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Um einen Beitrag zum besseren Verständnis der Rolle der Bienenwachse in der Kommunikation der Honigbienen leisten zu können, wurden Wabenwachse unterschiedlichen Alters und Kutikulawachse unterschiedlicher Kasten,Geschlechter und Berufsgruppen mit Hilfe von Gaschromatographie, Massenspektroskopie und FTIR-Spektroskopie untersucht. Die chemischen Analysen zeigten mittels Diskriminantenfunktionsanalysen hochsignifikante Unterschiede in den aliphatischen Kohlenwasserstoffen zwischen Wabenwachsen unterschiedlichen Alters und Kutikulawachsen unterschiedlicher Kasten und Geschlechter. Erstmals konnte für ein komplexes Substanzgemisch (Bienenwachs) eine lineare Abhängigkeit zwischen dem Schmelzverhalten und der chemischen Zusammensetzung der Wachse nachgewiesen werden.Mit Hilfe von Verhaltensversuchen wurde der Frage nachgegangen, ob die chemischen Unterschiede für die Bienen überhaupt relevant sind. Mit Hilfe der differentielle Konditionierung des Rüsselreflexes wurde getestet, inwieweit Bienen die verschiedenen Wachse unterscheiden können. Eine Diskriminierung der Wachse aufgrund der aliphatischen Kohlenwasserstoffe war den Honigbienen nicht möglich. Dies ergab einen neuen und interessanten Einblick in die Kommunikation der Honigbienen
The proventriculus regulates the food passage from crop to midgut. As the haemolymph provides a constantly updated indication of an insect’s nutritional state, it is assumed that the factor controlling the proventri-culus activity is to be found in the haemolymph. The purpose of this doctoral thesis was to investigate how output (metabolic rate), input (food quality and food quantity) and internal state variables (haemolymph osmolarity and haemolymph sugar titer) affect each other and which of these factors controls the activity of the proventriculus in the honeybee. Therefore free-flying foragers were trained to collect con-trolled amounts of different sugar solutions. Immediately after feeding, metabolic rates were measured over different periods of time, then crop-emptying rates and haemolymph sugar titers were measured for the same individual bees. Under all investigated conditions, both the sugar transport rates through the proventriculus and the haemolyph sugar titers depended mainly on the metabolism. For bees collecting controlled amounts of 15 per cent, 30 per cent or 50 per cent sucrose solution haemolymph trehalose, glucose and fructose titers were constant for metabolic rates from 0 to 4.5 mlCO2/h. At higher metabolic rates, trehalose concentration decreased while that of glucose and fructose increased with the exception of bees fed 15 per cent sucrose solution. As the supply of sugar from the crop via the proventriculus was sufficient to support even the highest metabolic rates, the observed pattern must result from an upper limit in the capacity of the fat body to synthesise trehalose. The maximal rate of conversion of glucose to trehalose in the fat body was therefore calculated to average 92.4 µg glucose/min. However, for bees fed 15 per cent sucrose solution both the rate of conversion of glucose to trehalose and the rate of sugar transport from the crop to the midgut were limited, causing an overall decrease in total haemolymph sugar titers for metabolic rates higher than 5 mlCO2/h. Haemolymph sucrose titers were generally low but increased with increasing metabolic rates, even though sucrose was not always detected in bees with high metabolic rates. Though foragers were able to adjust their sugar transport rates precisely to their metabolic rates, a fixed surplus of sugars was transported through the proventriculus under specific feed-ing conditions. This fixed amount of sugars increased with increasing concentration and in-creasing quantity of fed sugar solution, but decreased with progressing time after feeding. This fixed amount of sugars was independent of the metabolic rates of the bees and of the molarity and viscosity of the fed sugar solution. As long as the bees did not exhaust their crop content, the haemolymph sugar titers were unaffected by the sugar surplus, by the time after feeding, by the concentration and by the viscosity of fed sugar solution. When bees were fed pure glucose (or fructose) solutions, un-usually little fructose (or glucose) was found in the haemolymph, leading to lower total haemolymph sugar titers, while the trehalose titer remained unaffected. In order to investigate the mechanisms underlying the regulation of the honeybee proven-triculus, foraging bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable sugars (sorbose). Bees reacted to injections of metabolisable sugars with reduced crop-emptying rates, but injection of non-metabolisable sugars had no influence on crop emptying. Therefore it is concluded that the proventriculus regulation is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemo-lymph osmolarity. A period of 10min was enough to observe reduced crop emptying rates after injections. It is suggested that glucose and fructose have an effect on the proventriculus activity only via their transformation to trehalose. However, when the bees were already in-jected 5min after feeding, no response was detectable. In addition it was investigated whether the overregulation is the result of feed-forward regulation for the imminent take-off and flight. In a first experiment, we investigated whether the bees release an extra amount of sugar solution very shortly before leaving for the hive. In a second experiment, it was tested whether the distance covered by the bees might have an influence on the surplus amount released prior to the take-off. In a third experiment, it was investigated if walking bees fail to release this extra amount of sugars, as they do not have to fly. Though we were not able to demonstrate that the overregulation is the result of feed-forward regulation for the imminent take-off and flight, it is conceivable that this phenome-non is a fixed reaction in foragers that can not be modulated. To investigate whether regulated haemolymph sugar titers are also observed in honeybee foragers returning from natural food sources, their crop contents and haemolymph sugar titers were investigated. While the quantity of the collected nectar was without influence on the haemolymph sugar titers, foragers showed increasing haemolymph sugar titers of glucose, fructose and sucrose with increasing sugar concentration of the carried nectar. In contrast no relationship between crop nectar concentrations and haemolymph trehalose titers was observed. We are sure that the regulation of food passage from crop to midgut is controlled by the trehalose titer. However, under some conditions the balance between consumption and income is not numerically exact. This imprecision depends on the factors which have an impact on the foraging energetics of the bees but are independent of those without influence on the foraging energetics. Therefore we would assume that the proventriculus activity is modulated by the motivational state of the bees.
In this thesis, I examined honey bee nectar foraging with emphasis on the communication system. To document how a honey bee colony adjusts its daily nectar foraging effort, I observed a random sample of individually marked workers during the entire day, and then estimated the number and activity of all nectar foragers in the colony. The total number of active nectar foragers in a colony changed frequently between days. Foraging activity did not usually change between days. A honey bee colony adjusts its daily foraging effort by changing the number of its nectar foragers rather than their activity. I tested whether volatiles produced by a foraging colony activated nectar foragers of a non-foraging colony by connecting with a glass tube two colonies. Each colony had access to a different green house. In 50% of all experiments, volatile substances from the foraging colony stimulated nectar foragers of the non-foraging colony to fly to an empty feeder. The results of this study show that honey bees can produce a chemical signal or cue that activates nectar foragers. However, more experiments are needed to establish the significance of the activating volatiles for the foraging communication system. The brief piping signal of nectar foragers inhibits forager recruitment by stopping waggle dances (Nieh 1993, Kirchner 1993). However, I observed that many piping signals (approximately 43%) were produced off the dance floor, a restricted area in the hive where most waggle dances are performed. If the inhibition of waggle dances would be the only function of the brief piping signal, tremble dancers should produce piping signals mainly on the dance floor, where the probability to encounter waggle dancers is highest. To therefore investigate the piping signal in more detail, I experimentally established the foraging context of the brief piping signal, characterized its acoustic properties, and documented for the first time the unique behavior of piping nectar foragers by observing foragers throughout their entire stay in the hive. Piping nectar foragers usually began to tremble dance immediately upon their return into the hive, spent more time in the hive, more time dancing, had longer unloading latencies, and were the only foragers that sometimes unloaded their nectar directly into cells instead of giving it to a nectar receiver bee. Most of the brief piping signals (approximately 99%) were produced by tremble dancers, yet not all tremble dancers (approximately 48%) piped. This suggests that piping and tremble dancing have related, but not identical functions in the foraging system. Thus, the brief piping signals may not only inhibit forager recruitment, but have an additional function both on and off the dance floor. In particular, the piping signal might function 1. to stop the recruitment of additional nectar foragers, and 2. as a modulatory signal to alter the response threshold of signal receivers to the tremble dance. The observation that piping tremble dancers often did not experience long unloading delays before they started to dance gave rise to a question. A forager’s unloading delay provides reliable information about the relative work capacities of nectar foragers and nectar receivers, because each returning forager unloads her nectar to a nectar receiver before she takes off for the next foraging trip. Queuing delays for either foragers or receivers lower foraging efficiency and can be eliminated by recruiting workers to the group in shortage. Short unloading delays indicate to the nectar forager a shortage of foragers and stimulate waggle dancing which recruits nectar foragers. Long unloading delays indicate a shortage of nectar receivers and stimulate tremble dancing which recruits nectar receivers (Seeley 1992, Seeley et al. 1996). Because the short unloading delays of piping tremble dancers indicated that tremble dancing can be elicited by other factors than long unloading delays, I tested whether a hive-external stimulus, the density of foragers at the food source, stimulated tremble dancing directly. The experiments show that tremble dancing can be caused directly by a high density of foragers at the food source and suggest that tremble dancing can be elicited by a decrease of foraging efficiency either inside (e.g. shortage of receiver bees) or outside (e.g. difficulty of loading nectar) the hive. Tremble dancing as a reaction to hive-external stimuli seems to occur under natural conditions and can thus be expected to have some adaptive significance. The results imply that if the hive-external factors that elicit tremble dancing do not indicate a shortage of nectar receiver bees in the hive, the function of the tremble dance may not be restricted to the recruitment of additional nectar receivers, but might be the inhibition or re-organization of nectar foraging.
3. Zusammenfassung Ein noch immer unvollständig verstandenes Problem sind die exakten Mechanismen der Arbeitsteilung und Koordination innerhalb von Bienenvölkern Apis mellifera. Auf der einen Seite muss die sensorische und neuronale Ausstattung jedes Individuums das Potential zur Kommunikation und Aufgabenbewältigung enthalten, zum anderen müssen jedem Bienenvolk Mechanismen zur Steuerung zur Verfügung stehen, die auch so weit in die Zukunft reichenden Notwendigkeiten wie Wintervorbereitungen zuverlässig durchführen. Die vorliegende Arbeit beleuchtet daraus ausgewählte Aspekte. Zum einen werden Aspekte der kognitiven Fähigkeiten der Einzelbienen untersucht, die im Hinblick auf ihre Rolle als sammelnde Arbeiterinnen eine wichtige Rolle spielen. Das Erkennen und Verarbeiten von Mustern spielt eine wichtige Rolle beim Auffinden von potentiellen Nahrungsquellen. Hier konnte mittels des DMTS – Paradigma ein hoher Abstraktionsgrad der Musterverarbeitung sowie eine Speicherung auch komplexer Muster gezeigt werden. Zum anderen wird die Bruttemperatur als ein Einfluss auf die Puppenentwicklung und dessen mögliche Folgen auf kognitive Fähigkeiten und Lebenshistorie untersucht. Variation der Bruttemperatur wurde in verschiedenen Zusammenhängen als starker Einfluss auf unterschiedliche Aspekte der Entwicklung gezeigt. In der vorliegenden Arbeit kann diese Bruttemperatur als möglicher Faktor der nachfolgend unterschiedlichen Ausprägung von Verhaltensmustern gezeigt werden. Dabei wird ebenso auf die Unterschiede im Verhaltensmuster von täglichen Stocktätigkeiten wie auf die resultierenden Unterschiede in der Lebensgeschichte und –spanne eingegangen, die aus unterschiedlichen Brutaufzuchtstemperaturen resultieren können. Als Aufzuchtstemperaturen werden dabei 32°C, 35°C sowie 36°C verwendet, um eine Vari ation zwischen der an anderer Stelle berichteten mittleren, der niedrigsten und der höchsten Temperatur für morphologisch vollständig entwickelte Bienen zu erreichen und die daraus resultierenden Arbeiterinnen zu untersuchen. Sowohl die Ergebnisse der Verhaltensuntersuchungen von Stockbienen wie auch der Vergleich von Lebensaktivität und –spanne zeigen dabei signifikante Unterschiede zwischen den bei unterschiedlichen Temperaturen aufgezogenen Arbeiterinnen in deren analysiertem Verhalten.
Honigbienen (Apis mellifera carnica) regulieren die Temperatur ihrer Brut in einem sehr engen Temperaturfenster, da vor allem die gedeckelte Brut sehr temperaturempfindlich reagiert (Groh et al. 2004). Die Thermoregulation ist nicht – wie lange angenommen – Beiprodukt von alltäglichen Arbeiten der Bienen im Brutbereich, sondern eine aktive und Energie- und Zeitaufwändige eigene Tätigkeit. Arbeiterinnen ziehen sich mit ihren Beinen an die Brutoberfläche, drücken ihren warmen Thorax auf die Brutdeckel und verharren so für einige Minuten um mit der eigenen Körperwärme die Brut zu temperieren (Bujok et al. 2002). Wie erwartet korrelierte die Thoraxtemperatur einer Arbeiterin mit der Frequenz der abdominalen Atembewegungen, bei sehr hohen Thoraxtemperaturen (über 40°C) erreichten die Bienen Atemfrequenzen von über 8Hz. Eine weitere Methode die Brut effektiv zu wärmen übten Bienen aus, die leere Zellen im gedeckelten Brutbereich besuchen (Kleinhenz et al. 2003). Arbeiterinnen gingen dabei bevorzugt in Zellen, die von möglichst vielen gedeckelten Zellen umgeben waren. Sowohl die Dauer der Zellbesuche, als auch die mittlere Thoraxtemperatur bei Ein- und Austritt der Zelle korrelierten mit der Anzahl der benachbarten Brutzellen – je mehr Brutzellen eine leere Zelle in ihrer direkten Nachbarschaft hatte umso länger dauerte der Besuch einer Biene und umso höher ist die Ein- bzw. Austrittstemperatur der Biene. Mindestes 48 Stunden alte Bienen unterschieden sich signifikant in ihrem Wärmeverhalten von jüngeren Bienen. Tote gedeckelte Brut wurde in manchen Fällen über viele Tage (durchgehend bis 10 Tage) gewärmt, sie unterschied sich in ihrer Temperatur nicht von unbehandelter gedeckelter Brut. In weiteren Versuchen lag die Bruttemperatur von toter Brut zwar unter der eines Kontrollbereiches, die Temperatur lag aber weiterhin im optimalen Bereich von 33,5 bis 35°C (Groh et al. 2004). In diesen Versuchen wurde die tote Brut vor dem Einsetzen in den Beobachtungsstock wieder auf 35°C erwärmt. Wachskegel in gedeckelten Zellen wurden erkannt und ausgeräumt. Aktive Signale, die von der Brut ausgehen scheinen also nicht notwendig für die effektive Bruttemperaturregulierung zu sein. Untersuchungen mittels Laser-Doppler-Vibrometrie zeigten auch keine Hinweise auf eine mechanische Kommunikation zwischen den Puppen und den Arbeiterinnen. Das Brutwärmen scheint eine Aktion zu sein, die von den Bienen nur in Gemeinschaft sinnvoll durchgeführt werden kann. In einigen Fällen kam es während der Puppenphase zu unerklärlichen Abfällen in der Bruttemperatur, die nur durch einen positiven Rückkopplungseffekt seitens der Arbeiterinnen erklärt werden kann. Beim Brutwärmen spielen die Antennen der Arbeiterinnen wahrscheinlich eine wichtige Rolle. Während sich die Bienen beim aktiven Brutwärmen den Brutdeckel annähern sind die Antennenspitzen immer auf die Brutdeckel gerichtet. Fehlen den Arbeiterinnen die Antennen, dann ist die Thermoregulation eingeschränkt oder unzureichend. Die Bruttemperatur korreliert mit der Anzahl der abgetrennten Antennensegmente, je mehr Antennensegmente fehlen, desto weniger gut wird die Temperatur im Brutbereich hoch und konstant gehalten. Zusätzlich scheint es eine Lateralität in der Antennenfunktion zu geben, wurde die rechte Antenne gekürzt wärmten die Bienen die Brut signifikant schlechter, als beim Kürzen der linken Antenne. Durch das Kürzen der Antennen änderte sich auch das Verhalten der Tiere: Kontrollbienen verharrten ruhig im Brutbereich, während Bienen mit gekürzten Antennen teilweise ähnlich warm waren, aber nicht mehr das oben beschriebene aktive Brutwärmeverhalten zeigten.
Olfaction plays an important role in a variety of behaviors throughout the life of the European honeybee. Caste specific, environmentally induced and aging/experiencedependent differences in olfactory behavior represent a promising model to investigate mechanisms and consequences of phenotypic neuronal plasticity within the olfactory pathway of bees. This study focuses on the two different female phenotypes within the honeybee society, queens and workers. In this study, for the first time, structural plasticity in the honeybee brain was investigated at the synaptic level. Queens develop from fertilized eggs that are genetically not different from those that develop into workers. Adult queens are larger than workers, live much longer, and display different behaviors. Developmental trajectory is mainly determined by nutritional factors during the larval period. Within the subsequent post-capping period, brood incubation is precisely controlled, and pupae are incubated close to 35°C via thermoregulatory activity of adult workers. Behavioral studies suggest that lower rearing temperatures cause deficits in olfactory learning in adult bees. To unravel possible neuronal correlates for thermoregulatory and caste dependent influences on olfactory behavior, I examined structural plasticity of developing as well as mature olfactory synaptic neuropils. Brood cells were reared in incubators and pupal as well as adult brains were dissected for immunofluorescent staining. To label synaptic neuropils, I used an antibody to synapsin and fluophore-conjugated phalloidin which binds to filamentous (F-) actin. During development, neuronal F-actin is expressed in growing neurons, and in the mature nervous system, F-actin is most abundant in presynaptic terminals and dendritic spines. In the adult brains, this double labeling technique enables the quantification of distinct synaptic complexes microglomeruli [MG]) within olfactory and visual input regions of the mushroom bodies (MBs) prominent higher sensory integration centers. Analyses during larval-adult metamorphosis revealed that the ontogenetic plasticity in the female castes is reflected in the development of the brain. Distinct differences among the timing of the formation of primary and secondary olfactory neuropils were also revealed. These differences at different levels of the olfactory pathway in queens and workers correlate with differences in tasks performed by both female castes. In addition to caste specific differences, thermoregulation of sealed brood cells has important consequences on the synaptic organization within the MB calyces of adult workers and queens. Even small differences in rearing temperatures affected the number of MG in the olfactory calyx lip regions. In queens, the highest number of MG in the olfactory lip developed at 1°C below the temperature where the maximum of MG is found in workers (33.5 vs. 34.5°C). Apart from this developmental neuronal plasticity, this study exhibits a striking age-related plasticity of MG throughout the extended life span of queens. Interestingly, MG numbers in the olfactory lip increased with age, but decreased within the adjacent visual collar of the MB calyx. To conclude, developmental and adult plasticity of the synaptic circuitry in the sensory input regions of the MB calyx may underlie caste- and age-specific adaptations and long-term plasticity in behavior.
With the progress in sequencing of the honey bee genome new data become available which allows the search and identification of genes coding for homologous proteins found in other organism. Two genes coding for c-type lysozymes were identified in the genome of A. mellifera through an online-based BLAST search. Expression of both intron-less genes seems not to be under the regulatory control of either of the two pathways involved in humoral insect immunity, i.e. Toll and Imd, since no NF-κB transcription factor binding sites are found upstream of the genes. The encoded Lys-1 and Lys-2 are 157 and 143 amino acid long, respectively, and share a sequence similarity of 90%. Further in silico analysis revealed a signal peptidase cleavage site at the N-terminus of each amino acid sequence, strongly suggesting a secretion of the enzymes into the surrounding environment of the producing cells. Sequence alignments of both amino acid sequences with other c-type lysozymes identified the highly conserved active site glutamic acid (Glu32) as well as eight highly conserved cysteine residues. However, an important aspartic acid (Asp50) in the active site that helps to stabilize a substrate intermediate during catalysis is replaced by a serine residue in the lysozymes of A. mellifera. The replacement of the active site aspartic acid in the honey bee lysozymes suggests a different catalytic mechanism and/or a different substrate-specificity in respect to other c-type lysozymes. Furthermore, 3D-models of Lys-1 and Lys-2 were generated based on the sequence similarity of A. mellifera lysozymes with other c-type lysozymes. The published 3D structure of the lysozyme from the silkmoth Bombyx mori, which shares the highest sequence similarity of all available structures with A. mellifera lysozymes, was used as template for the construction of the 3D-models. The models of Lys-1 and Lys-2 suggest that both enzymes resemble, in large part, the structure of B. mori lysozyme. In order to identify the set of AMPs in the hemolymph of A. mellifera, hemolymph of immunized bees was analyzed. Applying SDS-polyacrylamide gel electrophoresis and mass spectrometry on hemolymph from immunized bees, three out of the four peptides were identified, i.e. abaecin, defensin 1 and hymenoptaecin. Furthermore, Lys-2 was identified in the hemolymph by mass spectrometry, conclusively demonstrating the presence of a lysozyme in the hemolymph of A. mellifera for the first time. However, the protein levels of Lys-2 were not affected by bacterial injection, suggesting that the gene expression of the putative antibacterial protein is not under the regulatory control of the Imd and/or Toll pathway. Besides the abovementioned antimicrobial peptides, the 76 kDa large transferrin was also identified. Transferrin is an iron-binding protein that has been implicated in innate immunity in the honey bee. Furthermore, the effect of pathogenic dose, the timeline of peptide induction and the age-related accumulation of the aforementioned AMPs were studied. The intensity of expression of the antimicrobial peptides, abaecin, defensin 1, and hymenoptaecin as well as transferrin increased proportionally with the amount of bacteria injected into the hemocoel. No such effect was observed for the protein levels of Lys-2. Furthermore, up-regulation of the three antibacterial peptides and transferrin was observed within the first 24 h following infection with E. coli (gram-). Infection with the gram+ bacterium Micrococcus flavus resulted in high and moderate protein levels for transferrin and abaecin, respectively, whereas hardly any accumulation of hymenoptaecin was observed, indicating that the gene expression of abaecin and transferrin is somehow positively correlated, and would suggest a shared regulatory pathway that differs from that of hymenoptaecin. Although bacterial infections didn’t seem to stimulate the production of Lys-2, different concentrations in the hemolymph were observed in bees of different ages, suggesting a correlation between the expression of Lys-2 and the age-related division of labor of adult worker honey bees, also known as age polyethism. The results further allow a proposed causal connection between the age-dependent accumulation of Lys-2 and the hemolymph titer of the gonotrophic hormone juvenile hormone, which is the “behavioral pacemaker” in adult honey bees.
In dieser Arbeit untersuche ich das Verhalten von Arbeiterbienen beim Brutwärmen, die Wärmeübertragung von den Bienen auf die gedeckelte Brut, die thermophysikalischen Eigenschaften des Brutnests und spezielle Aspekte des Brutnestaufbaus, die für dieses Thema relevant sind und bisher nicht untersucht wurden. Meine Arbeit umfasst Verhaltensbeobachtungen und thermografische Messungen an individuellen Bienen, die Simulation des Heizverhaltens von Arbeiterinnen und das Messen der Temperaturänderungen in der Wabe, die Messung der thermophysikalischen Eigenschaften der Brutwabe und der Zellwände (Wärmeleitfähigkeit und Durchlässigkeit für Wärmestrahlung), die Auswertung von Brutzelltemperaturen als Ergebnis des Verhaltens von Arbeiterbienen, die Analyse der Anzahl und der räumlichen Verteilung von Brutlücken (Auswertung in 2-D und 3-D bezüglich beider Wabenseiten) und die Entwicklung spezifischer Computersoftware, die zur Erarbeitung dieser Ergebnisse unverzichtbar ist. Ein wichtiges Ergebnis dieser Arbeit ist die Entdeckung und Beschreibung eines bemerkenswerten, bislang unbekannten Verhaltens der Honigbiene: Die Aufrechterhaltung hoher Thoraxtemperaturen (TTh) bei Langzeitbesuchen in offenen Zellen („Lücken“) die verstreut in der gedeckelten Brutfläche vorkommen. Hier zeige ich, dass die Aufrechterhaltung der hohen TTh nicht auf den Zellinhalt (z. B. offene Brut) bezogen ist - in den meisten Fällen waren die besuchten Zellen ohnehin leer - sondern auf die direkt benachbarte gedeckelte Brut, mit der diese Zellen über gemeinsame Zellwände in Kontakt stehen. Dieses Verhalten liefert eine Erklärung für Langzeitzellbesuche von sehr langer Dauer ohne erkennbare Aktivität, die in früheren Arbeiten beschrieben aber nicht völlig verstanden wurden, und es rehabilitiert die scheinbar „faulen“ Bienen im Zellinnern. Diesem Verhalten kommt eine große Bedeutung für das Brutwärmen zu, da sich der aufgeheizte Thorax tief in der Wabe (fast an der Mittelwand) befindet wo der Wärmeverlust an die Luft minimiert ist und von wo bis zu 6 umliegende Puppenzellen gleichzeitig gewärmt werden können. Im Vergleich zum Brutwärmeverhalten an der Wabenoberfläche (Andrücken des Thorax an die Brutdeckel), wo nur 1 oder Teile von 3 Brutdeckeln mit dem Thorax in Berührung stehen, ist das Wärmen im Zellinnern mit derselben TTh bis zu 2,6-fach effizienter. Die Messung der thermophysikalischen Eigenschaften der Brutwabe und die Simulation des Brutwärmeverhaltens unter kontrollierten Bedingungen zeigen, dass sich die Wabe langsam aufwärmt und eher ein lokal begrenztes Wärmen als eine rasche Wärmeausbreitung über eine große Fläche begünstigt. Der Einflussbereich eines einzelnen Zellbesuchers hängt von seiner TTh und der Dauer des Zellbesuchs ab. Anstiege der Bruttemperatur in bis zu 3 Zellen Abstand zum Zellbesucher sind nachweisbar. Das hier beschriebene Brutwärmeverhalten im Innern von Lücken (offenen Zellen) bietet nicht nur neue Einsichten in das Bienenverhalten. Es ermöglicht auch eine Neubewertung der Lücken und ihrer Nützlichkeit für die Bienen. Eine von mir entwickelte Computersoftware („CombUse 2.0“) ermöglicht es, das Vorkommen und die räumliche Verteilung von Lücken mit hoher Genauigkeit auf der Ebene einzelner Zellen zu erfassen und auszuwerten. Die räumliche Verteilung der Lücken in der gedeckelten Brutfläche zeigt, dass schon bei geringen Lückenhäufigkeiten von ca. 4 bis 10 %, die in gesunden Kolonien normal sind, eine überraschend große Zahl gedeckelter Brutzellen (88 % bis 99 %, wenn die dreidimensionale Verteilung berücksichtigt wird) im Einflussbereich von Brut wärmenden Zellbesuchern sind. Obwohl das Brutwärmeverhalten im Zellinnern schwer zu entdecken und zu beobachten ist, führen die in dieser Arbeit präsentierten Daten zu dem Schluss, dass es sich dabei um einen wichtigen Bestandteil der Nestklimatisierung bei Honigbienen handelt.
Like many other social insect societies, honeybees collectively share the resources they gather by feeding each other. These feeding contacts, known as trophallaxis, are regarded as the fundamental basis for social behavior in honeybees and other social insects for assuring the survival of the individual and the welfare of the group. In honeybees, where most of the trophallactic contacts are formed in the total darkness of the hive, the antennae play a decisive role in initiation and maintenance of the feeding contact, because they are sensitive to gustatory stimuli. The sequences of behaviors performed by the receiver bees at the beginning of a feeding contact includes the contact of one antenna with the mouthparts of a donor bee where the regurgitated food is located. The antennal motor action is characterized by behavioral asymmetry, which is novel among communicative motor actions in invertebrates. This preference of right over left antenna is without exception even after removal of the antennal flagellum. This case of laterality in basic social interaction might have its reason in the gustatory asymmetry in the antennae, because the right antenna turns out to be significantly more sensitive to stimulation with sugar water of various concentrations than the left one. Trophallactic contacts which guarantee a constant access to food for every individual in the hive are vitally important to the honeybee society, because honeybees are heterothermic insects which actively regulate their thoracic temperature. Even though the individual can regulate its body temperature, its heating performance is strictly limited by the amount of sugar ingested. The reason for this is that honeybees use mostly the glucose in their hemolymph as the energy substrate for muscular activity, and the heat producing flight muscles are among the metabolically most active tissues known. The fuel for their activity is honey; processed nectar with a sugar content of ~80% stored in the honeycomb. The results show that the sugar content of the ingested food correlates positively with the thoracic temperature of the honeybees even if they are caged and show no actual heating-related behavior as in brood warming or heating in the centre of the winter cluster. Honeybees actively regulate their brood temperature by heating to keep the temperature between 33 °C to 36 °C if ambient temperatures are lower. Heating rapidly depletes the worker’s internal energy; therefore the heating performance is limited by the honey that is ingested before the heating process. This study focused on the behavior and the thoracic temperature of the participants in trophallactic food exchanges on the brood comb. The brood area is the centre of heating activity in the hive, and therefore the region of highest energy demand. The results show that the recipients in a trophallactic food exchange have a higher thoracic temperature during feeding contacts than donors, and after the feeding contact the former engage in brood heating more often. The donor bees have lower thoracic temperature and shuttle constantly between honey stores and the brood comb, where they transfer the stored honey to heating bees. In addition, the results show a heat-triggered mechanism that enables donor and recipient to accomplish trophallactic contacts without delay in the total darkness of the hive in the brood area as the most energy consuming part of the hive. Providing heat-emitting workers with small doses of high performance fuel contributes to an economic distribution of resources consistent with the physiological conditions of the bees and the ecological requirements of the hive, resulting in a highly economical resource management system which might be one of the factors favouring the evolution of perennial bee colonies in temperate regions. The conclusion of these findings suggests a resource management strategy that has evolved from submissive placation behavior as it is seen in honeybees, bumblebees and other hymenopterans. The heat-triggered feedback mechanism behind the resource management of the honeybee´s thermoregulatory behavior reveals a new aspect of the division of labor and a new aspect of communication, and sheds new light on sociality in honeybees.
This study was conducted to determine the influence of different stress factors on the honeybee Apis mellifera. The investigation was motivated by previous experiments that suggested the existence of an unspecific defense mechanism causing a generalized change of flight behavior after the onset of different diseases. This mechanism is thought to impede the ability of flight bees to return to their respective colonies thereby removing the disease from the colony over time. During the last years, the existence of such a “suicidal behavior” was supported by further studies. Thus, an unnoticed, potentially highly effective defense mechanism of social insects was revealed whose spectrum of activity and physiological basics require further investigation. Suggesting that the reaction by the bees is unspecific to different diseases as well as to other potential stress factors, this study was designed to investigate the influence of pathogens, insecticides, and different brood rearing temperatures on different parameters like lifespan, foraging activity, and foraging trip duration of worker bees.
Honeybee foragers frequently fly several kilometres to and from vital resources, and communicate those locations to their nest mates by a symbolic dance language. Research has shown that they achieve this feat by memorizing landmarks and the skyline panorama, using the sun and polarized skylight as compasses and by integrating their outbound flight paths. In order to investigate the capacity of the honeybees’ homing abilities, we artificially displaced foragers to novel release spots at various distances up to 13 km in the four cardinal directions. Returning bees were individually registered by a radio frequency identification (RFID) system at the hive entrance. We found that homing rate, homing speed and the maximum homing distance depend on the release direction. Bees released in the east were more likely to find their way back home, and returned faster than bees released in any other direction, due to the familiarity of global landmarks seen from the hive. Our findings suggest that such large scale homing is facilitated by global landmarks acting as beacons, and possibly the entire skyline panorama.
Bees are subject to permanent threat from predators such as ants. Their nests with large quantities of brood, pollen and honey represent lucrative targets for attacks whereas foragers have to face rivalry at food sources. This thesis focused on the role of stingless bees as third party interactor on ant-aphid-associations as well as on the predatory potential represented by ants and defense mechanisms against this threat. Regular observations of an aphid infested Podocarpus for approaching stingless bees yielded no results. Another aim of this thesis was the observation of foraging habits of four native and one introduced ant species for assessment of their predatory potential to stingless bees. All species turned out to be dietary balanced generalists with one mostly carnivorous species and four species predominantly collecting nectar roughly according to optimal foraging theory. Two of the species monitored, Rhytidoponera metallica and Iridomyrmex rufoniger were considered potential nest robbers. As the name implies, stingless bees lack the powerful weapon of their distant relatives; hence they specialized on other defense strategies. Resin is an important, multipurpose resource for stingless bees that is used as material for nest construction, antibiotic and for defensive means. For the latter purpose highly viscous resin is either directly used to stick down aggressors or its terpenic compounds are included in the bees cuticular surface. In a feeding choice experiment, three ant species were confronted with the choice between two native bee species - Tetragonula carbonaria and Austroplebeia australis - with different cuticular profiles and resin collection habits. Two of the ant species, especially the introduced Tetramorium bicarinatum did not show any preferences. The carnivorous R. metallica predominantly took the less resinous A. australis as prey. The reluctance towards T. carbonaria disappeared when the resinous compounds on its cuticle had been washed off with hexane. To test whether the repulsive reactions were related to the stickiness of the resinous surface or to chemical substances, hexane extracts of bees’ cuticles, propolis and three natural tree resins were prepared. In the following assay responses of ants towards extract treated surfaces were observed. Except for one of the resin extracts, all tested substances had repellent effects to the ants. Efficacy varied with the type of extract and species. Especially to the introduced T. bicarinatum the cuticular extract had no effect. GCMS-analyses showed that some of the resinous compounds were also found in the cuticular profile of T. carbonaria which featured reasonable analogies to the resin of Corymbia torelliana that is highly attractive for stingless bees. The results showed that repellent effects were only partially related to the sticky quality of resin but were rather caused by chemical substances, presumably sesqui- and diterpenes. Despite its efficacy this defense strategy only provides short time repellent effects sufficient for escape and warning of nest mates to initiate further preventive measures.
Das angeborene Immunsystem von Insekten besteht aus einer humoralen Komponente, einer zellulären Komponente und dem Prophenoloxidase-aktivierenden System. Fast alle Erkenntnisse über das angeborene Immunsystem stammen von Arbeiten mit Modellorganismen wie z.B. Drosophila oder Anopheles gambiae. Wie genau das Immunsystem der Honigbiene (Apis mellifera) funktioniert, ist jedoch noch relativ unbekannt. In der vorliegenden Arbeit wurden die unterschiedlichen Immunreaktionen aller drei Entwicklungsstadien der Honigbiene nach artifizieller Infektion mit Gram-negativen und Gram-positiven Bakterien (Escherichia coli und Micrococcus flavus) und dem Akuten Bienen Paralyse Virus (ABPV) untersucht und verglichen. Eine E. coli-Injektion zeigt bei Larven und adulten Arbeiterinnen nur wenig Auswirkung auf das äußere Erscheinungsbild und die Überlebensrate. In beiden Entwicklungsstadien wird die humorale Immunantwort stark induziert, erkennbar an der Expression der antimikrobiellen Peptide (AMPs) Hymenoptaecin, Defensin1 und Abaecin. Zusätzlich werden allein in Jungbienen nach bakterieller Infektion vier weitere immunspezifische Proteine exprimiert. Unter anderem eine Carboxylesterase (CE1) und das Immune-Responsive Protein 30 (IRp30). Die Expression von CE1 und IRp30 zeigt dabei den gleichen zeitlichen Verlauf wie die der AMPs. In Jungbienen kommt es zudem nach E. coli-Injektion zu einer raschen Abnahme an lebenden Bakterien in der Hämolymphe, was auf eine Aktivierung der zellulären Immunantwort schließen lässt. Ältere Bienen und Winterbienen zeigen eine stärkere Immunkompetenz als Jungbienen. Selbst nicht-infizierte Winterbienen exprimieren geringe Mengen der immunspezifischen Proteine IRp30 und CE1. Die Expression von IRp30 kann dabei durch Verwundung oder Injektion von E. coli noch gesteigert werden. Eine weitere Besonderheit ist die im Vergleich zu Jungbienen raschere Abnahme an lebenden Bakterien in der Hämolymphe bis hin zur vollständigen Eliminierung. Die Reaktion von Puppen auf eine bakterielle Infektion war völlig unerwartet. Nach Injektion von E. coli-Zellen kommt es innerhalb von 24 h p.i. zu einem tödlichen Kollaps, der sich in einer Graufärbung des gesamten Puppenkörpers äußert. Da keine Expression von AMPs nachzuweisen war, wird die humorale Immunantwort offensichtlich nicht induziert. Auch die zelluläre Immunantwort scheint nicht aktiviert zu werden, denn es konnte keine Abnahme an lebenden E. coli-Zellen beobachtet werden. Aufgrund dieser fehlenden Immunreaktionen vermehrt sich E. coli im Hämocoel infizierter Puppen und scheint damit deren Tod herbeizuführen. Nach viraler Infektion wurden in allen drei Entwicklungsstadien der Honigbiene gänzlich andere Reaktionen beobachtet als nach bakterieller Infektion. Bei dem verwendeten Akuten Bienen Paralyse Virus (ABPV) handelt es sich um ein Picorna-ähnliches Virus, dessen Vermehrung in der Hämolymphe über die massive Synthese der Capsidproteine verfolgt werden kann. Eine Injektion von sehr wenigen ABPV-Partikeln ins Hämocoel hat dramatische Auswirkungen auf Larven. Nach Virusinjektion kommt es innerhalb weniger Stunden zu einer raschen Virusvermehrung und schon 24 h p.i. zum Tod, häufig begleitet von einer Schwarzfärbung der gesamten Larve. Kurz vor dem Ableben kommt es neben dem Abbau hochmolekularer Speicherproteine zur Expression zahlreicher Proteine, die u.a. an der Translation oder dem Schutz vor oxidativem Stress beteiligt sind. Auf Jungbienen hat eine ABPV-Infektion keine so dramatischen Auswirkungen wie auf Larven. Sie zeigen lediglich Zeichen von Paralyse, zudem überleben sie länger bei höheren injizierten Partikelzahlen, die Virusvermehrung ist langsamer und es kommt zu keiner starken Veränderung des Hämolymph-Proteinmusters. Es konnte gezeigt werden, dass es in ABPV-infizierten Larven oder adulten Bienen zu keiner erkennbaren Aktivierung des humoralen Immunsystems in Form von exprimierten AMPs kommt. Zudem scheint die humorale Immunantwort auch nicht unterdrückt zu werden, denn nach gleichzeitiger Injektion von E. coli und ABPV kommt es neben der Expression viraler Capsidproteine auch zur Expression von AMPs. Zusätzlich konnte in Jungbienen nach Infektion mit ABPV eine zelluläre Immunantwort in Form von Nodulation ausgeschlossen werden. Ältere Bienen scheinen nicht nur mit bakteriellen Infektionen, sondern auch mit einer ABPV-Infektion besser zurechtzukommen. Bei einer Menge an ABPV-Partikeln, die in Jungbienen spätestens 72 h p.i. zum Tod führt, ist in Winterbienen eine Virusvermehrung erst ab 96 h p.i. erkennbar und diese beeinträchtigt die Überlebensrate kaum. Puppen sind einer Virusinfektion genauso schutzlos ausgeliefert wie einer Bakterieninfektion. Es kommt zwar zu keiner starken Änderung des äußeren Erscheinungsbildes, jedoch bleiben Puppen in ihrer Entwicklung komplett stehen. Das Virus muss sich daher stark vermehren, allerdings nicht überwiegend - wie bei Larven und adulten Bienen - in der Hämolymphe.
Honeybees (Apis mellifera) forage on a great variety of plant species, navigate over large distances to crucial resources, and return to communicate the locations of food sources and potential new nest sites to nest mates using a symbolic dance language. In order to achieve this, honeybees have evolved a rich repertoire of adaptive behaviours, some of which were earlier believed to be restricted to vertebrates. In this thesis, I explore the mechanisms involved in honeybee learning, memory, numerical competence and navigation. The findings acquired in this thesis show that honeybees are not the simple reflex automats they were once believed to be. The level of sophistication I found in the bees’ memory, their learning ability, their time sense, their numerical competence and their navigational abilities are surprisingly similar to the results obtained in comparable experiments with vertebrates. Thus, we should reconsider the notion that a bigger brain automatically indicates higher intelligence.
Non-target effects of a multiple insect resistant Bt-maize on the honey bee (Apis mellifera L.)
(2011)
Honey bee pollination is an ecologically and economically important ecosystem service. New methodological developments are needed to research the underlying factors of globally observed bee losses. The honey bee (Apis mellifera) is a key non-target arthropod species for environmental risk assessment of genetically modified (GM) crops. For GM-crop risk assessments, mainly methods for monitoring adult honey bees under laboratory conditions are documented. However, protocols with robust methods for standardized colonies or in vitro reared honey bee larvae are currently lacking. Within the research, presented in this this dissertation, multiple methodological developments are achieved; a mortality trap (Chapter II), a ‘full life cycle test’ (III), a novel in vitro rearing methodology (IV), a standardized in vitro test for Bt-pollen (V), a mixed toxicity test for purified transgenic proteins (VI), and a bacterial flora test with pollen digestion rate monitoring (VII). Overall, the studies did not indicate a detrimental effect caused by Bt-maize pollen, or by purified Bt-proteins at worst case exposure levels. Considering the risk for honey bees and larvae, we conclude that the tested Bt-maize Mon89034xMon88017 is not likely to cause harm to honey bee colonies. The study methods presented are highly recommended for future environmental risk assessment studies testing GM-crop biosafety on honey bees.
Soziale Insekten wie die Honigbiene (Apis mellifera) besitzen ein breites Spektrum an Abwehrmechanismen gegen Pathogenbefall, sowohl auf der Ebene der Kolonie (soziale Immunität) als auch auf der Stufe des Individuums (angeborenes Immunsystem). Die Hauptaufgabe der relativ kurzlebigen Drohnen besteht in der Begattung von Jungköniginnen. Daher stellte sich die Frage, ob auch die Drohnen ähnlich den Arbeiterinnen mit energieaufwendigen Immunreaktionen auf Infektionen reagieren. Wie im Folgenden beschrieben, konnte ich nachweisen, dass Drohnen eine ausgeprägte Immunkompetenz besitzen. Das angeborene Immunsystem setzt sich aus humoralen und zellulären Abwehrreaktionen zusammen. Bei der humoralen Immunantwort werden bestimmte evolutionär konservierte Signalkaskaden aktiviert, an deren Ende die Expression einer Vielzahl von antimikrobiellen Peptiden (AMPs) und immunspezifischen Proteinen (IRPs) steht. Zur Analyse der humoralen Immunantwort wurden von mir zum einen Hemmhoftests durchgeführt, um die gesamte antimikrobielle Aktivität der Haemolymphe nach artifizieller Infektion zu ermitteln und zum anderen spezifische AMPs bzw. IRPs identifiziert. Hierzu wurden die Haemolymphproteine in ein- oder zwei-dimensionalen Polyacrylamidgelen aufgetrennt und ausgewählte Proteinbanden bzw. -spots mittels nano HPLC/Massenspektrometrie analysiert. Die Hauptkomponenten des zellulären Immunsystems sind Wundheilung, Phagozytose, Einkapselung und Nodulation. In meiner Arbeit habe ich zum ersten Mal Noduli bei infizierten Drohnen nachweisen können. Frisch geschlüpfte adulte Drohnen (1d) weisen ein breites Spektrum an Immunreaktionen auf, das sowohl humorale als auch zelluläre Immunantworten umfasst. Nach Infektion mit dem Gram-negativen Bakterium E.coli und verschiedenen bakteriellen Zellwandbestandteilen wie Lipopolysaccharid (LPS), Peptidoglycan (PGN) und 1,3ß-Glucan (Bestandteil von Pilzzellwänden), werden die AMPs Hymenoptaecin, Defensin 1 und Abaecin induziert. Desweiteren exprimieren junge adulte Drohnen eine Reihe hochmolekularer immunspezifischer Proteine (IRPs) wie z.B. Carboxylesterase (CE 1), eine Serinprotease, die möglicherweise an der Prozessierung der Prophenoloxidase beteiligt ist, ein Peptidoglycan-interagierendes Protein (PGRP-S2) und zwei Proteine unbekannter Funktion, IRp42 und IRp30. Parallel zu bekannten bienenspezifischen AMPs wurde ein animales Peptidtoxin (APT) in Drohnenlarven, adulten Drohnen und adulten Hummeln nach E.coli Infektion in der Haemolymphe nachgewiesen. Von dem als OCLP 1 (ω-conotoxin-like protein 1) benannten Peptid war bereits bekannt, dass es in Fischen paralytische und damit toxische Effekte auslöst. Meine Beobachtungen lassen vermuten, dass es sich bei OCLP 1 um ein Peptidtoxin mit antimikrobiellen Eigenschaften und damit um eine neue Klasse von AMPs handelt. Die allgemeine humorale Immunkompetenz scheint während der gesamten Lebensspanne adulter Drohnen (~ 7 Wochen) konstant zu bleiben, wie durch die gleichbleibende antimikrobielle Aktivität im Hemmhoftest gezeigt wurde. Junge Drohnen reagieren auf eine E.coli Infektion mit der Bildung zahlreicher Noduli (~1000 Noduli/Drohn), die vor allem entlang des Herzschlauches zu finden sind. Diese zelluläre Immunantwort nimmt mit dem Alter der Drohnen ab, so dass bei 18 d alten Drohnen nur noch rund 10 Noduli/Drohn gefunden werden. Auf der anderen Seite nimmt die phagozytotische Aktivität bei älteren Drohnen scheinbar zu. In einer Reihe von parallel laufenden Versuchsreihen konnte ich eindrucksvoll zeigen, dass zelluläre Immunreaktionen wie Phagozytose und Nodulation unmittelbar nach bakterieller Infektion einsetzen. Hierbei erreicht die Nodulibildung 8-10 h p.i. eine Plateauphase, wohingegen die humorale Immunantwort erst 6 h p.i. schwach einsetzt, danach stetig zunimmt und noch 72 h p.i. nachweisbar ist. Es ist mir gelungen, eine Methode zur künstlichen Aufzucht von Drohnenlarven zu etablieren. Diese ermöglichte konstante und sterile Versuchsbedingungen zur Untersuchung der Immunreaktionen von Larven. Nach Infektion mit E.coli reagieren Drohnenlarven mit einer starken Aktivierung ihrer humoralen Immunantwort durch die Expression von AMPs, jedoch werden keine hochmolekularen IRPs wie in adulten Drohnen hochreguliert. Zudem ist die Nodulibildung in Larven nur schwach ausgeprägt. Völlig unerwartete Beobachtungen wurden beim Studium der Immunkompetenz von Drohnenpuppen gemacht. Nach Injektion lebender E.coli Zellen in Drohnenpuppen stellte ich eine dramatische Veränderung im Aussehen der Puppen fest. Die Puppen verfärbten sich gräulich schwarz. Genauere Untersuchungen haben dann gezeigt, dass die Drohnenpuppen, wie auch die der Arbeiterinnen, offensichtlich keine zelluläre Abwehrreaktion aktivieren können und die humorale Immunantwort nur sehr schwach ausfällt und viel zu spät einsetzt.
In their natural environment animals face complex and highly dynamic olfactory input. This requires fast and reliable processing of olfactory information, in vertebrates as well as invertebrates. Parallel processing has been shown to improve processing speed and power in other sensory systems like auditory or visual. In the olfactory system less is known about olfactory coding in general and parallel processing in particular. With its elaborated olfactory system and due to their specialized neuroanatomy, honeybees are well-suited model organism to study parallel olfactory processing. The honeybee possesses a unique neuronal architecture - a dual olfactory pathway. Two mirror-imaged output projection neuron (PN) pathways connect the first olfactory processing stage, the antennal lobe (analog to the vertebrates olfactory bulb, OB), with the second, the mushroom body (MB) known to be involved in orientation and learning and memory, and the lateral horn (LH). The medial antennal lobe-protocerebral tract (m-APT) first innervates the MB and thereafter the LH, while the other, the lateral-APT (l-APT) projects in opposite direction. The neuroanatomy and evolution of these pathways has been analyzed, yet little is known about its physiology. To analyze the function of the dual olfactory pathway a new established recording method was designed and is described in the first chapter of this thesis (multi-unit-recordings). This is now the first time where odor response from several PNs of both tracts is recorded simultaneously and with high temporal precision. In the second chapter the PN odor responses are analyzed. The major findings are: both tracts responded to all tested odors but with differing characteristics. Since recent studies describe the input to the two tracts being rather similar, the results now indicate differential odor processing along the tracts, therefore this is a good indicator for parallel processing. PNs of the m-APT process odors in a sparse manner with delayed response latencies, but with high odor-specificity. PNs of the l-APT in contrast respond to several odor stimuli and respond in general faster. In some PN originating from both tracts, characteristics of odor-identity coding via response latencies were found. Analyzing the over-all dynamic range of the PNs both l- and m-APT PNs were tested over a large odor concentration range (10-6 to 10-2) (3. chapter). The PNs responded with linear and non-linear correlation of the response strength to the odor concentration. In most cases the l-APT is comparatively more sensitive to low odor concentrations. Response latency decreases with increasing odor concentration in both tracts. Alternative coding principles and elaboration on the hypothesis whether the dual olfactory pathway may contribute coincidental innervation to the next higher-order neurons, the Kenyon cells (KC), is subject of the 4. chapter. Cross-correlations and synchronous responses of both tracts show that in principle odors may be coded via temporal coding. Results suggest that odor processing is enhanced if both tracts contribute to olfactory coding together. In another project the distribution of the inhibitory neurotransmitter GABA (gamma-aminobutyric acid) was measured in the bee’s MB during adult maturation (5. chapter). GABAergic inhibition is of high importance in odor coding. An almost threefold decrease in the total amount of GABAergic innervation was found during adult maturation in the l- and m-APT target region, in particular at the change in division of labor during the transition from a young nurse bee to an older forager bee. The results fit well into the current understanding of brain development in the honeybee and other social insects during adult maturation, which was described as presynaptic pruning and KC dendritic outgrowth. Combining anatomical and functional properties of the bee’s dual olfactory pathway suggests that both rate and temporal coding are implemented along two parallel streams. Comparison with recent work on analog output pathways of the vertebrate’s OB indicates that parallel processing of olfactory information may be a common principle across distant taxa.
Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males.
The ability to perceive the number of objects has been known to exist in vertebrates for a few decades, but recent behavioral investigations have demonstrated that several invertebrate species can also be placed on the continuum of numerical abilities shared with birds, mammals, and reptiles. In this review article, we present the main experimental studies that have examined the ability of insects to use numerical information. These studies have made use of a wide range of methodologies, and for this reason it is striking that a common finding is the inability of the tested animals to discriminate numerical quantities greater than four. Furthermore, the finding that bees can not only transfer learnt numerical discrimination to novel objects, but also to novel numerosities, is strongly suggestive of a true, albeit limited, ability to count. Later in the review, we evaluate the available evidence to narrow down the possible mechanisms that the animals might be using to solve the number-based experimental tasks presented to them. We conclude by suggesting avenues of further research that take into account variables such as the animals’ age and experience, as well as complementary cognitive systems such as attention and the time sense.