A spectrophotometric technique for measuring particle ingestion by black fly larvae Joseph P. Iburg*, Elmer W. Gray, Roger D. Wyatt & Raymond Noblet Department of Entomology, University of Georgia, 413 Biological Science Building, Athens, GA 30602, USA Accepted: 22 October 2012 Key words: Simulium vittatum, feeding, pigmented particles, Neon Red, DayGlo ® , Bacillus thuringiensis ssp. israelensis, Diptera, Simuliidae Abstract A spectrophotometric technique was developed to provide insight into the feeding behavior of Simu- lium vittatum Zetterstedt (Diptera: Simuliidae) larvae. Larvae were exposed to water insoluble Neon Red particles (NRP) (DayGlo ® ) in a controlled current. The insoluble particles were available for cap- ture by the cephalic fans of the larvae and subsequent ingestion. The length of gut occupied by the particles after a given exposure time was determined by visual inspection and measured with the aid of a dissecting microscope. Larvae were then homogenized in acetone to solubilize the particles. After filtration, the quantity of pigmented particles in the alimentary tract of the larvae was determined using spectrophotometric analysis. The quantity of particles per unit length of the alimentary tract was calculated. Experiments were conducted to determine the ideal concentration of NRP for obtain- ing an accurate measure of ingestion without interfering with normal larval feeding behavior. Larval mortality following ingestion of insecticidal proteins produced by Bacillus thuringiensis ssp. israelensis was used as an indirect measure of feeding behavior for these experiments. A concentration of 15 p.p.m. of NRP in the larval medium was the highest concentration used that did not interfere with larval mortality following exposure to the insecticidal proteins. Additional experiments demon- strated that components of the experimental matrix did not interfere with NRP absorbance. The final experiment revealed that the consumption of NRP and insecticidal proteins by larvae was influenced by clay and cellulose in the larval medium. Introduction Black fly (Diptera: Simuliidae) larvae are indiscriminate filter feeders that thrive throughout the world in streams and rivers (Adler et al., 2004). The majority of Simuliidae larvae feed by capturing seston (suspended biotic and abi- otic material) with their labral fans (Chance, 1970). The fans periodically close and are inserted into the cibarium where the mandibles and labrum remove captured mate- rial from the fans during fan retraction (Merritt et al., 1996). In areas where black flies thrive, populations can trans- mit a variety of pathogens to humans and livestock (Adler et al., 2004). The adult female flies can cause economic problems due to their nuisance and biting behavior (Gray et al., 1996). Currently, the only products used to suppress black fly larvae contain insecticidal crystalline proteins (ICPs) produced by Bacillus thuringiensis ssp. israelensis de Barjac (Bti). Because the ICPs must be ingested for the protoxin to become toxic to the larvae, a comprehensive understanding of black fly feeding behavior is necessary. Numerous methods have been used to characterize the feeding behavior of Simuliidae. In several studies, the flick rate of the labral fans has been examined in relation to par- ticle ingestion (Gaugler & Molloy, 1980; Hart & Latta, 1986; Merritt et al., 1996; Stoops & Adler, 2009). In other studies, various types of markers such as dyes, beads, and charcoal were added to the environment of the larvae and the movement of the marker through the gut was mea- sured (Mulla & Lacey, 1976; Ladle & Hansford, 1981; Thompson, 1987; Miller et al., 1998; Wotton, 2009). These methods were developed to determine particle size selec- tion and time needed for material to pass through the gut of the larvae. Hart & Latta (1986) estimated the quantity of material ingested by black fly larvae by calculating how many parti- cles of a marker could occupy the volume of space in the midgut that contained the marker. This estimate relied on *Correspondence: E-mail: jpiburg@gmail.com © 2012 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 146: 293–301, 2012 Entomologia Experimentalis et Applicata © 2012 The Netherlands Entomological Society 293 DOI: 10.1111/eea.12029