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.

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

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

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

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!

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

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

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

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.

Does S. vittatum exhibit “communal oviposition” behavior? Time A Wilcoxon matched-pairs signed rank test B UGA 2010. Sixty minute test runs C McCall et al. 1994. Coupled-twenty minute test runs P < 0.05 (unpaired t-test)

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

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

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)

PD Bioassay: lipophilic egg extract Fresh Eggs Hexane 31 (52%) Lipophilic Egg Extract Fisher’s Exact Test (* P = 0.0009)

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

PD Bioassay Results Fisher’s Exact Test (* P < 0.05, ** P < 0.005, *** P < 0.0005)

Electroantennogram (EAG)

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

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 #OPP1017870