1. Southeastern Society of Parasitology
Cracking the Egg for Black Fly Oviposition: A potential approach for controlling onchocerciasis
Tommy W. McGaha Jr., Ryan M. Young, Nathan D. Burkett-Cadena, Sayed Hassan, Eddie W. Cupp, Bill J. Baker, Raymond Noblet, and Thomas R. Unnasch
Southeastern Society of Parasitology
4/10/2014
Dr. Unnasch has already provided you with one part of the project concerning improvement of onchocerciasis surveillance by using host-seeking compounds from sweat to attract flies to a trap. So I‘m going provide the talk of the investigation on oviposition pheromones of black flies and how they can be used not only for surveillance but also control. Since Dr. Unnasch has already presented to you a large portion of the introduction to my talk, I will just give a brief overview and get right into it.

2. Onchocerciasis (river blindness)
Neglected Tropical Disease (NTD)
Caused by Onchocerca volvulus
Transmitted by black flies
(species of genus Simulium)
People want to know the life cycle….so REVIEW

3. Onchocerciasis Surveillance
Determine transmission rate with entomological approach = real time
Vector population sample (6,000 flies)
PCR
Current method: human landing collection
Inefficient
Ethically questionable
Improved surveillance: black fly trap
More efficient
Lure: host-seeking and oviposition compounds
Vector control

4. Oviposition Trap Improved Bellec design Physiologically specific
Optimized for attracting gravid black flies
Baited with volatile lure
Oviposition pheromone(s)
Ovicidal compounds
Kills eggs

5. Black Fly Oviposition Field Studies
“Communal oviposition” observed from Simulium damnosum Theobald
(Muirhead-Thompson 1956)
Simulium reptans was observed to
prefer ovipositing on substrates with
≤ 1-day old eggs
(Coupland 1991, 1992)
Simulium damnosum (McCall et al. 1994)
and Simulium ochraceum
(Rodriguez-Perez et al. 2004) prefer to
oviposit on a substrate with eggs than
without
Is it just me or is there something about these eggs!

6. Simulium tribulatum ovipositing on vegetation in Holston Sluice Fork
(Holston River), Kingsport, TN, USA

7. Oviposition Pheromone Study Objectives
Develop behavioral bioassay to assess oviposition preference behavior
Isolate chemical components associated with the bioactivity
Verify activity for extract
Characterize chemical components
Verify activity for each chemical compound

8. Model Species for Study
Simulium vittatum
University of Georgia (UGA) Colony
Large number of flies
Preferred physiological state
Autogenous

9. Binary Choice Chamber (BCC) Bioassay
20 gravid flies
2 oviposition substrates
(no eggs vs. eggs present)
Measure oviposition preference
1. (no. of ovipositing flies on substrate)
2. (no. of flies located on substrate)
BCC was used to observe the behavioral responses of 20 gravid black flies when presented with two oviposition substrates which have a control or test treatment.
Keep talk about experimental protocols to a minimum and keep it clear, to the point.

10. Does S. vittatum exhibit “communal oviposition” behavior?
Time
A Wilcoxon matched-pairs signed rank test
B UGA Sixty minute test runs
C McCall et al Coupled-twenty minute test runs
P < 0.05 (unpaired t-test)

11. Orientation Chamber (OC) Bioassay
20 gravid flies
Single oviposition substrate
no eggs or eggs present
Measure attraction response
no. of flies on substrate

12. Orientation Single Chamber (OSC) Bioassay
OC Experiment #3
no screen
No. of flies attracted to substrate (mean)
Experiment N
Control
(no eggs)
Test
(eggs) 2
(no screen) 15
0.8
2.5 * 3
(screen present)
1.9
3.1
OC Experiment #4
screen present
* P < 0.005, unpaired t-test

13. Petri Dish (PD) Bioassay
1 gravid fly per petri dish
Single treatment per petri dish
Control treatment = solvent only
Test treatment = solvent +
test substance
60 petri dishes per treatment
Measure oviposition response
(no. of ovipositing flies)
Light table
20 glass petri dishes
Cotton under filter paper
10 control and 10 test
Climate controlled room (25ºC and 85% R.H.)
40 µL of treatments on filter paper and 8.0 mL of water
Compare (unpaired t-test)

14. PD Bioassay: lipophilic egg extract
Fresh Eggs
Hexane
31 (52%)
Lipophilic Egg Extract
Fisher’s Exact Test (* P = )

15. Chemical Analysis of Lipophilic Extract from S. vittatum Eggs
Gas Chromatography coupled with Mass Spectrometry (GC-MS) 1) 3) 2) 4)

16. PD Bioassay Results
Fisher’s Exact Test (* P < 0.05, ** P < 0.005, *** P < )

17. Electroantennogram (EAG)

18. Conclusions
Non-polar compounds washed from surface of eggs induced oviposition
Direct contact = induced oviposition
Does volatile compounds have any role?
EAG experiments demonstrate elicited response
How do the flies sense the oviposition compounds?
“drumming” behavior (forelegs) = stimulation?
Y-tube assay now available!
Translate research to vector species

19. Acknowledgements
UGA Black Fly Lab members - Dr. Ray Noblet, Elmer Gray, Joe Iburg, Roger Wyatt, Ivey Pomazal, Kate Klein
Dr. Ed and Mary Cupp
USF – Dr. Thomas Unnasch, Dr. Bill Baker, Dr. Nathan Burkett-Cadena, Dr. Ryan Young, Dr. Jeremy Beau, Andrew Shilling
Auburn University – Dr. Rao Balusu, Dr. Henry Fadamiro
Union Juarez, Chiapas, Mexico – Dr. Mario Rodriguez-Perez, Juan Armando Rodriguez-Perez, and the entire Chiapas crew
Banfora, Burkina Faso – Dr. Laurent Toe, Koala Lassane, and
the entire Burkina Faso crew
UGA Environmental Analysis Lab – Dr. Sayed Hassan
UGA Instrument Shop
Bill and Melinda Gates Foundation Grant #OPP