Final Presentation Recovered

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

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Overall (Zeroed)

Control -Zero (TSB + Arthro)Test 1 - ZeroTest 3 -ZeroTest 5- ZeroTest 10- Zero

Reading Period

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MANDUCA SEXTA RESULTS BY EGG NUMBER

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3 Egg (Zeroed)

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TERMITE RESULTS OVERALL

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TERMITE RESULTS BY EGG NUMBER

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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.