Submitted to: Journal of Insect Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/25/2005
Publication Date: 5/5/2006
Citation: Blanco, C.A., Perera, O.P., Ray, J.D., Taliercio, E.W., Williams III, L.H. 2006. INCORPORATION OF RHODAMINE B INTO MALE TOBACCO BUDWORM MOTHS HELIOTHIS VIRESCENS TO USE AS A MARKER FOR MATING STUDIES. Journal of Insect Science. 6:05. AVAILABLE ONLINE: insectscience.org/6.05. Interpretive Summary: The U. S. Department of Agriculture, Agricultural Research Service in Stoneville, MS has received field-collected samples of pheromone trap-captured tobacco budworm (H. virescens) male moths from the North America cotton belt to test their susceptibility (resistance) to Bacillus thuringiensis (Bt) proteins expressed by Bt cottons. This collection method established in '1998 was an easy and logistically feasible way of obtaining a large number of moths per sample in space and time. In recent years it has become increasingly more difficult to collect this insect from cotton regions. Optimal mating conditions can ensure the greatest genetic representation of these field-collected samples and a correct interpretation of results can be evaluated through the intensity of moth copulations. Counting the sperm bundles (spermatophores) in the female’s bursa copulatrix, passed during copulation, is an established method of obtaining mating frequency. Due to the polyandrous nature of H. virescens, a bursa copulatrix can contain more than one spermatophore indicating multiple copulations but this fact does not necessarily indicate that mating occurred with different males. Discerning the male genetic contribution under mass mating conditions, where theoretically every moth has the same opportunity to mate, can be assessed by a) including a known proportion of marked males that could produce the same proportion of marked spermatophores or b) by elaborate, time-consuming and expensive detailed genetic studies. For this study, Rhodamine marker was incorporated into >82% of male’s spermatophores via feeding moths for 1-5 days. The use of this relatively inexpensive dye ($0.35 for >2,000 marked moths) did not significantly affect their reproductive biology and females that copulated with marked males produced the same number of offspring than those mated with unmarked males. This marking method can be a useful tool in biological, behavioral and genetic studies.
Technical Abstract: Rhodamine B, a dye commonly used in a variety of biological studies was incorporated into the bodies of tobacco budworm moths by feeding them 0.1% rhodamine in 10% sucrose solution. After one to three days of male moth exposure to this pigment it was clearly detectable in >82% of spermatophores from rhodamine-fed males extracted from untreated females. The intake of this dye did not affect the life span, the production of eggs or the capacity of moths to copulate when compared with moths fed only a sucrose solution or water. Rhodamine B was easily identifiable externally but more so internally in moths after only one day of exposure to the pigment. Even at this short feeding duration, rhodamine was detectable in >50% of moths 5 days after feeding stopped. Longer exposure to the dye significantly increased the percentage of moths stained. Detection of rhodamine was slightly enhanced by the use of ultraviolet light. The dye accumulation in internal abdominal contents was a better indicator of the presence of the pigment than external accumulation on the bodies. The use of the method described in this report for incorporating a low cost dye in tobacco budworm can be a tool in biological, behavioral and genetic studies. This method will discern the level of feral male genetic contribution to the USDA-ARS Southern Insect Management Research Unit’s tobacco budworm resistance to Bt cotton monitoring program.