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J U L I A H O L LO WAY , FA L L 2 0 1 4
ANTIBACTERIAL ACTIVITY OF MANDUCA SEXTA & TERMITE
EGGS
BACKGROUND INFORMATION – TRANSGENERATIONAL IMMUNITY
• Through their own exposure to pathogens, parents provide immune protection to offspring• Non-genetic factors
• Benefits both offspring and parents • Parents – increased reproductive success
and fitness• Tradeoffs• Especially true if offspring are likely to
encounter the same pathogens as parents
EXAMPLES OF TRANSGENERATIONAL IMMUNITY
• Vertebrates: birds, rodents• Invertebrates: honeybee, mealworm beetle• Lack specific antibodies• Lack of parental care – parents likely
pass down something• Traditionally thought of as maternal, but
fathers may play a role as well red flour beetle
WHAT WE KNOW
• Transgenerational immune priming (TGIP) occurs in Manduca• Effects vary depending on offspring
stage and type of immunity• Rosengaus Lab –• Evidence to suggest M. sexta eggs
contain antibacterial properties that decrease bacteria (Arthrobacter) growth
• No research on antibacterial properties of termite eggs
OUR EXPERIMENT
• Testing antibacterial properties of Manduca sexta and termite eggs• As a function of Arthrobacter growth
• Important implications for the understanding of the mechanisms of immunity in insects• Further knowledge of parent-offspring
relationship and dynamics• Understand if insects demonstrate levels of
immunocompetence from the beginning & possible effects at the individual and colony levels
HYPOTHESIS
• Both Manduca sexta and termite eggs will demonstrate antibacterial properties by means of decreased Arthrobacter growth• Antibacterial properties will increase as the number of pooled eggs increases
SAMPLE SPECIES
• Tobacco Hornworm, Manduca Sexta• Found throughout much of the American continent• Model species used in many neurobiology and
biochemistry experiments• Larvae known to have many innate immune mechanisms
http://mothphotographersgroup.msstate.edu/
Manduca moth
SAMPLE SPECIES
• Dampwood Termite, Zootermopsis angusticollis • Found in states along the Pacific
Coast • Social insect –high density
colonies; division of labor• Social immunity – increased
survivorship with group living; mutual grooming, removal of diseased individuals, communication about pathogens
http://www.termitesgonewild.com
Dampwood termite
BACTERIA
• Arthrobacter • Gram-positive and
rod shaped• Extremely numerous
in certain soils• Less hardy than
other bacteria, such as Serratia marcescens
https://microbewiki.kenyon.edu/images/1/17/1-2C.jpg
Arthrobacter
PROTOCOL OVERVIEW
• Grow Arthrobacter• Prepare egg samples• Crush eggs• Eggs homogenized via sonicator
• Controls and egg mixtures added to well plate• Plate read for twenty-four hours• Replicate
BACTERIAL GROWTH
• Grows ideally at room temperature to slightly above room temp.• Longer shaking
incubation time than with S. marcescens• 4 hours
SAMPLE COLLECTION & PREPARATION
• Manduca and termite eggs naïve• Unknown
mothers• Termite eggs
stage 1• Manduca: 1, 3, 5,
or 10 eggs pooled• Termite: 1, 2, 5, or
10 eggs pooled
http://www.carolina.com/teacher-resouces http://www.corkyspest.com/images/pests
Manduca eggs
Termite eggs
SAMPLE PREPARATION
• Samples kept on ice• Eggs crushed with tissue grinder and 250
microliters phosphate buffered saline (PBS)• Egg mixtures homogenized via use of the
sonicator (Epstein lab)• Three rounds, 5 second blasts• Amplitude 40%
http://www.omni-inc.com/images/Dounce%20Tissue%20Grinder.jpghttp://www.biologics-inc.com/images/products/probe-sonicator.jpg
Sonicator Tissue grinder
WELL PLATE & READER
• 94 well plate• Total volume: 200 microliters for Manduca samples, 100
microliters for termite samples• 100 microliters too small
• Plate read for 24 hours, absorbance measured every thirty minutes• Temperature = 27 Celsius, 600nm (Godoy Lab)
RESULTS
• For both termite and Manduca, growth of egg mixtures appears less than that of the controls
• Positive correlation between number of pooled eggs and antibacterial properties• No statistics calculated - need larger sample
size
• Data was manipulated before plotting:• “Zeroed” - account for any initial differences
in absorbance
MANDUCA SEXTA RESULTS OVERALL
1 2 3 4 5 6 7 8 9 10
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0
0.1
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0.9
Overall (Zeroed)
Control -Zero (TSB + Arthro)Test 1 - ZeroTest 3 -ZeroTest 5- ZeroTest 10- Zero
Reading Period
Abso
rban
ce
MANDUCA SEXTA RESULTS BY EGG NUMBER
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00.10.20.30.40.50.60.70.80.9
1 Egg (Zeroed)
Control1 Egg
Reading Period
Abso
rban
ce
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00.10.20.30.40.50.60.70.80.9
3 Egg (Zeroed)
Control3 Egg
Reading Period
Abso
rban
ce
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00.10.20.30.40.50.60.70.80.9
5 Egg (Zeroed)
Control5 Egg
Reading Period
Abso
rban
ce
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00.10.20.30.40.50.60.70.80.9
10 Egg (Zeroed)
Control10 Egg
Reading Period
Abso
rban
ce
TERMITE RESULTS OVERALL
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
Overall (Zeroed)
Control -Zero (TSB + Arthro)Test 1 - ZeroTest 2 -ZeroTest 5- ZeroTest 10- Zero
Reading Time
Absorb
ance
TERMITE RESULTS BY EGG NUMBER
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-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 Egg (Zeroed)
Control1 Egg
Reading Period
Abso
rban
ce
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748
-0.2
-0.15
-0.1
-0.05
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0.1
0.15
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0.25
2 Egg (Zeroed)
Control2 Egg
Reading Period
Abso
rban
ce
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-0.15
-0.1
-0.05
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0.25
5 Egg (Zeroed)
Control5 Egg
Reading Period
Abso
rban
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-0.2
-0.15
-0.1
-0.05
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0.15
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0.25
10 Egg (Zeroed)
Control10 Egg
Reading Period
Abso
rban
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DISCUSSION
• Evidence for antibacterial properties of eggs• Insight into mechanisms of immunity • At this point, cannot say it is due to
parents passing down something• Results consist with previous findings• Novel findings with regards to both
Manduca and termite species• No statistics run – no statistical
significance concluded
FUTURE STUDIES
• Test antibacterial properties of eggs as a function of maternal treatment• Naïve, ringer, vaccine, challenge
• Test eggs of different stages/ages • Increase sample size/number of replicates• Look for repeatability• Multiple mothers of same treatment
group• Run statistics and determine possible
significance
REFLECTION
• Research is challenging• Don’t plan on anything going the way
you want it to/think it will• Critical thinking is a must
• …But you learn a lot!• How to be self critical and think outside
of the box• Time management & lab skills• Scientific process
ACKNOWLEDGEMENTS
• Thank you to:• The Smith Lab – Manduca eggs• The Epstein Lab – use of the sonicator• The Godoy Lab – use of the plate reader• All members of the Rosengaus Lab • Professor Rosengaus and Erin Cole
REFERENCES
• Grindstaff, J.L, Hasselquist, D., Nilsson, J., Sandell, M., Smith, H.G., Stjernman, M. 2006. Transgenerational priming of immunity: maternal exposure to a bacterial antigen enhances offspring humoral immunity. Proceedings of the Royal Society 273: 2551-2557.
• Hasselquist, D., Nilsson, J. 2009. Maternal transfer of antibodies in vertebrates: trans-generational effects on offspring immunity. Proceedings of the Royal Society 364: 51-60.
• Kanost, M.R., Jiang, H., Yu, X.Q. 2004. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunological Reviews 198: 97-105.
• Lopez, J.H., Schuehly, W., Crailsheim, K., Reissberger-Galle, U. 2014. Trans-generational immune priming in honeybees. Proceedings of the Royal Society 281: 1-8.
• Moret, Yannick. 2006. Transgenerational immune priming: specific enhancement of the antimicrobial immune response in the mealworm beetle Tenebrio molitor. Proceedings of the Royal Society 273: 1399-1405.
• Roth, O., Joop, G., Eggert, H., Hilbert, J., Daniel, J., Schmid-Hempel, P., Kurtz, J. 2010. Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum. Journal of Animal Ecology 79: 403-413.
• Sadd, B.M., Kleinlogel, Y., Schmid-Hempel, R., Schmid-Hempel, P. 2005. Trans-generational immune priming in a social insect. Biology Letters 1: 386-388.
• Traniello, J.F.A., Rosengaus, R.B., Savoie, K. 2002. The development of immunity in a social insect: evidence for the group facilitation of disease resistance. PNAS 99: 6838-6842.
• Trauer, U., Hilker, M. 2013. Parental legacy in insects: variation of transgenerational immune priming during offspring development. PlOS ONE 8: 1-15.