Ulrike Riessberger-Gallé

Standard methods for artificial rearing of Apis mellifera larvae

Summary Originally, 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.

Trans-generational immune priming in honeybees

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.

Flight performance of artificially reared honeybees (Apis mellifera)

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.ZusammenfassungHonigbienen können im Labor künstlich aufgezogen werden. Dazu werden Larven aus Brutwaben in Plastikschälchen übersiedelt und die umfassende Brutpflege der Ammenbienen im Stock durch wenige definierte Fütterungen ersetzt sowie die Temperatur und die Luftfeuchtigkeit genau reguliert. Diese Methode ermöglicht es nun standardisierte Untersuchungen über die Auswirkungen von Chemikalien wie Pflanzenschutzmitteln oder Infektionen mit Krankheitserregern durchzuführen, ohne gesamte Völker in Kontakt mit den Schadstoffen oder Erregern zu bringen. Wir haben erstmals die Qualität von Arbeiterinnen die mit Hilfe dieser Technik aufgezogen wurden anhand ihrer Flugleistung in einem Karussell analysiert und mit der Leistung ihrer natürlich aufgezogener Schwestern verglichen (Abb. 1). Die durchschnittliche Fluggeschwindigkeit im Karussell betrug etwa 1 m/s und die maximale Fluggeschwindigkeit 1,4 m/s (Tab. I). Diese unterschieden sich bei künstlich und natürlich aufgezogenen Bienen nicht, wenn 10 μL einer 1-Molaren Glukoselösung gefüttert werden. Bei Fütterung von hochenergetischer 2-Molarer Glukoselösung flogen die Kontrollbienen, nicht aber die künstlich aufgezogenen, schneller als mit 1-Molarer Glukoselösung nämlich mit 1,2 m/sec Durchschnittsgeschwindigkeit. Die Maximalgeschwindigkeit (1,6 m/s) der Kontrollbienen war bei dieser Fütterung auch höher als die der künstlich aufgezogenen (1,4 m/s, Tab. I).Ein guter Indikator für die Qualität der Larvalernährung ist das Gewicht der Arbeiterinnen, und wir fanden, dass das Trockengewicht bei den von uns künstlich aufgezogenen Bienen niedriger als bei den Kontrollbienen war. Wir konnten auch zeigen, dass dieser Unterschied vor allem in einem leichteren Thorax, in dem sich die Flugmuskulatur befindet, der künstlich aufgezogenen Bienen begründet ist (Abb. 2). Außerdem fanden wir schwache Unterschiede in den Flächen der Vorder- und Hinterflügel: wiederum waren die der künstlich aufgezogenen etwas kleiner als die der Kontrollbienen.Unsere Ergebnisse zeigen, dass künstlich aufgezogene Bienen das Alter von Sammlerinnen (über 20 Tage) erreichen können. Trotz eines etwas leichteren Thoraxgewichtes, in dem sich die Flugmuskulatur befindet, und etwas kleinerer Flügel zeigten sie annähernd ähnliche Flugleistungen wie natürlich aufgezogene Honigbienen.

In vitro Growth Inhibition by Hypericum Extracts and Isolated Pure Compounds of Paenibacillus larvae, a Lethal Disease Affecting Honeybees Worldwide

The in vitro inhibitory potential of 50 extracts from various species of the flowering plant genus Hypericum was investigated using the KirbyBauer disk diffusion susceptibility test against Paenibacillus larvae, a spore‐forming, Gram‐positive bacterial pathogen that causes American foulbrood (AFB), a lethal disease affecting honeybee brood worldwide. Of the tested extracts, 14 were identified as highly active against P. larvae as compared to the activity of the positive control, indicating the presence of highly potent antibacterial compounds in the extracts. Examination of these extracts using TLC and HPLC/MS analyses revealed the presence of acylphloroglucinol and filicinic‐acid derivatives. Six pure compounds isolated from these extracts, viz., hyperforin (1), uliginosin B (2), uliginosin A (3), 7‐epiclusianone (4), albaspidin AA (5), and drummondin E (6), displayed strong antibacterial activity against the vegetative form of P. larvae (MIC ranging from 0.168–220 μM). Incubation of P. larvae spores with the lipophilic extract of Hypericum perforatum and its main acylphloroglucinol constituent 1 led to the observation of significantly fewer colony forming units as compared to the negative control, indicating that the acylphloroglucinol scaffold represents an interesting lead structure for the development of new AFB control agents.

Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae

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.

Cuticular hydrocarbon cues of immune‐challenged workers elicit immune activation in honey bee queens

Recently, evidence has shown that variations in the cuticular hydrocarbons (CHCs) profile allow healthy honeybees to identify diseased nestmates, eliciting agonistic responses in the former. Here, we determined whether these ‘immunologic cues’ emitted by diseased nestmates were only detected by workers, who consequently took hygienic measures and excluded these individuals from the colony, or whether queens were also able to detect these cues and respond accordingly. Healthy honeybee queens were exposed to (i) healthy, (ii) Ringer‐injected and (iii) lipopolysaccharide (LPS)‐injected nestmates by allowing direct body contact. Quantitative differences in the CHC profiles of these three groups were measured using GC‐MS. The transcript levels of the products of four genes that encode for antimicrobial peptides (AMPs), which are part of the queens immune response, were measured in bees exposed to direct contact using qPCR. A significant increase in the transcript levels of these AMP genes over baseline levels in queens was observed when body contact was allowed between the queens and the Ringer‐ and LPS‐injected nestmates. These results provide the first evidence that the detection of CHCs contributes to the initiation of an immune response in insects. In an additional experiment, CHCs were extracted from diseased workers and directly presented to queens, which also evoked a similar immune response. A potential mechanism that relied on volatile compounds could be ruled out by conducting a distance experiment. The study helps to expand our knowledge of chemical communication in insects and sheds light on a likely new mechanism of social immunity.

Immune responses of honeybees and their fitness costs as compared to bumblebees

Immune responses of invertebrates imply more than developing a merely unspecific response to an infection. Great interest has been raised to unveil whether this investment into immunity also involves fitness costs associated to the individual or the group. Focusing on the immune responses of honeybees, we use the well-studied insect bumblebee for comparison. Bumblebees are capable of producing specific immune responses to infections whereas this has not been assessed for honeybees so far. We investigated whether a prior bacterial encounter provides protection against a later exposure to the same or a different bacterium in honeybees. Additionally, we studied whether the foraging activities of honeybees and bumblebees are affected upon immune stimulation by assessing the flight performance. Finally, the acceptance behavior of nestmates toward immune-challenged honeybees was determined. Results show that despite stimulating the immune system of honeybees, no protective effects to infections were found. Further, honeybees were not affected by an immune challenge in their flight performance whereas bumblebees showed significant flight impairment. Immune-challenged honeybees showed lower survival rates than naive individuals when introduced into a regular colony. Here, we reveal different immune response-cost scenarios in honeybees and bumblebees for the first time.

Lysophosphatidylcholine acts in the constitutive immune defence against American foulbrood in adult honeybees.

Honeybee (Apis mellifera) imagines are resistant to the Gram-positive bacterium Paenibacillus larvae (P. larvae), causative agent of American foulbrood (AFB), whereas honeybee larvae show susceptibility against this pathogen only during the first 48 h of their life. It is known that midgut homogenate of adult honeybees as well as a homogenate of aged larvae exhibit strong anti-P. larvae activity. A bioactivity-guided LC-HRMS analysis of midgut homogenate resulted in the identification of 1-oleoyl-sn-glycero-3-phosphocholine (LPC) pointing to a yet unknown immune defence in adult honeybees against P. larvae. Antimicrobial activity of LPC was also demonstrated against Melissococcus plutonius, causative agent of European Foulbrood. To demonstrate an AFB-preventive effect of LPC in larvae, artificially reared larvae were supplemented with LPC to evaluate its toxicity and to assess whether, after infection with P. larvae spores, LPC supplementation prevents AFB infection. 10 μg LPC per larva applied for 3 d significantly lowered mortality due to AFB in comparison to controls. A potential delivery route of LPC to the larvae in a colony via nurse bees was assessed through a tracking experiment using fluorescent-labelled LPC. This yet undescribed and non-proteinous defense of honeybees against P. larvae may offer new perspectives for a treatment of AFB without the utilization of classic antibiotics.