Aleš Gregorc

A review of methods for discrimination of honey bee populations as applied to European beekeeping

Summary Here, scientists from 19 European countries, most of them collaborating in Working Group 4: “Diversity and Vitality” of COST Action FA 0803 “Prevention of honey bee COlony LOSSes” (COLOSS), review the methodology applied in each country for discriminating between honey bee populations. Morphometric analyses (classical and geometric) and different molecular markers have been applied. Even if the approach has been similar, however, different methodologies regarding measurements, landmarks or molecular markers may have been used, as well as different statistical procedures. There is therefore the necessity to establish common methods in all countries in order to have results that can be directly compared. This is one of the goals of WG4 of the COLOSS project.

PROGRAMMED CELL DEATH IN THE HONEY-BEE (APIS MELLIFERA L.) LARVAE MIDGUT

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra‐cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti‐histone associated DNA fragmentation indicative of programmed cell death.

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline.

A number of techniques were employed to assess cell death induced in honeybee larvae midgut afterper os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24h after treatment. Cell death reduced to 36% in the epithelial cells, 48h after treatment. In Oxytetracycline‐treated larvae, cell death was identified in 40% of midgut epithelial cells, 24h after inoculation and increased to 55% over the next 24h. In Paenibacillus ‐infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24h after Amitraz application. Cell death was reduced to 9% over the next 24h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.

Residues of Pesticides in Honeybee (Apis mellifera carnica) Bee Bread and in Pollen Loads from Treated Apple Orchards

Honey bee (Apis mellifera carnica) colonies were placed in two apple orchards treated with the insecticides diazinon and thiacloprid and the fungicide difenoconazole in accordance with a Protection Treatment Plan in the spring of 2007. Pollen and bee bread were collected from combs inside the hives. The residue of diazinon in pollen loads 10xa0days after orchard treatment was 0.09xa0mg/kg, and the same amount of residue was found in bee bread 16xa0days after treatment. In pollen loads 6xa0days after application 0.03xa0mg/kg of thiacloprid residues and 0.01xa0mg/kg of difenoconazole were found on the first day after application. Possible sub-lethal effects on individual honey bees and brood are discussed.

Histopathological and histochemical changes in honeybee larvae (Apis mellifera L.) after infection with Bacillus larvae, the causative agent of American foulbrood disease.

Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae. The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B. larvae. Many autolysosomes were positive for acid phosphatase. Non‐vacuolar acid phosphatase activity was also found in lysed cell compartments. No such activity was found in regenerative epithelial cells. Degradation of haemocytes, salivary glands and other tissues was also observed. Histochemical analyses after per cutaneous inoculation with B. larvae of three‐ and five‐day‐old honeybee larvae show intense non‐vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes.

Rotenone and oxalic acid as alternative acaricidal treatments for Varroa destructor in honeybee colonies.

This experiment assessed the efficacy of rotenone and oxalic acid (OA) in an aqueous sugar solution in controlling the honeybee mite Varroa destructor. Colonies were populated with mite-infested brood combs and worker bees. Three rotenone or OA treatments administered during the period with capped brood on 31 July, 14 and 18 August resulted in an average efficacy of 24.10%. In untreated colonies mite mortality averaged 5.40%. No significant differences (P>0.05) were found between the rotenone and OA treatments. Three OA treatments administered on 9, 12 and 18 September resulted in a 77.93% mite mortality. An increase in mite drop (P<0.05) was observed at 2 and 4 days after each treatment. OA applications in broodless colonies resulted in significantly (P<0.001) higher mite mortality rates (98.65% average) than the three treatments of rotenone or OA in colonies with capped brood. The dynamics of mite mortality after each rotenone or OA treatment are discussed in this study.

Heat shock proteins and cell death in situ localisation in hypopharyngeal glands of honeybee (Apis mellifera carnica) workers after imidacloprid or coumaphos treatment

Worker honeybees (Apis mellifera carnica Polm.) were treated with imidacloprid or coumaphos. Significant effects of treatment and treatment duration were found on hypopharyngeal glands (HPG) acinus diameter (P < 0.05). Differences in the size of acini were evident in all long term (48 h and 72 h) treatments. Short term (24 h) imidacloprid treatment induced heat shock protein 70 (Hsp 70) localisation in nuclei and cytoplasm and Hsp 90 activity was found in most cell cytoplasm. Coumaphos triggered an increased level of programmed cell death, and imidacloprid induced extended necrosis in comparison to coumaphos. In 7–12 day old workers, the level of cell death after 48 hours of imidacloprid treatment was approximately 50% and increased to all cells after 72 hours. Programmed cell death remained at the normal level of approximately 10%. Our results suggest that both pesticide treatments have an influence on the reduced size of HPG and also on the extended expression of cell death.ZusammenfassungArbeiterinnen der Honigbiene (Apis mellifera carnica Polm.), die in einem Brutschrank geschlüpft waren, wurden auf dem Thorax markiert und in weiselrichtige Völker gegeben. Danach wurden altersspezifische Gruppen aus jeweils acht markierten Arbeiterinnen gebildet und in Holzkäfigen gehalten. Die erste Gruppe bestand aus 1 bis 6 Tage alten Arbeiterinnen, die zweite aus 7 bis 12 Tage alten, die dritte Gruppe aus 13 bis 18 Tage alten und die vierte Gruppe aus 19 bis 32 Tage alten. Diese erhielten als Behandlung entweder: (1) eine Imidacloprid-Lösung (500 ng/kg in 35 % Zuckerwasser), (2) eine Coumaphos-Lösung oder (3) als Kontrolle 35 % (w/v) Zuckerwasser. Die Arbeiterinnen konnten diese Lösungen über 24, 48 oder 72 Stunden ad libitum aufnehmen. Am Ende der jeweiligen Behandlungsperioden wurden die Hypopharynxdrüsen dieser Bienen entnommen, fixiert und in Wachs eingebettet. Für alle Alters- und Behandlungsgruppen wurden immunhistologische Präparate angefertigt und ‘morphometrische Messungen durchgeführt, die statistisch ausgewertet wurden. Entwachste Schnitte wurden mit Primärantikörpern gegen die Hitzeschockproteine 70 und 90 (Hsp 70 und Hsp 90) inkubiert. Zelltodereignisse wurden mittels TUNEL Reagenz des ‘In situ cell death detection kit, AP’ (Roche) erfasst. Als zweites Detektionsverfahren für Zelltod verwendeten wir das Apop Tag in situ Apoptosis detection kit (Chemiocon). Damit erfassten wir die Prozentsätze an Zellen, die behandlungsbedingten Zelltod oder Hitzeschockproteine aufwiesen. Wir konnten signifikante Unterschiede im Acinus-Durchmesser der Drüsen in Abhängigkeit von der Behandlung und der Behandlungsdauer erkennen, wobei die Unterschiede in der Acinus-Größe vor allem bei den Langzeitbehandlungen (48 und 72 Std.) deutlich waren. Bei der Kurzzeitbehandlung (24 Std.) mit Imidacloprid wurde Hsp 70 induziert und war sowohl im Zytoplasma als auch im Zellkern zu finden, während Hsp 90 im Zytoplasma der meisten Zellen nachweisbar war. Die Hsp-Aktivität war bei Coumaphos behandelten Gruppen deutlicher als bei den Imidacloprid behandelten. Coumaphos führte außerdem zu höheren Zelltodraten als Imidacloprid, während nach Imidaclopridbehandlung mehr Nekroseschäden zu sehen waren. Bei den 7–12 Tage alten Arbeiterinnen lag die Zelltodrate nach 48 stündiger Imidacloprid-Behandlung bei 50 % und erfasste bei längerer Behandlung (72 Std.) nahezu alle Zellen. Die normalen Zelltodraten bei Kontrollen lagen bei 10 %. Wir schliessen daraus, dass beide Pestizide die Größe der Hypopharynxdrüsen negativ beeinflussen können und dass eine längere Exposition zum Zelltod führt. Unser Versuchsansatz erlaubt es auch, subletale Effekte auf bestimmte Organe erkennen zu können. Diese Nachweisverfahren können damit Hilfsmittel darstellen, um die zelluläre Antwort in Hypopharynxdrüsen nach Feldeinsätzen von Imidacloprid, bzw. von Coumaphos in Bienenvölkern zu erfassen.

Toxicological and immunohistochemical testing of honeybees after oxalic acid and rotenone treatments

Bees removed capped brood and young larvae from combs at a greater rate after a rotenone treatment than after an oxalic acid (OA)/sucrose treatment. Rotenone (1%) caused 75.2% of capped brood to be removed, OA (3%) 18.7% and a control treatment, 13.3%. Caged worker bees treated with a 1% rotenone powder, a 3% OA or with a control solution had mortality rates of 10.9%, 5.1% and 1.9% respectively. Rotenone (1%) significantly affected the mortality of brood and adult bees whereas OA (3%), did not. Solutions of 3% OA/32% sucrose, 3.4% OA/47.6% sucrose, 3.7% OA/27.1% sucrose (w/w) and a 32% sugar solution applied to adult bees resulted in death rates of 11%, 14%, 11.2% and 6.5% respectively. Individually treated bees consumed more of a 3% OA solution than solutions with higher OA concentrations. A TUNEL assay detected necrotic cell death in 69% of bee midgut cells 24 h after an OA treatment. Normal cell turnover is approximately 8%.ZusammenfassungWir führten vier unterschiedliche Experimente und eine immunohistochemische Analyse an Honigbienenvölkern durch, um die Effekte von zwei Akariziden besser zu charakterisieren. Sowohl Oxalsäure (OA) als auch Rotenon werden gängigerweise zur Kontrolle der ektoparasitischen Milbe Varroa destructor eingesetzt. Im ersten Experiment wurden 12 Apis mellifera Völker mit einer 3 % OA-Lösung oder mit Rotenon (1 %, in Pulverform) behandelt, um den Einfluss dieser Applikationen auf die Brutentwicklung zu untersuchen.Im zweiten Experiment wurden diese Akarizide an gekäfigten Arbeiterinnen (18 Versuchsansätze) im Hinblick auf ihre Toxizität (Mortalität) getestet. In einem dritten Experiment untersuchten wir ebenfalls die Mortalitätsraten in 19 Käfigen mit Arbeiterinnen, denen jeweils eine der folgenden OA/Sacharose-Lösungen verabreicht wurde: 3 %/32 %; 3,4 %/47,7 %; oder 3,7 %/27,l %. Im vierten Experiment erhielten 36 Arbeiterinnen in Einzelhaltung jeweils eine der folgenden OA/Sacharose-Lösungen: 3 %/32 %; 3,7 %/27,l % oder 4,5 %/31,3 %. Für diese ad libitum über eine 24-Stunden Periode verabreichten OA/Sacharose-Lösungen wurden die Aufnahmeraten bestimmt. Für die Bienen, die im vierten Experiment eine 3 % OA-Lösung erhalten hatten, bestimmten wir anschliessend den Grad der durch die Behandlung verursachte Gewebeschädigung im Mitteldarm. Dazu wurde der herauspräparierte Darm zwei immunohistochemischen Tests unterzogen. Spezifische Ziele dieser Arbeit waren die Bestimmung (a) der Effekte der OA- und Rotenon-Behandlungen auf die Brutstadien, (b) der Mortalitätsraten als Folge unterschiedlicher OA oder Rotenon-Behandlungen, (c) der Aufnahmeraten für unterschiedliche OA-Konzenrationen und (d) der Effekte einer OA-Behandlung auf Zelltod in Arbeiterinnen.Die Ergebnisse waren, dass nach der Rotenon-Behandlung mehr Brut entfernt wurde (75,2 %) als nach der 3 % OA-Behandlung (18,7 %) (Tab. I). Die Rotenon-Behandlung im zweiten Experiment zeigte eine signifikant erhöhte Mortalität dieser Bienen (Abb. 1). Im dritten Experiment fanden wir für die mit der 3,4 % und der 3,7 % OA-Lösung behandelten gekäfigten Bienen eine signifikant erhöhte Mortalität (Abb. 2). Im vierten Experiment zeigte sich, dass die Bienen die 3 % OA-Lösung den anderen bevorzugten (Tab. II). Die immunohistochemischen Untersuchungen an Bienen, die im vierten Experiment über 24 Stunden hinweg eine 3 % OA-Lösung erhalten hatten, zeigten Anzeichen von natürlichem (apoptotischem) oder verletzungsbedingtem (nekrotischem) Zelltod in 69 % der Zellen des Mitteldarms. Der normale Zellumsatz in diesem Gewebe liegt bei 8 % (Tab. III).Während die Wirksamkeit sowohl von Rotenon als auch OA zur Bekämpfung der Varroamilbe unbestritten ist, sollten dennoch die Faktoren Jahreszeit — d.h. ob Brut vorhanden ist oder nicht — und Konzentration der OA-Lösungen berücksichtigt werden, um eine ökonomisch optimale Wirkung an den Bienen eines Volkes zu erzielen. Wir denken, dass unsere Untersuchungen hierzu eine Beitrag leisten konnten.

In situ localization of heat-shock and histone proteins in honey-bee (apis mellifera l.) larvae infected with paenibacillus larvae

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%.

A review of methods used in some European countries for assessing the quality of honey bee queens through their physical characters and the performance of their colonies

Summary The term “quality” in relation to queens and drones refers to certain quantitative physical and/or behavioural characters. It is generally believed that a high quality queen should have the following physical characteristics: high live weight; high number of ovarioles; large size of spermatheca; high number of spermatozoa in spermatheca; and be free from diseases and pests. It is, however, also known that the performance of a honey bee colony is the result of its queens function as well as of that of the drones that mated with her. These two approaches are often considered together and give a general picture of the queen production technique and selection. Here we describe the most common and well known anatomical, physiological, behavioural and performance characters related to the queens, as measured in different European countries: the live weight of the virgin queen (Bulgaria); the live weight of the laying queen (Bulgaria, Italy); the diameter and volume of spermatheca (Bulgaria, Greece, Slovenia); the number of ovarioles (Greece, Italy, Slovenia); the weight of ovaries (Slovenia); the number of spermatozoa in spermatheca (Italy, Poland, Slovenia); the brood pattern (Bulgaria, Greece); the egg laying ability/fecundity (Bulgaria); the brood production (Croatia, Serbia); the colony population development (Croatia, Serbia, Slovakia); the honey production (Croatia, Denmark, Serbia, Slovakia); the hygienic behaviour (Croatia, Denmark, Serbia, Slovakia); the defence behaviour (Croatia); the calmness/sitting on the comb (Croatia, Denmark); and swarming (Croatia, Denmark). The data presented fit well with the findings of the same characters in the literature, and in general they support the argument for the term “quality characters”. Especially for the weight of the queen, the number of ovarioles, the volume of the spermatheca and the number of spermatozoa, data per country proved its own accuracy by repetition through the years. We also report that when instrumentally inseminated queens are kept under mass production conditions (in small cages in queen banks and with low number of attendants) they can transfer the semen to their spermatheca and clear their oviducts more efficiently when they have been inseminated with small amounts of semen in two or three sequences (but not four), compared to those inseminated with the same amount of semen at once (Poland). Furthermore, we had an inside view of the sanitary conditions of the colony: a. through the health status of the queen (nosema plus virus analysis) (Slovenia); and b. evaluating the nosema load of worker bees (Denmark) and of the queens (Greece). This is the first step to summarize this type of diverse data for such an important issue. The knowledge acquired can be used to fill in the existing gaps in the breeding or queen evaluation systems of each country in order to facilitate standardization of methodology for comparable results.