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.

-Artificially reared larvae are an ideal model for experiments involving brood diseases or testing pesticides. Because conditions during larval development can influence the general performance of adult honeybees, we created an evaluation method for the viability of artificially reared honeybees. We compared the flight performance of honeybees artificially reared in the laboratory with that of their sisters naturally reared in the colony. Fresh and dry weight, wing surface area, flight speed, flight duration, and distance covered by honeybee workers after feeding defined amounts of different sugar solutions were measured during tethered flight in a roundabout. Our results demonstrate that after artificial rearing, adult honeybees at the natural age of flight exhibit similar flight performances to their naturally reared sisters. The naturally reared honeybees, however, attained higher maximum flight speeds when fed energy-rich 2molar glucose solution.Apis mellifera / flight / in vitro rearing / larval nutrition / worker quality

Maternal immune experience acquired during pathogen exposure and passed on to progeny to enhance resistance to infection is called trans-generational immune priming (TgIP). In eusocial insects like honeybees, TgIP would result in a significant improvement of health at individual and colony level. Demonstrated in invertebrates other than honeybees, TgIP has not yet been fully elucidated in terms of intensity and molecular mechanisms underlying this response. Here, we immune-stimulated honeybee queens with Paenibacillus larvae (Pl), a spore-forming bacterium causing American Foulbrood, the most deadly bee brood disease worldwide. Subsequently, offspring of stimulated queens were exposed to spores of Pl and mortality rates were measured to evaluate maternal transfer of immunity. Our data substantiate the existence of TgIP effects in honeybees by direct evaluation of offspring resistance to bacterial infection. A further aspect of this study was to investigate a potential correlation between immune priming responses and prohaemocytes–haemocyte differentiation processes in larvae. The results point out that a priming effect triggers differentiation of prohaemocytes to haemocytes. However, the mechanisms underlying TgIP responses are still elusive and require future investigation.

Hydroxymethylfurfural (HMF) is a heat-formed, acid-catalyzed contaminant of sugar syrups, which find their way into honey bee feeding. As HMF was noted to be toxic to adult honey bees, we investigated the toxicity of HMF towards larvae. Therefore we exposed artificially reared larvae to a chronic HMF intoxication over 6 days using 6 different concentrations (5, 50, 750, 5000, 7500 and 10,000 ppm) and a control. The mortality was assessed from day 2 to day 7 (d7) and on day 22 (d22). Concentrations ranging from 5 to 750 ppm HMF did not show any influence on larval or pupal mortality compared to controls (p > 0.05; Kaplan-Meier analysis). Concentrations of 7500 ppm or higher caused a larval mortality of 100%. An experimental LC50 of 4280 ppm (d7) and 2424 ppm (d22) was determined. The calculated LD50 was 778 µg HMF per larva on d7 and 441 µg HMF on d22. Additionally, we exposed adult honey bees to high concentrations of HMF to compare the mortality to the results from larvae. On d7 larvae are much more sensitive against HMF than adult honey bees after 6 days of feeding. However, on d22 after emergence adults show a lower LC50, which indicates a higher sensitivity than larvae. As toxicity of HMF against honey bees is a function of time and concentration, our results indicate that HMF in supplemental food will probably not cause great brood losses. Yet sublethal effects might decrease fitness of the colony.

Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.