This study tested the antibacterial properties of eggs from Manduca sexta tobacco hornworm moths and Zootermopsis angusticollis dampwood termites. Eggs were crushed and mixed with the bacterium Arthrobacter in increasing quantities from 1 to 10 eggs. Absorbance readings over 24 hours showed bacterial growth was lower in egg mixtures compared to controls, indicating eggs have antibacterial properties. As more eggs were added, antibacterial effects increased. This provides evidence that parental immunity can be transmitted to offspring in insects through eggs. Further studies controlling for maternal treatment are needed.

1. J U L I A H O L L O WA Y , F A L L 2 0 1 4
ANTIBACTERIAL ACTIVITY OF
MANDUCA SEXTA & TERMITE
EGGS

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. BACTERIAL GROWTH
• Grows ideally at room
temperature to
slightly above room
temp.
• Longer shaking
incubation time than
with S. marcescens
• 4 hours

12. 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

13. 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

14. 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)

15. 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

16. MANDUCA SEXTA RESULTS OVERALL
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47
Absorbance
Reading Period
Overall (Zeroed)
Control -Zero (TSB + Arthro)
Test 1 - Zero
Test 3 -Zero
Test 5- Zero
Test 10- Zero

17. MANDUCA SEXTA RESULTS BY EGG
NUMBER
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
1 Egg (Zeroed)
Control
1 Egg
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
3 Egg (Zeroed)
Control
3 Egg
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
5 Egg (Zeroed)
Control
5 Egg
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
10 Egg (Zeroed)
Control
10 Egg

18. TERMITE RESULTS OVERALL
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47
Absorbance
Reading Time
Overall (Zeroed)
Control -Zero (TSB + Arthro)
Test 1 - Zero
Test 2 -Zero
Test 5- Zero
Test 10- Zero

19. TERMITE RESULTS BY EGG NUMBER
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
1 Egg (Zeroed)
Control
1 Egg
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
2 Egg (Zeroed)
Control
2 Egg
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
5 Egg (Zeroed)
Control
5 Egg
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Absorbance
Reading Period
10 Egg (Zeroed)
Control
10 Egg

20. 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

21. 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

22. 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

23. 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

24. REFERENCES
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Transgenerational priming of immunity: maternal exposure to a bacterial antigen
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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
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• Sadd, B.M., Kleinlogel, Y., Schmid-Hempel, R., Schmid-Hempel, P. 2005. Trans-
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• Trauer, U., Hilker, M. 2013. Parental legacy in insects: variation of transgenerational
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