Gudrun Koeniger

High degree of polyandry in Apis dorsata queens detected by DNA microsatellite variability

Workers of six colonies of the giant honeybee Apis dorsata from Sabah, Malaysia (five colonies) and Java (one colony) were genotyped using single locus DNA fingerprinting. The colonies from Sabah nested in colony aggregations of 5 and 28 nests respectively on two trees. Three DNA microsatellite loci (A14, A76, A88) with a total of 27 alleles provided sufficient genetic variability to classify the workers into distinct sub-families revealing the degree of polyandry of the queens. Queens mated on average with 30.17 ± 5.98 drones with a range from 19 to 53. The average effective number of matings per queen was 25.56 ± 11.63. In the total sample of 192 workers, 22 individuals were found that were not offspring of the colonys queen. Three of these were potentially drifted offspring workers from genotyped queens of colonies nesting on the same tree.

Multiple nuptial flights, sperm transfer and the evolution of extreme polyandry in honeybee queens

The honeybee, Apis mellifera, has an extremely polyandrous mating system, which often involves multiple nuptial flights by its queens. To understand the evolution of extreme polyandry, we investigated the cost of multiple nuptial flights in relation to potential benefits. We analysed, with eight DNA microsatellite loci, the paternity of worker offspring of naturally mated queens. Queens that were restricted to one nuptial flight, but may have taken additional nuptial flights if allowed to do so, had significantly fewer matings than queens that started oviposition after a single nuptial flight. Furthermore, the number of sperm stored in a spermatheca increased significantly with the number of matings. We suggest that queens adjust their nuptial flight frequencies according to their mating success in their previous nuptial flights. The number of copulations seems to serve as a signal for the initiation of oviposition. In the light of these findings, we reconsider and discuss the significance of the sperm limitation hypothesis for the evolution of extreme polyandry in A. mellifera.

Standard methods for behavioural studies of Apis mellifera

Summary In this BEEBOOK paper we present a set of established methods for quantifying honey bee behaviour. We start with general methods for preparing bees for behavioural assays. Then we introduce assays for quantifying sensory responsiveness to gustatory, visual and olfactory stimuli. Presentation of more complex behaviours like appetitive and aversive learning under controlled laboratory conditions and learning paradigms under free-flying conditions will allow the reader to investigate a large range of cognitive skills in honey bees. Honey bees are very sensitive to changing temperatures. We therefore present experiments which aim at analysing honey bee locomotion in temperature gradients. The complex flight behaviour of honey bees can be investigated under controlled conditions in the laboratory or with sophisticated technologies like harmonic radar or RFID in the field. These methods will be explained in detail in different sections. Honey bees are model organisms in behavioural biology for their complex yet plastic division of labour. To observe the daily behaviour of individual bees in a colony, classical observation hives are very useful. The setting up and use of typical observation hives will be the focus of another section. The honey bee dance language has important characteristics of a real language and has been the focus of numerous studies. We here discuss the background of the honey bee dance language and describe how it can be studied. Finally, the mating of a honey bee queen with drones is essential to survival of the entire colony. We here give detailed and structured information how the mating behaviour of drones and queens can be observed and experimentally manipulated. The ultimate goal of this chapter is to provide the reader with a comprehensive set of experimental protocols for detailed studies on all aspects of honey bee behaviour including investigation of pesticide and insecticide effects.

The role of the mating sign in honey bees, Apis mellifera L.: does it hinder or promote multiple mating?

Abstract In a two-choice test, drones preferred queen models marked by a distinctly coloured ‘mating sign’ (secretions of the drones copulatory organs) to unmarked models. This was true also for models marked by aluminium foil. Dark-coloured drones preferred to copulate with light-coloured models and vice versa. Models carrying copulating drones were more attractive than single models. Thus a copulating drone as well as the mating sign and other coloured markers increased the attractiveness of a queen. The mating sign may indicate a form of cooperation between drones by marking a queen after sperm transfer to facilitate the identification of a queen by the following drones.

Evidence for Convergent Nucleotide Evolution and High Allelic Turnover Rates at the complementary sex determiner Gene of Western and Asian Honeybees

Our understanding of the impact of recombination, mutation, genetic drift, and selection on the evolution of a single gene is still limited. Here we investigate the impact of all these evolutionary forces at the complementary sex determiner (csd) gene that evolves under a balancing mode of selection. Females are heterozygous at the csd gene and males are hemizygous; diploid males are lethal and occur when csd is homozygous. Rare alleles thus have a selective advantage, are seldom lost by the effect of genetic drift, and are maintained over extended periods of time when compared with neutral polymorphisms. Here, we report on the analysis of 17, 19, and 15 csd alleles of Apis cerana, Apis dorsata, and Apis mellifera honeybees, respectively. We observed great heterogeneity of synonymous (piS) and nonsynonymous (piN) polymorphisms across the gene, with a consistent peak in exons 6 and 7. We propose that exons 6 and 7 encode the potential specifying domain (csd-PSD) that has accumulated elevated nucleotide polymorphisms over time by balancing selection. We observed no direct evidence that balancing selection favors the accumulation of nonsynonymous changes at csd-PSD (piN/piS ratios are all <1, ranging from 0.6 to 0.95). We observed an excess of shared nonsynonymous changes, which suggest that strong evolutionary constraints are operating at csd-PSD resulting in the independent accumulation of the same nonsynonymous changes in different alleles across species (convergent evolution). Analysis of csd-PSD genealogy revealed relatively short average coalescence times ( approximately 6 Myr), low average synonymous nucleotide diversity (piS < 0.09), and a lack of trans-specific alleles that substantially contrasts with previously analyzed loci under strong balancing selection. We excluded the possibility of a burst of diversification after population bottlenecking and intragenic recombination as explanatory factors, leaving high turnover rates as the explanation for this observation. By comparing observed allele richness and average coalescence times with a simplified model of csd-coalescence, we found that small long-term population sizes (i.e., N(e) < 10(4)), but not high mutation rates, can explain short maintenance times, implicating a strong historical impact of genetic drift on the molecular evolution of highly social honeybees.

Die Füllung der Spermatheka der Bienenkönigin

SummaryWhen the spermathecal glands are removed from young unmated queens and they are then naturally mated or artificially inseminated, only a small number of spermatozoa reach the spermatheca. Turgor of the spermatheca is at the same time reduced. Removal of one branch of the gland has very little effect.When intact queens are inseminated with damaged spermatozoa, the spermatheca remains almost empty. Paralysis of the skeletal muscles in the queens abdomen caused by Microbracon poison results in a sharp reduction of sperm entering the spermatheca. Under such conditions, the sperm is not removed from the oviducts.From these experiments it is concluded, that transfer of spermatozoa out of the oviducts into the spermatheca is a complex process in which the muscles of the queen, as well as the fluid of the spermatheca and its glands, and the individual movements of the spermatozoa take part.ZusammenfassungWerden bei unbegatteten Jungköniginnen die Spermathekaldrüsen operativ entfernt, dann gelangt nach natürlicher Paarung oder instrumenteller Insemination nur eine geringe Zahl von Spermatozoen in die Spermatheka, außerdem sinkt der Turgor der Samenblase; die Entfernung nur eines Drüsenastes hat kaum einen Effekt. Werden intakte Königinnen mit geschädigten Spermatozoen besamt, dann bleibt die Spermatheka fast leer.Nach Lähmung der Skelettmuskulatur der Königin durch Microbracon-Gift wird ebenfalls die Füllung der Spermatheka stark reduziert, und das Sperma wird nicht aus den Ovidukten entleert.Aus diesen Experimenten ergibt sich, daß der Transfer der Spermatozoen aus den Ovidukten in die Spermatheka als ein komplexer Vorgang zu verstehen ist, an dem sowohl die Muskulatur der Königin, die Flüssigkeit der Spermatheka und ihrer Drüse sowie die Eigenbewegungen der Spermatozoen beteiligt sind.

Home-site fidelity in migratory honeybees.

Home-site fidelity is well known in migratory animals, but not in social insects. Here we show that colonies of the giant honeybee Apis dorsata are able to find the same nest location even after seasonal migration. As worker bees do not have first-hand knowledge of the old nest site, the swarms must be guided by some form of genetic mechanism.

Assortative mating in a mixed population of European Honeybees,Apis mellifera ligustica andApis mellifera carnica

SummaryThe drone population at a natural drone congregation area (DCA) was composed of 50%Apis mellifera ligustica (L) and 50%Apis mellifera carnica (C) drones. Both kind of drones were monitored by frequent sampling. Significant differences in the vertical drone distribution were detected: at 4 meters the portion of L drones was larger and C drones were relatively more frequent in 20 meters. Virgin queens (L and C) were naturally mated in the DCA and mating frequencies for L and C drones were established. Their worker offspring was classified as pure Ls. pure Cs or hybrids. A significant tendency for assortative mating was found for both groups of queens. One of the mechanisms of mate choice may involve the differences in vertical distribution of the drones.ZusammenfassungDie Drohnenpopulation eines natürlichen Drohnensammelplatzes wurde zu 50% aus Drohnen vonApis mellifera ligustica (L) and zu 50% ausApis mellifera carnica (C) Drohnen zusammengesetzt. Durch kontinuierlich durchgeführte Drohnenfänge wurde die Relation beider Drohnenrassen überprüft. Es wurden signifikante Unterschiede in der Höhenverteilung der Drohnen gefunden: In 4 m Höhe war der relative Anteil der L-Drohnen größer als in 20 m und entsprechend waren die C-Drohnen in 20 m Höhe relativ häufiger als in 4 m. Jungfräulichen Königinnen (L und C) wurde der ungehinderte Hochzeitsflug auf dem Drohnensammelplatz gestattet. Die von diesen Königinnen produzierten Arbeiterinnen konnten als reine L-, reine C-Bienen oder als Hybriden bestimmt werden. Auf diese Weise waren Aussagen über die Paarungsfrequenz möglich. Sowohl die C- als auch die L-Königinnen hatten mehr rassereine Nachkommen als Hybriden. Daraus ergab sich für beide Rassen eine Bevorzugung von Paarungen innerhalb der Rasse. Eine rassespezifisch unterschiedliche Flughöhe beim Hochzeitsflug der Königinnen könnte zu diesen Paarungsergnis beigetragen haben.

Mating flight duration of Apis mellifera queens: As short as possible, as long as necessary*

Polyandry in queen honey bees (Apis mellifera) prolongs the duration of nuptial flights which increases costs and risks. Under conditions of limited drone numbers the hypothesis was tested whether or not there is a threshold for successful mating during mating flight. In 29 queens we found a significant negative correlation between mating flight duration and number of spermatozoa in the spermatheca (Pearson r = −0.38, P = 0.04). This negative correlation supports the idea that queens continuously get information on her mating success during flight and return to the colony as soon as they have met a sufficient number of drones. In case of normal availability of drones queens fly from 10 to 30 minutes, so we compared 2 groups of queens (flight duration less than 30 versus more than 30 minutes). Sperm numbers differed significantly between the two groups (3.0 ± 0.77 and 1.1 ± 1.04 million, Wilcoxon, P < 0.001). These results further indicate that queens monitor mating success during flight.ZusammenfassungDie Mehrfachpaarung der Bienenkönigin führt zu einer Verlängerung des Paarungsfluges. Dadurch steigen die „Kosten“ und vor allem auch die Risiken des Paarungsfluges erheblich und einer Optimierung der Flugdauer im Sinne eines Selektionsvorteils kommt zweifelsfrei eine wesentliche evolutive Bedeutung zu.Wir haben in einem Gebiet, in dem die Anzahl der vorhandenen Drohnen auf 2500 experimentell vermindert war, die Frage untersucht, ob Königinnen den Paarungserfolg überwachen und heimkehren, sowie die Schwelle für erfolgreiche Paarungen erreicht ist. Bei einer Drohnenanzahl von 2500 dauerten die Paarungsflüge von 29 Königinnen im Durchschnitt 21, 8±9, 67 Minuten, während wir früher bei mehr als 10 000 Drohnen eine signifikant kürzere Flugdauer von 13,7 ± 6, 1 Minuten (Koeniger et al., 1989) gemessen hatten. Das war eine Bestätigung für unsere Annahme, dass die Anzahl der Drohnen die Dauer des Paarungsfluges der Königin beeinflusst und zugleich ein erster Hinweis darauf, dass Königinnen ihren Paarungserfolg während des Fluges „überwachen“.Bei 29 Königinnen ergab sich eine negative Korrelation zwischen der Flugdauer und der Anzahl der in der Spermatheka gefundenen Spermien (Pearson r = −0,38, P = 0,04). Diese negative Korrelation spricht für die Idee, dass die Königin während des Fluges kontinuierlich Information über den Paarungserfolg erhält und sofort zurück fliegt, wenn sie genügend Drohnen getroffen hat.Sind genügend Drohnen vorhanden so streut die Dauer der Hochzeitsflüge, wie wir am gleichen Ort in den Vorjahren feststellten, zwischen 10 und 30 Minuten. Auf dieser Grundlage (Apriori — Annahme!) wurden die Daten in 2 Gruppen unterteilt:Zum ersten in die Gruppe der Königinnen, die weniger als 30 Minuten ausgeflogen waren und zum zweiten in die Gruppe von Königinnen, die länger als 30 Minuten gebraucht hatten. Die Spermienzahlen zwischen den beiden Gruppen unterschieden sich erheblich. Die kürzer als 30 Minuten geflogenen Königinnen hatten im Durchschnitt 3,0 ± 0,77 Millionen Spermien in der Spermatheka während die länger als 30 Minuten geflogenen Königinn mit nur 1, 1 ± 1, 04 Millionen signifikant weniger Spermien hatten (Wilcoxon, P < 0,001). Bei längerem Flug der Königin könnte der Honigvorrat im Kropf zu Ende gegangen sein. Die betroffene Königin wäre dann gezwungen sofort den Heimflug anzutreten und könnte nicht weiterfliegen, bis sie eine ausreichende Zahl von Paarungen erzielt hätte.Unsere Ergebnisse sprechen dafür, dass die Königin während des Hochzeitsfluges den Paarungserfolg kontrolliert, der mit der Anzahl der Kopulationen (Schlüns et al., 2005) sowie auch mit der Anzahl der Spermien in der Spermatheka (Woyke, 1964) zusammenhängt und zurück fliegt sobald die Reizschwelle erreicht ist.

Crossfostered drones of Apis cerana (Fabricius, 1793) and Apis koschevnikovi (Buttel-Reepen, 1906) fly at their species specific mating times

SummaryReciprocal transfer of sealed drone brood between colonies ofApis cerana andApis koschevnikovi was successful and resulted in four colonies (two of each species) with a mixed drone population. Flights ofApis cerana drones occurred between 14.00 and 16.15 regardless whether they were in a conspecific or alien colony.Apis koschevnikovi drones also flew at their species specific time from 16.45 to 18.30. A variance estimation revealed that 99.4% of the total variance depended on the species of the drone. In contrast to theApis drones general biological dependence upon the colony, crossfostered drones ofApis cerana andApis koschevnikovi showed an unexpected autonomy in chosing their mating flight time.