Incorporation of rhodamine B into male tobacco budworm moths Heliothis virescens to use as a marker for mating studies

Abstract

Rhodamine B, a dye commonly used in a variety of biological studies was incorporated into the bodies of male tobacco budworm moths, Heliothis virescens (Lepidoptera: Noctuidae), by allowing them to feed freely on 0.1% rhodamine dissolved in a 10% sucrose solution. After exposing males for one to three days to this pigment, rhodamine was clearly detectable in >82% of spermatophores 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 was more apparent internally in males after only one day of exposure to the pigment. Even at this short feeding duration, rhodamine was detectable in >50% of males 5 days after feeding stopped. Longer exposure to the dye significantly increased the percentage stained. Detection of rhodamine was slightly enhanced by the use of ultraviolet light. The dye accumulation in internal abdominal organs was a better indicator of the presence of the pigment than external contamination of the moth. The use of the method described in this report can be a tool for the rapid incorporation of a low cost dye in the tobacco budworm for biological, behavioral and genetic studies.

An MTA phosphorylase gene discovered in the metagenomic library derived from Antarctic top soil during screening for lipolytic active clones confers strong pink fluorescence in the presence of rhodamine B

“…This is the first report describing the pink fluorescence phenomenon of the recombinant E. coli cells expressing the MTA phosphorylase gene growing on media containing rhodamine B. Rhodamine B is a fluorescent dye used in a variety of biological studies, for example, as a marker and tracer in animal and insect studies (Lindsey, 1983;Blanco et al, 2006), as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential (Reungpatthanaphong et al, 2003), as a marker in multi-drugresistant protein activity determination (Smital & Kurelec, 1998), as an extremely specific stain for cornification (Liisberg, 1968) and as a fluorescent dye in the complex with free fatty acids in a plate assay for bacterial lipases (Kouker & Jaeger, 1987).…”

Paternity Allocation in a Mutant Heliothis virescens1Colony

“…Newly emerged albino-type and wild-type males (<24 hours) were divided into two feeding groups: a) half of the emerging males was fed 10% sucrose solution for 2 days in a 3.8-liter carton capped with cloth, and b) the other half was fed 0.1% rhodamine B (Sigma-Aldrich ® , St. Louis, MO) dissolved in 10% sucrose solution for 2 days in a 3.8-liter carton capped with cloth. This procedure produced 'marked' males (Blanco et al 2006). After the 2-day feeding period, moths were paired in 473-ml-cartons (Neptune ® , Newark, NJ) capped with cloth and free access to sucrose solution.…”

“…Removed males were frozen and discarded. Dead females were dissected, and their spermatophores examined under fluorescent light to detect the presence of rhodamine B (Blanco et al 2006) that originated from the marked males. This method determined whether females had mated with a marked and an unmarked male.…”

Offspring From Sequential Matings Between <I>Bacillus thuringiensis</I>-Resistant and <I>Bacillus thuringiensis</I>-Susceptible <I>Heliothis virescens</I> Moths (Lepidoptera: Noctuidae)

“…ARS male moths were obtained from the ARS colony and were fed in the same manner. Rhodamine presence in marked male spermatophores provides a measure to determine the origin of these marked reproductive structures and conÞrm that a female has copulated with two different males (Blanco et al 2006a).…”

“…When two or more spermatophores were present in a femaleÕs bursa copulatrix, these structures were inspected under ßu-orescent light (Blanco et al 2006a) to conÞrm the presence of a marked and an unmarked spermatophore. Only females that had marked and unmarked spermatophores were used for data analysis.…”