Standard methods for chemical ecology research inApis mellifera

Abstract

SummaryThis paper describes basic methods essential in elucidating chemically-mediated behavioural interactions among honey bees, and between honey bees and other arthropods. These range from bioassay methods used to demonstrate the role of specific behaviours, techniques and equipment used to collect and analyse semiochemicals (both volatiles and non-volatiles e.g. cuticular hydrocarbons) from individual honey bees, groups of bees or an entire colony in its native environments. This paper covers: collection and analysis of honey bee volatiles in the natural environment, collection and analysis of bee volatiles out of their natural environment and their antennal detection, collection and analysis of non-volatile cuticular hydrocarbons, bioassays with queen pheromone and finally a section focusing on in vitro bioassays as a tool for elucidation of mechanisms regulating pheromone gland activity. Métodos estándar para la investigación en la ecología química en Apis mellifera ResumenEste artículo describe los métodos esenciales básicos para dilucidar las interacciones de comportamiento mediadas por la química entre las abejas y entre éstas y otros artrópodos. Estos van desde los métodos de bioensayo usados para demostrar el rol de comportamientos específicos, hasta las técnicas y equipamientos usados para colectar y analizar semioquímicos (tanto volátiles como no volátiles, por ejemplo hidrocarburos cuticulares) en abejas al nivel individual, en grupos de abejas o en una colonia entera en su ambiente natural. Este artículo engloba: colección y análisis de los volátiles de la abeja de la miel en su ambiente natural, colección y análisis de los volátiles de la abeja fuera de su ambiente natural y con su detección por las antenas, colección y análisis de los hidrocarburos cuticulares no volátiles, bioensayos con la feromona real y finalmente una sección enfocada a los bioensayos in vitro como herramienta para dilucidar los mecanismos que regulan la actividad de la glándula que produce feromonas.

Standard methods forApis melliferabeeswax research

“…In many cases GC-MS and GC-FID complement each other. The GC-MS allows identification of unknown compounds based on the mass spectra without using standards, as described by Torto et al (2013) in the BEEBOOK chapter on chemical ecology. It is mainly used for qualitative analysis of beeswax composition (Jim enez et al, 2003, Jim enez, Bernal, Aumente et al, 2004; Serra Bonveh ı and Ornantes Bermejo (2012), while GC-FID is preferable for quantification (Jim enez, Bernal, Aumente et al, 2004, Jim enez et al, 2007; Serra Bonveh ı and Ornantes Bermejo (2012).…”

“…The rules of identification of unknown compounds using GC-MS were also described in details by Torto et al (2013).…”

“…The quantitative analysis of n-alkanes is conducted using an internal standard method. General advantages resulting from using of internal standard in quantitative analysis are listed by Torto et al (2013). The standard mixture of n-alkanes (C 8 H 18 -C 40 H 82 ) is used with the internal standard -squalane (C 30 H 62 ) at 10 mg/L concentration.…”

“…General analytical properties of CG-FID technique is provided in the BEEBOOK chapter on chemical ecology research in A. mellifera; for details see Section 2.2.4. "Detection and analysis of volatiles" (Torto et al, 2013). Using the methods described by Jim enez et al 2004, and for the analysis of beeswax by GC-MS and GC-FID, odd number hydrocarbons (with 17-35 carbon atoms), even number hydrocarbons (with 22-34 carbons), and monounsaturated hydrocarbons with an odd number of carbon atoms (from 21 to 35) containing isomers with a different position of the double bond (although from the mass spectra the position of the double bond cannot be identified) can be determined qualitatively and quantitatively in a single run.…”

Standard methods for fungal brood disease research

“…All bees can perform uncapping and removal behaviours, but bees that detect abnormal brood odours at a low stimulus level may rapidly initiate uncapping behaviour, resulting in the removal of diseased brood before it becomes infectious (Wilson-Rich et al ., 2009). Individual bees from rapid-hygienic line breeds exhibited significantly increased sensitivity to the odour of chalkbrood disease at lower concentrations compared with bees from the slow-hygienic line, based on electrophysiological recordings of nerve impulses from the antennae (EAG), by proboscis-extension response conditioning (PER), and by isolation of volatiles from chalkbrood–infected larvae for use in field bioassays (see detailed methods in: Masterman et al ., 2000, 2001; Gramacho and Spivak, 2003; Spivak et al ., 2003; Swanson et al ., 2009; and general methods of EAG and collection of volatiles in the BEEBOOK paper on chemical ecology methods (Torto et al ., 2013) and of PER in the BEEBOOK paper on behavioural methods (Scheiner et al ., 2013)).…”