Standard methods for artificial rearing ofApis melliferalarvae

Abstract

SummaryOriginally, a method to rear worker honey bee larvae in vitro was introduced into the field of bee biology to analyse honey bee physiology and caste development. Recently, it has become an increasingly important method in bee pathology and toxicology. The in vitro method of rearing larvae is complex and can be developed as an art by itself, especially if the aim is to obtain queens or worker bees which, for example, can be re-introduced into the colony as able members. However, a more pragmatic approach to in vitro rearing of larvae is also possible and justified if the aim is to focus on certain pathogens or compounds to be tested. It is up to the researcher(s) to decide on the appropriate experimental establishment and design. This paper will help with this decision and provide guidelines on how to adjust the method of in vitro rearing according to the specific needs of the scientific project. Métodos estándar para la cría in vitro de larvas de Apis mellifera ResumenOriginalmente, el método para la cría in vitro de larvas de obreras de abejas melíferas se introdujo en el campo de la biología de las abejas para analizar la fisiología y el desarrollo de las castas. Recientemente, se ha convertido en un método cada vez más importante para la patología y la toxicología de la abeja. El método de cría in vitro de larvas es complejo y constituye un arte en sí mismo, especialmente si el objetivo es obtener reinas o abejas obreras que, por ejemplo, puedan ser re-introducidas en la colonia como miembros activos. Sin embargo, un enfoque más pragmático de la cría de larvas in vitro también es posible y justificado si el objetivo es centrarse en ensayos con ciertos patógenos o compuestos. Corresponde al investigador (es) decidir sobre el adecuado establecimiento experimental y el diseño. Este artículo ayudará con esta decisión y proporcionará directrices sobre cómo ajustar el método de cría in vitro en función de las necesidades específicas del proyecto científico.

Protocol for the in vitro rearing of honey bee (Apis mellifera L.) workers

“…One of the main differences between our larval diet recipe and those listed in previous protocols (Aupinel et al, 2005;Crailsheim et al, 2013) are the increase in the water content and a reduction in the royal jelly content of diets A and B. We found that the diets noted in the literature (Aupinel et al, 2005;Crailsheim et al, 2013) were prone to drying out in the experimental conditions under which we reared larvae. Consequently, we increased the amount of water added to our diets over that recommended by Aupinel et al (2005) and Crailsheim et al (2013).…”

“…We provide an updated protocol with a revised method in a step-by-step format to outline how to rear honey bee workers artificially. The Crailsheim et al (2013) COLOSS BEEBOOK publication for the artificial rearing of honey bees provides guidance for understanding the historical context of the methodology and the different rearing approaches that are utilized by various researchers. The "standardized methods" are, however, difficult to follow and interpret for a researcher who is not familiar with the methodology.…”

“…The advancements in the in vitro rearing protocol have given government, industry, and academia a new tool for accessing the risks pesticides and other chemicals pose to immature honey bee development and survival (Aupinel et al, 2005(Aupinel et al, , 2007(Aupinel et al, , 2009Crailsheim et al, 2013;Hendriksma, Härtel, & Steffan-Dewenter, 2011b;Huang, 2009;Peng et al, 1992;Zhu, Schmehl, Mullin, & Frazier, 2014). Honey bee in vitro rearing protocols have allowed researchers to standardize environmental conditions (e.g.…”

“…Substantial progress has been made with bioassays that can be used to rear immature honey bees in the laboratory over the past few decades (Aupinel et al, 2005;Crailsheim et al, 2013;Huang, 2009;Peng, Mussen, Fong, Montague, & Tyler, 1992;Rembold & Lackner, 1981;Vandenberg & Shimanuki, 1987). The in vitro methodology for workers was initially used to investigate queen/worker differentiation, nutrition, and pathogen impacts on immature bees (Rembold & Lackner, 1981;Vandenberg & Shimanuki, 1987).…”

Resistance of developing honeybee larvae during chronic exposure to dietary nicotine

“…Two day old worker honeybee larvae were grafted onto larval food in 48-well microtiter plates (Thermo Fisher Scientific, Rochester, New York), using a protocol slightly modified from Aupinel et al (2005); see also Crailsheim et al (2013). The grafted larvae were kept in an incubator (Memmert HCP 108, GmbH + Co. KG, Schwabach, Germany) under constant darkness at 34°C and 95% relative humidity (days 1 to 6).…”