funding Provided by:USDA (NRI)

Ron Lin

Laurent Clauda Jayne Michalek

Rebecca Michalek

Joerg Schmidt-Bailey

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Thank you for your attention.

Bees on Protea, Mt Tomah BG, April 7, 2009 www.beetography.com

Bee on a gum flo on honey bee Outline - Puget Sound …...Nosema apis affected food glands and blood...

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