across and within Hymenoptera
—
The leaf-cutting ant Atta vollenweideri as a case study
Dissertation submitted for the degree of Doctor of Natural Sciences
Presented by Sarah Koch
at the
Faculty of Sciences Department of Biology
Date of the oral examination: 24. October 2014 First supervisor: Dr. Christoph J. Kleineidam
Second supervisor: Dr. Ewald Grosse-Wilde
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-285152
Arthur Weasley in the Chamber of Secrets from JK Rowling [1]
Summary 1
Zusammenfassung 3
Introduction 5
Eusocial insects . . . 5
Division of labor . . . 6
Reproductive division of labor in the honeybee A. mellifera . . . 7
Age-polyethism in the honeybee A. mellifera . . . 8
Developmentally induced polymorphism . . . 9
The biology of the ant Atta . . . 10
Collective behavior . . . 12
The olfactory system in insects . . . 13
Complexity of the olfactory system in Hymenoptera . . . 16
Aim of the study . . . 20
1 Phylogenetic comparisons of sensory modality bias in eusocial insects 23 1.1 Introduction . . . 23
1.2 Materials and Methods . . . 27
Animals . . . 27
Fixation and staining procedures . . . 27
Quantitative neuroanatomy . . . 28
Phylogenetic principle component analysis . . . 28
Linear model and phylogenetic independence . . . 29
Phylogenetic multivariate allometry analysis . . . 31
Ancestral reconstruction . . . 32
1.3 Results and Discussion . . . 34
AL/OL ratio as sensory bias measure independent from brain volume . 34 Linear model on the sensory modality bias of eusocial Hymenoptera . . 39
Olfactory processing capacity is taxon independent . . . 40
Common ancestor of bees and ants is not sensory specialized . . . 43
iii
2 Caste-specific expression of antennal genes in A. vollenweideri 49
2.1 Introduction . . . 49
2.2 Materials and Methods . . . 52
Ethic Statement . . . 52
Animals . . . 52
Extraction of total RNA . . . 52
Sequencing and Assembly . . . 53
GO analysis . . . 53
Homology based search and sequence alignments for more conserved genes (eg TRP channels, biogenic amine receptors) . . . 54
Identification of genes of interest (chemosensory genes and immune re- sponse genes) . . . 54
Phylogenetic analysis . . . 55
Gene expression analysis . . . 56
2.3 Results and Discussion . . . 58
Initial sequencing and transcriptome . . . 58
Caste and subcaste specific GO . . . 60
Differential gene expression on the antenna of castes and subcastes apart from olfaction . . . 60
AL-phenotypes are reflected in differential gene expression of chemosen- sory genes . . . 74
Discussion 85 Complex olfactory system in a common ancestor . . . 85
Complex olfactory system and food provisioning . . . 86
Complex olfactory systems and complex olfactory signaling . . . 87
Changes in pheromone communication on the sender side and the evo- lution of pheromone receptor candidates . . . 91
Olfactory systems in Hymenoptera and how they are used for pheromone processing . . . 94
Supplementary Data 97
Bibliography 143
Acknowledgements 179
Author contributions 183
1.1 Phylogenetic tree of eusocial species with collected brain parameters . . 31
1.2 Phylogenetic tree of hymenopteran species used for ancestral reconstruc- tion of glomerular number . . . 33
1.3 AL and OL volume in relation to MB size . . . 35
1.4 Size of sensory neuropils along the vision-olfaction axis . . . 36
1.5 Correlation of the AL volume with the number of glomeruli in the AL . 38 1.6 Number of glomeruli along the vision-olfaction axis . . . 40
1.7 Phylogenetic tree of ant and bee species with their sensory modality bias 42 1.8 Ancestral reconstruction of glomerular number and bias for a small set of species . . . 44
1.9 Ancestral reconstruction of glomerular number for 56 hymenopteran in- sect species . . . 45
2.1 GO-term analysis in A. vollenweideri . . . 59
2.2 Heatmap of differentially expressed genes across castes and subcastes . 61 2.3 Phylogenetic relationship of the OPB protein sequences across different hymenopteran species . . . 76
2.4 Phylogenetic relationship of the IR protein sequences across different hymenopteran species and D. melanogaster. . . 79
2.5 Phylogenetic relationship of the OR protein sequences across different hymenopteran species . . . 81
2.6 Comparison of OR gene expression between males versus queens and between large versus tiny workers . . . 83
S1 Residual plots of the linear model . . . 97
S2 Caste and subcaste-specific GO-term analysis in A. vollenweideri . . . 104
S3 Alignment file of predicted protein sequences of painless genes . . . 105
S4 Alignment file of predicted protein sequences of hymenopteran-specific TRP channel genes . . . 108
S5 Alignment file of predicted protein sequences of Hsc70-4 genes . . . 109
S6 Alignment file of predicted protein sequences of Hsc70-3 genes . . . 110
S7 Alignment file of predicted protein sequences of Hsc70-5 genes . . . 111
S8 Alignment file of predicted protein sequences of Hsp70 genes . . . 112
v
S9 Alignment file of predicted protein sequences ofNompC genes . . . 113
S10 Alignment file of predicted protein sequences ofNompA genes . . . 114
S11 Alignment file of predicted protein sequences of beta-adrenergic-likeoc- topamine receptor 1 genes . . . 115
S12 Alignment file of predicted protein sequences of beta-adrenergic-likeoc- topamine receptor 2 genes . . . 116
S13 Alignment file of predicted protein sequences of beta-adrenergic-likeoc- topamine receptor 3 genes . . . 117
S14 Alignment file of predicted protein sequences of dopamine transporter genes . . . 118
S15 Alignment file of predicted protein sequences ofserotonin receptor genes 119 S16 Alignment file of predicted protein sequences of muscarinicacetylcholine receptor genes . . . 120
S17 Alignment file of predicted protein sequences of acetylcholine esterase genes . . . 121
S18 Alignment file of predicted protein sequences ofGABAb receptor subunit 1 genes . . . 122
S19 Alignment file of predicted protein sequences ofGABAb receptor subunit 2 genes . . . 123
S20 Alignment file of predicted protein sequences ofperiod genes . . . 124
S21 Alignment file of predicted protein sequences oftimeless genes . . . 125
S22 Alignment file of predicted protein sequences ofcycle genes . . . 126
S23 Alignment file of predicted protein sequences ofCLOCK genes . . . 127
S24 Alignment file of predicted protein sequences of JHEpoxide hydroxylase genes . . . 128
S25 Alignment file of predicted protein sequences of JHEsterase genes . . . 129
S26 Alignment file of predicted protein sequences of MRJP genes . . . 130
S27 Alignment file of predicted protein sequences ofVg receptor genes . . . 131
S28 Alignment file of predicted protein sequences ofmalvolio genes . . . 132
S29 Alignment file of predicted protein sequences offoraging genes . . . 133
S30 Phylogenetic relationship of the OPB protein sequences across different hymenopteran species. . . 134
S31 Phylogenetic relationship of the CSP protein sequences across different hymenopteran species . . . 135
S32 Phylogenetic relationship of the SNMP protein sequences across different hymenopteran and dipteran species . . . 136
S33 Phylogenetic relationship of the IR protein sequences across different hymenopteran species andD. melanogaster . . . 137
S34 Phylogenetic relationship of the GR protein sequences across different hymenopteran and dipteran species . . . 138
1.1 Volumes of total brain and brain regions in eusocial insects . . . 29
1.2 Loadings of the PCA . . . 37
1.3 Bivariate allometric coefficients . . . 43
2.1 Assembly output of the antennal transcriptome data. . . 58
2.2 Overview of chemosensory-related gene fragments and genes (used for phylogenetic analysis) in A. vollenweideri. . . 74
S1 Mean values of differentially expressed genes across castes and subcastes. 98 S2 p-values of differentially expressed genes across castes and subcastes. . 101
S3 Differentially expressed genes and gene fragments between males, work- ers and queens. . . 106
S4 Differentially expressed genes and gene fragments between large and tiny workers. . . 107
S5 Differentially expressed OR-genes and gene fragments between males versus queens and between large versus tiny workers. . . 139
ix
In eusocial insects, social organization is mainly based on olfactory communication via pheromones which are used to convey different information content. Nestmates can alert other nestmates in case of intruders, recruit them to profitable food sources, and queens use pheromones to attract mating partners during the nuptial flight. The olfac- tory systems of eusocial Hymenoptera are highly developed and the primary olfactory processing center, the antennal lobe (AL), contains a high number of functional units, called glomeruli. Vision and olfaction are unequally important for eusocial insects and previous studies have revealed volumetric differences of sensory neuropiles across ant and bee species where flying insects were shown to have larger eyes and optic lobes (OLs) than ground living insects. In the present thesis, I focus on the complexity and evolution of olfactory systems in different hymenopteran species under the assumption that the number of glomeruli can serve as a measure for the odor processing capac- ity of a species. Thus, I reconstructed the ancestral state for the olfactory system in Hymenoptera and found that the common ancestor of Hymenoptera already had a high number of glomeruli (∼200) in the AL indicating a high olfactory processing capacity. Based on allometric relations of sensory neuropiles and higher information processing centers, I could show that the number of glomeruli is a well-suited measure for the olfactory processing capacity, and can be used to assess the sensory modality bias of a species, i.e. the ratio between olfactory and visual brain areas. In my stud- ies, I could show that the sensory modality bias of ants and bees is not necessarily biased towards a specific sensory modality and thus species with a high number of glomeruli can be found in both clades most likely as adaptations to their ecological niches which could be subject to subsequent studies. Within the order Hymenoptera, however, one ant clade, the highly derived Attini, has a particularly high number of glomeruli (∼440-630). This clade is characterized by having an obligate symbiosis with a fungus they grow inside their nests and feed on. The most derived fungus-growing species are the leaf-cutting ants (Atta and Acromyrmex) which are evolutionary suc- cessful due to their enormous sized colonies with millions of workers. They show a complex form of division of labor which is linked to a pronounced size polymorphism, especially across worker subcastes. InAtta, even within the same species, the number of glomeruli differs in castes and worker subcastes. Large workers (foragers) have a higher number of glomeruli than tiny workers (brood and fungus carer). Also, queens
1
have more glomeruli than males (AL-phenotypes). These AL-phenotypes support the hypothesis that increased specialization, especially in the polymorphic workers, can lead to a loss in neuronal processing capacity. Furthermore, large workers have a single enlarged glomerulus in their AL, a so called macroglomerulus (MG) and males have 3 MGs. Functional imaging studies have revealed that the trail pheromone is represented in the MG of large workers and presumably sex pheromone components are represented in the MGs of males. I used the AL-phenotypes to identify receptors involved in olfac- tory communication. I could identify pheromone receptor candidates in large workers and males of A. vollenweideri by using antennal transcriptome data in combination with caste and subcaste-specific microarrays. Furthermore, I compared chemosensory related genes of A. vollenweideri with other eusocial insects in order to gain new in- sights into the evolution of pheromone communication systems in Hymenoptera. The trail and sex pheromone receptor candidates are not members of the same subfamily of odorant receptor coding genes. Because these different pheromone receptors are so diverse, I suggest that pheromone receptors have been recruited by changes in ligands and therefore the sender side of the communication system. Moreover, I presume that the high number of glomeruli in the leaf-cutting ants has facilitated the evolution of their elaborate farming system and potentially their complex form of social organiza- tion. Whether complex forms of social organization are accompanied by high olfactory processing capacities still needs to be elucidated.
Die soziale Organisation von eusozialen Insekten basiert hauptsächlich auf olfaktorischer Kommunikation, bei der Pheromone für viele verschiedene Informationszwecke genutzt werden. Nestgenossen können ihre Nestgenossen vor Eindringlingen warnen oder sie zu ergiebigen Futterquellen rekrutieren, wohingegen Geschlechtstiere (Königinnen und Männchen) Pheromone dazu nutzen, Paarungspartner während des Hochzeitsfluges ausfindig zu machen. Das olfaktorische System der eusozialen Hymenopteren (Haut- flügler) ist hoch entwickelt mit einer hohen Anzahl von Glomeruli. Diese sind die funktionellen Einheiten des Antennallobus (AL), der duftverarbeitenden Gehirnregion.
Der Seh- und der Duftsinn spielen unterschiedlich große Rollen im Leben der eusozialen Insekten. Vorherige Studien haben gezeigt, dass Ameisen und Bienen unterschiedlich große sensorische Neuropile haben. Es wurde auch gezeigt, dass fliegende Insekten größere Augen und größere optische Loben haben als am Boden lebende Insekten.
In meiner Studie konzentriere ich mich auf die Komplexität und Evolution des olfak- torischen Systems in verschiedenen Hymenopteren, basierend auf der Annahme, dass die Anzahl an Glomeruli als Maß für die Leistungsfähigkeit der Duftverarbeitung im Gehirn einzelner Arten verwendet werden kann. Daher habe ich den Urzustand des olfaktorischen Systems der Hymenopteren rekonstruiert. Ich konnte zeigen, dass der hypothetische gemeinsame Vorfahre der Hymenopteren bereits eine hohe Anzahl von Glomeruli (∼200) im AL hatte, was darauf hindeutet, dass eine hohe Leistungsfähigkeit für Duftverarbeitung bereits vorhanden war. Aufgrund von allometrischen Beziehun- gen der sensorischen Neuropile und höher verarbeitenden Gehirnzentren, konnte ich zeigen, dass die Anzahl von Glomeruli ein geeignetes Maß für die Leistungsfähigkeit der Duftverarbeitung im Gehirn ist. Die Anzahl der Glomeruli kann dazu genutzt wer- den, die sensorische Tendenz einer Art in Richtung Geruchssinn oder Sehvermögen fest zu stellen (das Verhältnis von optischem und olfaktorischem Gehirnareal). Mit meinen Studien konnte ich zeigen, dass Bienen und Ameisen in dieser Hinsicht nicht grund- sätzlich verschieden sind. Arten mit einer hohen Anzahl an Glomeruli sind in beiden Stämmen zu finden, wahrscheinlich als Anpassungen an deren ökologische Nischen, was in weiteren Studien untersucht werden könnte.
Innerhalb der Ordnung der Hymenopteren, weisen die Attini, ein hoch abgeleiteter Ameisenstamm, eine außergewöhnlich hohe Anzahl an Glomeruli im AL auf (∼440- 630). Dieser Ameisenstamm zeichnet sich durch eine bindende Symbiose mit einem Pilz
3
aus, den sie in ihrem Nest züchten und von welchem sie sich ernähren. Die am höchsten abgeleiteten pilz-züchtenden Ameisenarten innerhalb der Attini, sind die Blattschnei- derameisen (Atta undAcromyrmex). Evolutionär gesehen sind diese Ameisenarten sehr erfolgreich, da sie riesige Kolonien mit Millionen von Arbeiterinnen bilden. Darüber hinaus zeichnen sie sich durch einen komplexe Art der Arbeitsteilung aus, welche mit ausgeprägten Größenunterschieden verknüpft ist, dies besonders unter den Subkasten der Arbeiterinnen. Innerhalb des Genus Atta weisen sogar die Kasten und Subkasten derselben Arten unterschiedliche Anzahlen von Glomeruli auf. Große Arbeiterinnen (Sammlerinnen) besitzen eine höhere Anzahl an Glomeruli als winzige Arbeiterinnen, die sich um Brut und Pilz kümmern oder als Königinnen. Die Königinnen selbst be- sitzen mehr Glomeruli in ihrem AL als die Männchen (AL-Phänotypen). Diese sogenan- nten AL-Phänotypen unterstützen die Hypothese, dass eine höhere Spezialisierung des Individuums zu einem Verlust von neuronaler Leistungsfähigkeit führen kann. Darüber hinaus haben große Arbeiterinnen einen einzelnen vergrößerten Glomerulus in ihrem AL, einen sogenannten Makroglomerulus (MG). Männchen hingegen haben sogar drei MGs im Antennallobus. Das Spurpheromon der Blattschneiderameisen aktiviert den MG im Antenallobus der großen Arbeiterinnen, wie funktionale Bildgebung bereits of- fenlegte. Vermutlich werden die drei MGs der Männchen durch Komponenten der Sex- pheromone aktiviert. In meiner Studie habe ich die AL-Phänotypen dazu genutzt, um Rezeptoren zu identifizieren, die an der olfaktorischen Kommunikation beteiligt sind.
Basierend auf den Daten des antennalen Transkriptoms in Kombination mit Kasten- und Subkasten-spezifischen Microarrays, war ich in der Lage Kandidaten für Pheromon- rezeptoren in großen Arbeiterinnen und Männchen zu identifizieren. Phylogenetische Analysen der Duftrezeptoren haben es ermöglicht, neue Erkenntnisse über die Evolu- tion der Kommunikationssysteme mittels Pheromonen in Hymenopteren zu gewinnen.
Die Rezeptorkandidaten für Spurpheromone und Sexpheromone gehören nicht zu ein und derselben Unterfamilie der Duftrezeptoren. Da die Pheromonrezeptor-Kandidaten so verschieden sind, komme ich zu dem Schluss, dass die Pheromonrezeptoren von den verschiedenen Liganden rekrutiert wurden und damit jegliche Veränderung des Pheromonsystems von der Senderseite des Kommunikationssystems ausging. Darüber hinaus nehme ich an, dass die hohe Anzahl der Glomeruli in den Blattschneiderameisen die Evolution ihrer ausgeklügelten Pilzkultivierung begünstigt hat und möglicherweise auch die Evolution von solch komplexen sozialen Organisationsstrukturen. Ob kom- plexe Organisationsstrukturen mit einer hohen Leistungsfähigkeit der Duftverarbeitung einhergehen, muss allerdings noch geklärt werden.
Eusocial insects
The class of insects is one of the most diverse classes in the animal kingdom with ∼1 Mio species worldwide [2]. Insects can be found in all parts of the world and some of the insect families show amazing adaptations to the ecological niches they live in.
2% of insect species are social insects like wasps, bees, ants and termites [2]. Within the family of ants alone, an immense diversity of species can be found with ∼15.000 species worldwide and 170 species described for central Europe [3]. Eusocial insects are considered to be evolutionary very successful not only because of the species diversity but also due to the large amount of biomass they constitute. Eusocial insects live in colonies and some of the species form colonies with millions of individuals living in it.
These eusocial insects are believed to make up 50% of the biomass of all insects and in total the biomass of ants alone is considered to be equal to the biomass of humans [2].
Most eusocial insects are members of the order Hymenoptera where eusociality evolved at least 9 times independently [2].
Insects are considered eusocial if they meet three criteria which distinguishes them from solitary or non-social insects:
First, overlapping generations live together in one colony and secondly older individuals raise the next generation together [4]. Third, there is reproductive division of labor, for example only a single individual or a subset of individuals in the colony repro- duces whereas the other individuals are sterile or reproduce only to a smaller degree.
While most hymenopteran insects are eusocial there is a huge variation in the size of the colonies and the way the colonies are organized. Some social insects live in small colonies (10-100 individuals) whereas some of the more derived species live in large colonies containing millions of workers. There is a great variety of different nest types, ranging from nests formed out of the bodies of colony members, so-called bivouacs, to complex nest constructions including intricate ventilation systems [5, 6].
Many advantages of the eusocial life style relate to the division of labor that necessi- tates a certain group size. A joint and organized food search (foraging) in numerous small groups enables the colony to cover a larger area than can be covered by one individual alone. This increases the chances of locating a patch of food by one of
5
the foraging groups. Furthermore, lucrative food sources can be harvested more effec- tively by the colony members, because more individuals can carry bigger pieces and are therefore faster in harvesting the food. As foraging trips are rather dangerous in terms of exposure to predators or infections, the loss of a forager in a colony is less drastic than losing the reproductive unit of the colony. The division of labor allows non-reproductive individuals to perform the most dangerous tasks, while the members of the colony who take care of reproduction remain safe.
Division of labor
There is a huge variation in the degree of eusociality and division of labor across dif- ferent eusocial insects species. Different types of reproductive division of labor exist with colonies being either polygynous or monogynous [2]. Polygynous colonies have multiple queens living in one colony, reproducing offspring together (a basal trait). In derived eusocial insects monogynous colonies are predominant, and the reproductive unit is a single individual laying eggs. Although only one or few individuals of a colony reproduce, the reproductive potential of the workers was lost in the course of evolution only in some species.
Most eusocial insects have a haplo-diploid sex determination system (for review see [7]). Males are haploid and develop from unfertilized eggs whereas all females of a colony are diploid and develop from fertilized eggs. The development of an egg into a queen or a worker female mostly depends on larval nutrition (for review see [8]).
Queens of eusocial insects vary greatly in the number of mating partners. While basal eusocial insects mate with a single male, queens of more derived species, like A. mel- lifera, mate with 10-18 males during the nuptial flight (polyandrous mating system, [9]).
Some comparative studies suggest that the complexity of social systems evolved with the order Hymenoptera [2]. Along with the evolution of the eusocial mating system, more derived species show a more complex organized social system whereas basal ant species for example show a diminished division of labor where workers are capable of laying eggs of nutritional value or unfertilized eggs (male eggs) [10]. Primitively euso- cial insects, like the ant Pachycondyla sublaevis, establish dominance hierarchies where some individuals of the group are the elite and one is the most superior individual which produces the offspring [11]. These hierarchic structures are established through aggressive behavior with the most aggressive individual becoming the queen. Social organizations which are based on such dominance hierarchies tend to have rather small colonies [2] and workers can often not be distinguished from queens due to morphology.
The size of the colonies seems to correlate with the complexity of the social organiza-
tion and larger colonies tend to show a more pronounced queen-worker polymorphism where queens can be easily distinguished from workers due to their size or body shape.
Furthermore, dominance hierarchies play only minor roles in ant species with increased colony sizes.
Apart from the reproductive division of labor, a non-reproductive division of labor can be found in many eusocial insects where the different tasks in a colony are allocated to different specialized workers.
Reproductive division of labor in the honeybee A. mellifera
Besides dominance hierarchies where queens and workers can hardly be distinguished, there are other mechanism of how the reproductive division of labor is established, leading to a distinct queen worker dimorphism. Traditionally, the honeybee A. mel- lifera was one of the first species on which the phenomenon of reproductive division of labor was studied. Ever since, most studies revealing the molecular basis for the reproductive division of labor were conducted inA. mellifera and therefore this section will exemplarily focus on this model organism.
Queens, males and workers ofA. mellifera can be very different in terms of body size, behavior and life span. They exhibit a dimorphism where queens are for example larger than workers. This dimorphism is established during larval development and mostly based on larval nutrition [12]. Although many different substances and hormones are found to correlate with differentiation towards queens and workers, the exact mecha- nism how these different substances orchestrate the different developmental pathways is largely unknown due to the complexity of the underlying developmental pathways.
In most cases, the different substances and hormones were found to regulate each other, showing that the developmental regulation is a very complex interplay of a multitude of mutual regulators.
All female larva are fed with royal jelly (RJ), a highly proteinacous secretion from the workers’ hypopharyngeal glands, but quantitative and qualitative differences in RJ were found determining either queen or worker development [12]. RJ contains major royal jelly proteins (MRJPs) and most importantly royalactin [13, 14, 12, 15] which has recently been found to be involved in the switch from worker to queen develop- ment. Increased levels of epidermal growth factor receptor (Egfr, [16, 17]) and juvenile hormone (JH) which might depend on royalactin feeding, influence the growth and the development time of the female larva and ultimately lead to a queen-destined de- velopment of the larva [12, 16]. Although some of the molecular machinery of queen
worker differentiation is already investigated, it is not clear how the different pathways influence each other or whether both pathways are substitutes and constitute a backup pathway.
Age-polyethism in the honeybee A. mellifera
Not only is A. mellifera a model organism for reproductive division of labor, most of the research regarding the molecular basis for non-reproductive division of labor has been done on the honeybee as well. Therefore, this section will focus on A. mellifera and how worker task allocation is established. Workers of A. mellifera and similarly in most ants species, perform tasks like nursing, guarding and foraging [18]. In A. mel- lifera the age of the workers correlates with the task that they perform. Bees switch their tasks during their life span and this type of non-reproductive division of labor is called temporal castes or age-polyethism. Newly hatched workers stay close to the queen and brood and act as cell-cleaners and nurses [19, 20]. Middle aged bees (MAB) perform tasks like nest building, guarding, or nectar processing where they take up the nectar from foragers entering the nest [21]. Foragers are the oldest workers of a colony and they actively search for pollen, nectar or water and recruit other nest-mates to food sources [18].
Many different mechanisms and models have been suggested throughout the years which propose nutritional, hormonal, behavioral feedback from nest-mates or pheromone communication as causes for task switching [22, 23]. More and more research on this topic is revealing that most of the causes are linked physiologically and orchestrate caste switching in honeybee workers together. Early studies on the division of labor in A. mellifera found antagonistic levels of JH and Vitellogenin (Vg) in the hemolymph of nurse and forager bees [24, 25, 26]. Both hormones are suggested to repress each other so that only one of the hormones is highly expressed at any given time [25, 27]. The reason why these hormones were the subject of intense studies is that their expression levels were reflected in behavior which was linked to the different temporal castes. For example JH and Vg were found to be involved in the timing of the onset of foraging or brood food production [28, 29]. Further studies revealed that hormone levels were influenced by the exposure of workers to different pheromones. One of the pheromones is the brood pheromone (BP) produced by larvae [30] and mostly nurses are exposed to BP while feeding the brood. A second pheromone involved in caste differentiation, the queen mandibular pheromone (QMP) is released by the queen herself. QMP and BP both seem to be involved in the switch from nursing to foraging trough regulation of the expression of the key players in Vg and JH. Hence the exposure to BP was found to
stimulate RJ secretion or delays the onset of foraging [31] whereas QMP was found to inhibit ovary development in workers [32] and the rearing of new queens in the colony [33].
It was suggested by Johnson et al [34] that the exposure to BP and QMP is lim- ited by the fact that more and more young bees hatch and push the older bees out of the broodzone where they are exposed to these pheromones. Released from the suppression of the pheromones, JH levels in the hemolymph of older workers change and simultaneously a multitude of different genes are upregulated leading to different behavioral output [35, 36]. Most of the genes upregulated in older workers are reg- ulated by a transcription factor called ultraspiracle (ups, [37]) which forms a protein complex with other transcription factors and transcription is activated upon JH bind- ing [37, 38, 39, 40]. These differences in the expression levels of some genes like for example foraging or malvolio [41, 42, 43] lead to altered behavioral output and bees performing more tasks of MABs than nurse bees. Because MABs are suggested to be fully capable of performing foraging tasks, an inhibitory mechanism of foragers actively preventing MABs from foraging is proposed through a primer pheromone ethyloleate [44]. Yet another pheromone present on the foragers’ cuticule is found to alter work- ers’ behavioral output [45] and thus contributes to the coordination of division of labor.
Developmentally induced polymorphism
Besides temporal specialization for task allocation, a developmentally induced form of specialization can be found in some eusocial insects, the physical castes [2]. In this system, workers perform different tasks and can be classified in behavioral phenotypes or subcastes similar to the tasks that are performed by honeybee workers. In many ant species, polymorphic workers can be found which differ in body size and are a product of distinct developmental pathways. The morphological polymorphism described in the workers is not plastic with age, it is rather fixed and developmentally induced. Mostly the workers’ task allocation is then linked to size-polymorphism of the workers which is called alloethism. This means that workers of different sizes perform different tasks.
Only little is known about the molecular basis of this subcaste differentiation whereas morphological and behavioral descriptions of workers have been published for numer- ous ant species [10, 2]. The rare studies that have been performed in ant species have revealed that JH is involved in the morphological polymorphism similar to the age- polymorphism in honeybees [46]. JH in solitary insects is known to be involved in the maturation of the insect whereas in A. mellifera JH is recruited for division of labor
in worker bees. In more derived eusocial insects like Pheidole ants, JH is found to be involved in subcaste differentiation. Pheidoleants are the only ants with a developmen- tally induced polymorphism where the influence of JH on subcaste development has been studied. Workers of the species rich genus Pheidole are either small and perform tasks inside the nest (minors) or large and guard the nest (soldiers) [47]. Soldiers are much larger than minors and in contrast to the minors they have wing vestiges on the mesothorax [46]. In very few cases, even a third subcaste is found in small percentages in natural populations of some Pheidole species, the supersoldiers [48, 49]. These are even larger than soldiers and have wing vestiges on their thorax as well [48, 49]. In contrast to the honeybee caste system, ant worker subcastes do not switch tasks when getting older.
At fixed time points during larval development there is a subcaste-specific switch in the developmental program of workers. JH is particularly important in minor-soldier differentiation [50, 51]. In honeybees, JH is important in queen-worker differentiation very early in larval development. Very late (third instar) in the larval development of Pheidole workers there is an additional JH-sensitive phase during which variations in JH levels induce either minor or soldier development [51]. In Pheidole species with naturally occurring supersoldiers, a third JH-dependent switch is suggested to be in- volved in the supersoldier-soldier differentiation [46]. Interestingly, this supersoldier development seems to be an ancient potential as the subcaste can be induced in other Pheidole species where supersoldiers are absent in natural colonies [46].
The biology of the ant Atta
Species of the genus Atta exhibit the most complex social organization of eusocial in- sects with some of them building the largest colonies with millions of adult workers [2].
Full-grown colonies not only exhibit an immense queen worker dimorphism but also ex- hibit a worker size-polymorphism with a 200 fold difference in body size [52]. This size polymorphism is strongly linked to task allocation which makes leaf-cutting ants a good model organism. Workers specialized in specific tasks can easily be selected based on body size in order to perform differential expression studies across castes and subcastes.
This section focuses on the biology of Atta in general and the different morphological Atta worker phenotypes, since leaf-cutting ants are the topic of this thesis.
The huge Atta colonies are founded by only a single queen after a nuptial flight. This behaviorally complex event happens once a year when the sexual individuals are pro-
duced. The nuptial flight is a precisely timed succession of events in which males aggregate at the nest entrances of their home colony before they take off and spread, shortly followed by queens [53]. Nuptial flights of colonies in an area as large as 100 km2 are synchronized in order to decrease inbreeding. Key factors in the determination of synchronization are the season and weather conditions [53]. Unfortunately, mating has never been observed because sexuals tend to fly very high into the air which makes it impossible to observe mating itself. Sex pheromones play an important role dur- ing the nuptial flight in general and for locating potential mating partners in the air in particular. During the nuptial flight, each queen mates multiple times [54]. Af- ter mating, the queen sheds off her wings and founds a new colony all on her own.
Each queen takes fungus mycelia from her mother colony with her when she leaves for her nuptial flight, and uses this to start her own fungal garden in her newly founded colony [10]. In the beginning she feeds the fungus with her feces, but after the first workers have been produced, they take over the fungus care and start collecting leaves [55]. Atta ant species live in a mutualism with a fungus mostly from the basidomycete family [56, 57], which provides the colony with lipids and carbohydrates [58] while the ants collect leaves, the fungus can feed on. The fungus is the only food source for the brood, and they take special care because if the fungus dies there is no replacement.
Workers inside the nest are rather small and perform different fungus or brood car- ing tasks. They cut large leaf material into smaller pieces, place the small leaf pieces on the fungus, remove old fungus pieces and take care to protect the precious fungus from infection [2]. The largest workers, so called soldiers, guard the nest and protect the nest from predators. large workers forage for leaf or grass material and cut and transport the plant pieces [2, 59]. Further, they build and maintain nest sites. In fully grown colonies, containing up to 20 million adult workers, up to several hundred fun- gus chambers exist in the nest in order to provide enough food for the colony [10]. In order to rear enough fungus, workers have to provide a huge amount of grass material for the fungus to grow on. Foraging is well organized and takes place on trails which are chemically marked with trail pheromone in order to recruit nestmates to profitable food sources [60]. The trail pheromone is a secretion from the poison gland which is composed of several components in a species-specific ratio [61, 62, 63].
The non-reproductive division of labor of leaf-cutting ants is by far the most complex in all eusocial insects. In some Atta species, there is even evidence for task partition- ing among workers foraging together. With an increased distance between the foraging sites and the nest, larger workers show a tendency towards cutting the grass rather than transporting it. Smaller workers on the other hand seem to have an increased tendency towards transporting the leave pieces to the nest [59]. Additionally, when leaf
material is transported back to the nest, it can be observed that smaller ants are riding on the backs of larger workers like hitchhikers [64, 65]. Although the exact function of this hitchhiking behavior has not been revealed so far, many hypotheses about its advantage have been postulated. Two likely explanations are that either smaller work- ers start cleaning the leaf material from pathogenic bacteria which could be a threat to the fungus garden or the smaller ants are preventing phorid flies, a parasitoid, from laying their eggs on the larger workers [64, 65, 66]. In either case, it seems that the hitchhiking actually has an advantage and that the smaller workers have a purpose and are not just being carried to the nest.
Other evidence for task partitioning among foragers comes from behavioral studies.
foragers of A. vollenweideri differ in their sensitivity to the trail pheromone and their discriminatory ability when being challenged with conspecific and heterospecific trails [67]. Workers with middle-sized head width (<1.4 mm) are better in discriminating conspecific from heterospecific trails than workers with larger heads (>1.4 mm, [67]).
These larger workers on the other hand are much more sensitive to the releaser compo- nent of the trail pheromone (personal communication C. Kelber). It is assumed that these different sized workers differ in their behavioral response threshold, as smaller amounts of the trail pheromone already elicit trail-following behavior in the larger ants but we do not know the molecular basis for caste and subcaste differentiation in A.
vollenweideri assuming that the same key players might be involved.
Collective behavior
Collective behavior is the self-organized behavior of an entire group or colony of animals which emergences from simple, repeated behavior of individuals [68]. The social orga- nization including division of labor and task allocation in colonies of eusocial insects is a self organizing process and several models try to describe this phenomenon (for review see [69]). Response threshold models are based on the assumption that every individual worker in a colony has an internal threshold and the variation of this internal threshold makes the castes and subcastes act differently to a certain stimulus [70, 71].
Individuals with the lowest threshold would be recruited to respond to that stimulus with a stereotyped behavioral response earlier than the other individuals of the colony with higher thresholds [72, 73]. This threshold is a behavioral response threshold-not a sensory threshold: Individuals do not respond behaviorally though they detect the stimulus. The variation in the behavioral thresholds can have multiple origins (for review see [23]) for example variations can result from individual experiences of colony members [74]. Workers from the antA. vollenweideri for example develop an avoidance of specific leaf material if the fungus gets harmed by that [75, 76]. Variation in behav-
ioral threshold can also result from differences in in larval nutrition or in the genetic make-up of the individuals. Differences in the genetic make-up of individuals is for example achieved in derived eusocial species through multiple matings [54]. Some of the partilines in the honey beeA. mellifera for instance seem to be favored for rearing queens [77]. In A. vollenweideri , workers seem to differ in their behavioral response thresholds for trail-following (C. Kelber personal communication). Beyond individual differences in behavioral thresholds, the coordination of collective behavior or division of labor requires effective communication among members of an insect colony. In all eu- social insect colonies, olfaction is the most important communication channel. In ants, for example pheromones are used as olfactory signals in sexuals for locating mating partners or in workers for nestmate recruitment to profitable food sources. Therefore, the key to understand the organization of the division of labor is the olfactory system of the eusocial insects.
The olfactory system in insects
Morphology of the olfactory system
Odors are volatile substances and are passively distributed through the air or water.
Natural occurring odors are mostly mixtures of different monomolecular odorants in varying concentrations. Insects detect odors with odorant receptors (ORs) which are located in the antennae, maxillary and/or labial palps [78, 79]. The antenna is the main olfactory organ of insects, and in addition to olfactory sensitive neurons (OSNs) it houses gustatory, temperaturesensitive and hygrosensitive receptor neurons [80, 81].
Receptor neurons are located in sensilla on the antenna’s surface. Sensilla are often hair-like structures which are specialized units for the reception of the different modal- ities. Olfactory sensilla (the hair-like trichoid, basiconic, coeloconic sensilla or the plate-shaped sensilla placodea) all have pores through which odor molecules can enter (for review see [81]). The cell bodies of OSNs are located at the sensillar base and the dendrites stretch into the sensillar lymph [82]. Each OSN expresses one or a few different OR coding genes which are generally sensitive to multiple different odorants [83]. OSNs transfer the odorant information via action potentials to the first olfac- tory neuropil, the antennal lobe (AL, [84]). The OSNs project into defined spherical substructures in the AL, the processing units called glomeruli. OSNs with the same OR often converge on the same glomerulus [83]. The number of ORs expressed in an insect is reflected in the number of glomeruli in the AL. This one receptor – one glomerulus hypothesis was only confirmed forDrosophila so far so that the high num- ber of glomeruli found in some ant species (630 glomeruli in Apterostigma cf mayeri) questioned the hypothesis as being true for all insects [85]. However, recent genome publications confirmed that indeed high number of OR coding genes can also be found
in the genome data of the ants matching the glomerular number [86].
Inside a glomerulus the numerous OSN input neurons converge onto a smaller number of output neurons, called projection neurons (PNs, [87]). Within the AL the odor information is processed in a network of local interneurons (LNs) that interconnect the different glomeruli. Many different types of LNs can be found in the AL of insects and they vary in their morphology as well as their neuromodulatory equipment [88, 89, 90, 91, 92] so that they can work as gain control or improve the signal to noise ratio [93, 94, 95]. PNs, convey the information to higher order brain areas like the mushroom bodies (MBs) or the lateral horn [96, 91, 97]. There the PNs diverge on numerous brain region specific neurons. Odor information is evaluated and categorized or integrated with other sensory modalities in these higher order brain areas (for review see [98]). In all insect species the ALs are equipped with the same basic types of neurons (OSNs, LNs, PNs) but numbers of neurons and neuron subtypes differ greatly across the insect species.
Molecular basis of odor transduction
Odor molecules enter the sensillum through the numerous sensillar pores. Because odor molecules are mostly hydrophobic, odorant binding proteins (OBPs) ferry the odor molecules through the aqueous sensillar lymph to their receptors, the odorant receptors (ORs, [99, 100]). ORs are embedded in the dendritic membranes of OSNs which protrude into the sensillar lymph. Most of the studies on the olfactory transduc- tion and the perireceptor events were conducted inD. melanogaster or several different moth species.
OBPs are small molecules which are secreted into the sensilla lymph by support cells which surround the cell bodies of OSNs at the base of the sensillum [101, 102, 103].
OBPs are able to bind odor molecules with some OBPs showing a narrow binding spectrum to a small subset of odorants with different binding affinities to the odor- ants [104, 105, 106], (for review see [107, 108]). However, the exact mechanism of how the odorants are bound and released is not well understood. OBPs dimerize in vitro and bind up to three odorant molecules with different affinities for each binding site [109, 110]. Whether this is true for in vivo OBPs is unknown. Upon binding to an odor- ant molecule, some OBPs undergo a conformational change so that the OBPs in the complex surround the odorant molecule and therefore supposedly protect the odor from being degraded in the sensilla lymph by odorant degrading enzymes [111, 112, 113].
Odorant degrading enzymes are secreted into the sensilla lymph where they are con- sidered to clear the lymph from odorants so that ORs are not constantly activated and thus preventing ORs from adaptation [111, 112, 101, 113]. It is believed that an
odorant which is bound by an OBP cannot be degraded by odorant degrading enzymes.
OBPs not only function as odorant molecule transporters, they are also increasing the sensitivity to specific odors [114, 115, 116, 104, 105, 106, 117]. In D. melanogaster for example, the OBP LUSH is required for the reception of low concentrations of the aggregation and sex pheromone, cis-vaccenyl acetate [118, 119, 120, 121, 122], although LUSH itself is not necessary to stimulate the cVA-sensitive neurons [123].
In order to activate an OR, the odorant is released from the OBP/odor complex. In vitro studies showed that the conformational change of the OBP/odor complex can be reversed upon pH-change so that the odor is released again [124], (for review see [108]).
It has been suggested that there is no constant pH in the lymph in vivo but close to the membrane the pH seems to be more acidic [125, 126] which favors the release of the odorant molecule and binding to the OR for odor signaling [127, 126, 115, 100, 128, 129].
ORs are 7-TM proteins [130, 131] embedded in the dendritic membrane of OSNs [132].
They belong to the family of GPCRs (G-protein coupled receptors) with an inverted topology in comparison to vertebrate ORs [133]. Members of the OR protein fam- ily are very diverse across different insect species and even within the same genus.
However, one OR, the odorant receptor co-receptor, orco [134], is rather conserved throughout the different insect species [135, 136]. Orco is expressed in all OSNs and has two major functions. On the one hand orco works as a chaperone in helping to traffic ORs to the membrane [133]. On the other hand orco forms functional het- eromers [137] with ligand-specific ORs (OR-x), for olfactory transduction [138, 136].
Until now the exact stoichiometry of the protein complex is not revealed neither is the olfactory transduction mechanism. The heteromeric OR-orco complex could form a ligand-gated ion channel which mediates a purely ionotropic odor transduction [136], or the heteromeric OR-orco complex could mediate both a ionotropic and metabotropic transduction [139, 140, 141, 142]. So far, we know that both proteins are needed in order to form a functional OR and that different types of OR-x can alter the ion se- lectivity [136, 143, 144, 145]. Furthermore, it was shown that orco phosphorylation increases odor responses [141] and Drosophila mutants for G-proteins, which are in- volved in metabotropic signaling cascades, show decreased sensitivity towards odorants [142].
Although the mechanism of olfactory transduction is still enigmatic, a lot is known about OR-ligand specificity. Studies in D. melanogaster [146, 147, 148, 149] showed that ORs vary in the number of ligands that they are capable to bind where some ORs are narrowly tuned and only bind to small amounts of odorants and others being more
broadly tuned [147]. The functional reason for some of the ORs being narrowly tuned is only under speculation. One possibility is that pheromone receptors, important for courtship behavior might be narrowly tuned, as the pheromones are often species spe- cific. In contrast, the specificity of broadly tuned ORs in moths can be increased by adding pheromone binding proteins [104, 105].
In conclusion, each odorant can bind to a set of different ORs and therefore activate a set of different OSNs. Odor information is therefore encoded in the combinatorial activation of OSNs. OSNs expressing the same odorant-specific OR converge onto the same glomerulus in the AL [150, 151, 152, 86] with only few exceptions known so far from Drosophila [153]. Therefore, the odor specific combinatorial pattern of OSN ac- tivity is maintained in an odor specific combinatorial activity pattern of glomeruli in the AL [154].
ORs are not the only receptors involved in insect olfaction. Ionotropic receptors (IRs) are the oldest class of chemosensory receptors which are found throughout the Pro- tostomia [155, 156]. This class of receptors evolved from non-NMDA iGluRs and can be divided in IRs that are expressed on the antenna (antennal IRs) and divergent IRs which are mostly expressed in gustatory organs like labellum and pharynx for example [156]. Antennal IRs are expressed in coeloconic OSNs and as 3-TM proteins they are located in the dendritic membrane of the OSNs [155, 157]. Similar to ORs, IRs form heteromeric complexes consisting of a ligand-specific IR and one of the two co-receptors (IR25a or IR8a, [157, 158]). The stochiometry of the IR complexes is suggested to be in a 2:2 ratio of ligand-specific IR and co-receptor. The IR co-receptors are highly conserved throughout the insects with the IR25a being present throughout all Proto- stomian species analyzed so far and the IR8a identified in all insect species. In contrast to the ORs, IRs are ligand-gated ion channels with no metabotropic component being involved in signal transduction [156].
Neither OR nor IR function was specifically studied in eusocial insects and therefore we only can assume that the physiology of the receptors in the ant is similar to that described in other insect species. The number of chemosensory gene family members differs greatly throughout the eusocial insect species with expansions in several differ- ent subfamilies. The number of OR genes in ants is in general very large in comparison to other hymenopteran species likeA. mellifera with 160 OR coding genes for example [159]. All ant species with identified ORs from genome data revealed at least twice as many OR coding genes (H. saltator: 347, Linepithema humile: 337, C. floridanus: 352, Pogonomyrmex barbatus: 344, [86, 160, 161]). These findings show that the euso- cial species show adaptations of their olfactory system to the different environmental conditions they are exposed to or their individual life styles.
Complexity of the olfactory system in Hymenoptera
Odor information processing in insects starts in the OSNs and continues in the AL and then in multiple higher brain areas. While the olfactory systems of all insect species share the same basic organization, they differ in their complexity. The olfactory path- way of Hymenoptera (ants, bees) appears to be particularly complex as compared to Diptera (flies) or Lepidoptera (moth). The high complexity of hymenopteran olfactory systems becomes already visible on the periphery. Hymenopteran species have multiple OSNs housed in a single sensillum with 5-35 OSNs per sensillum in honeybees [162]
up to 130 OSNs in Camponotus ants [163] whereas Diptera and Lepidoptera generally house 2-4 OSNs per sensillum [125]. OSNs compartmentalized in the same sensillum are supposed to interact with each other through ephaptic coupling [164, 165, 166]
where adjacent cells are influenced by each other through their electric fields without synapses being involved (for review see [167]). So far, functional ephaptic interactions have been described in only one study in D. melanogaster in a sensillum with two OSNs [165]. In this study, the effect of ephaptic coupling, i.e. an inhibition between the two neighbouring OSNs, was symmetric and gradually increased with increasing odor concentration. In Hymenoptera, olfactory sensilla house 10 to 100 times more OSNs. Accordingly, the effect of ephaptic coupling might be stronger and more com- plex, e.g. not symmetrically between OSNs.
The total number of OSNs in Hymenoptera antenna are generally high and vary a lot across the eusocial insects studied. For example, there are ∼100.000 OSNs in the antenna ofA. mellifera workers [168, 169] and as well ∼100.000 OSNs in the antenna of E. berlandi females [168]. A. vollenweideri workers, in contrast, have only ∼40.000 OSNs [170]. In line with the variation in OSN number across hymenopteran species, variations in the number of glomeruli in the AL were found which exceed the variation in neuronal numbers by far. The highest number of glomeruli found in a more derived fungus-growing antA. cf mayeri is 630 glomeruli [85] which is nearly 4 times the num- ber of glomeruli in the AL ofA. mellifera [171].
The OSNs project in the antennal nerve to the glomeruli in the AL. Before the an- tennal nerve enters the brain, it splits up into several tracts which innervate the AL and other neuropils. This subdivision of sensory tracts varies across eusocial insects with 4 tracts in A. mellifera (T1-T4, [91]) to 6 tracts in A. vollenweideri (T1-T6, [170]) to 7 tracts in C. floridanus (T1-T7, [172]). These tracts project to distinct clustered populations of glomeruli. Thus, more sensory tracts leads to more cluster- like arrangement of the glomeruli in the AL of ants [67]. This division of the AL into distinct clusters is reflected by the division of PNs into different tracts which are composed of PNs with distinct morphologies. PNs can be classified into two separate
morphological neuron types. The uniglomerular PNs innervate only one glomerulus in a dense manner and leave the AL via either of the two pathways, the medial or lateral antenno-protocerebral tract (m- and l-APT, [173, 174, 91]) and project to the MB calyces and the lateral protocerebral lobe where they remain separated [173, 172].
On the other hand multiglomerular PNs innervate many or all glomeruli of the AL and leave the AL in the mediolateral antenno-protocerebral tract (ml-APT) projecting only to the lateral protocerebral lobe [91]. In conclusion, this means that the glomeruli are either innervated by m- or l-APT uniglomerular PNs subdividing the AL into two hemilobes. Subdivision of the AL into two heimlobes seems to be a basal trait of hy- menopteran insects [175], but the assignment of antennal tracts to the APTs seems to vary greatly at least across the eusocial insects studied so far [91, 173, 172]. Dividing the OSN output into distinct clusters of glomeruli which themselves are innervated by distinct populations of PNs opens the possibility for parallel processing of odor information. Indeed, there are several studies which suggest that different aspects of odor information are processed in parallel in the honey bee AL [176, 177, 178, 179, 180].
Glomeruli in the AL are interconnected through several thousand LNs (almost 4000 LNs inA. mellifera, [181]) which take part in the processing of odor information. These LNs can be classified in several subtypes based on either their arborization patterns within the AL or the neuromodulatory equipment. While classification based on neu- romodulators is rather complicated due to co-expression in single LNs, classification based on morphology is clear cut. All LNs innervate several glomeruli but they differ in the density of arborization in glomeruli. So called homogeneous LNs have a similar density of arborization in all glomeruli they innervate whereas heterogeneous LNs al- ways innervate one or few glomeruli with a higher density than the rest [92, 182]. Half of the LNs in the AL of A. mellifera were found to express GABA as a neuromodula- tor [183] and additional studies revealed that histamine, allatostatin or tachykinin are either co-expressed with GABA or in separate LN populations [184, 185, 186]. Until today the morphological classes of LNs in the AL could not be matched with neuro- modulator expression, leaving the possibility that there are more classes to find and even more neuromodulators to test for expression in LNs.
The functional significance of the complexity on the different levels of the olfactory pathways is not well understood and subject to ongoing research projects. This com- plexity is evident for every level of information processing within the pathway and the AL has more secrets to reveal about how the separation of the input and output and the organization of glomeruli is used for odor processing. As long as we do not have full data sets measuring the OR response profiles in eusocial insects with differ- ent number of glomeruli or genes, we can only speculate about the adaptive value of such a high number of processing units. One possibility of understanding the adaptive
value are comparative studies and former studies already revealed that high number of glomeruli in the AL of ants correlate with small eyes [187]. Studies like this are rare and need to implement phylogenetic relationships. In order to understand the adaptive value of specific traits of the olfactory pathway in Hymenoptera we need to perform phylogenetic based comparative studies. In chapter one of my thesis, I will focus on how the complexity of the olfactory system can be estimated and how we can use that information to reveal the ancestral state of a specific trait.
Variations in the number of neurons and glomeruli across Hymenoptera are thought to be adaptations to the environment or specific life styles [85]. In eusocial hymenopteran species the number of neurons and glomeruli differs between different species but also within the same species between the sexes and workers [172, 188, 171, 189, 168]. Males of the honey bee A. mellifera or the long-horned bee E. berlandi for example have al- most 3 times more OSNs in the antenna compared to females of the same species [168], while the number of glomeruli is lower in males (male glomeruli/female glomeruli:
E. berlandi: 100/133; A. mellifera: 106/164 [168, 171, 189]). The lower number of glomeruli in males as compared to females can be found in most eusocial insects. In some ant species, males have only half the number of glomeruli in comparison to fe- males (male glomeruli/female glomeruli: H. saltator: 78/178, C. floridanus: 258/434, A. vollenweideri: 250/350; [188, 172, 190]).
Males are not involved in performing social tasks as their main task is reproduction [10]. The reduced number of glomeruli in males might reflect this difference in lifehis- tory. The olfactory system of males might be predominantly adapted to sex pheromone communication in the context of locating females for mating. Correspondingly, many males of eusocial insects have increased glomeruli volumes, so called macroglomeruli (MG), which are innervated by OSNs that are sensitive for sex pheromones (A. vollen- weideri: [190]; A. mellifera and E. berlandi: [168]; A. mellifera: [171]). Male-specific macroglomeruli also occur in many moth species, where several macroglomeruli form a so called macroglomerular complex (for review see [191]). Physiological measurements revealed that the macroglomerular complex is exclusively activated by pheromones (for review see [191] and [192]). Thus, it seems likely that male-specific MGs in males of A. vollenweideri are involved in sex pheromone processing as well. Indeed in honey bee drones a macroglomerulus specifically responds to the honey bee sex pheromone 9-ODA [193].
The only species were a MG is described in any other caste or subcaste than males, isA. vollenweideri [194, 190]. Large workers of A. vollenweideri have one MG at the entrance of the AL which is not present in queens and tiny workers [190]. Ca imaging
studies revealed that the releaser component of the trail pheromone is represented in this region of the AL so that the MG is most probably involved in trail pheromone processing [190]. The subcastes ofA. vollenweideri also vary in the number of glomeruli in the AL [190, 170]. Tiny workers have∼380 glomeruli whereas large workers have 440 glomeruli in the AL [190]. These distinct AL phenotypes across worker subcastes can be linked to the already described differences in odor-guided behavior [67]. Large workers are more sensitive to the releaser component of the trail pheromone probably due to the MG present in the AL (C. Kelber personal communication). The phenotypes of workers are strongly linked to size polymorphism and can therefore be selected based on their size. Therefore, this system is well suited to study differential expression of genes involved in olfaction across workers which are specialized in performing a particular task. Beyond that the MG and its functional implication make it possible to identify sex and trail pheromone receptor candidates. MGs are probably innervated by a larger number of OSNs which all express one type of OR coding gene along with orco. High expression levels of OR coding genes are therefore good candidates for pheromone receptors. These questions I address in the second chapter of my thesis.
Aim of the study
Sensory modality bias in eusocial insect species and how it can be predicted by brain morphology
The aim of chapter 1 was to evaluate the relationship between brain morphology and olfactory vs. visual bias.
Therefore, I analyzed the volumes of neuropils and functional units of selected brain areas across different eusocial Hymenoptera (bees and ants). I used multivariate corre- lation analysis to collect evidence that the mushroom bodies (MBs) limit the amount of sensory information that is processed. I compared visual and olfactory brain areas to establish a measure for the sensory modality bias of a species. Beyond that a linear model on complexity of the olfactory system was established trying to explain which parameters (neuropil volumes or functional units) can predict the sensory modality bias of a species. Principle component analysis revealed the allometry of the different neuropils and with the aid of phylogenetic information, the evolutionary history of the sensory modality bias was reconstructed for several hymenopteran species.
Gene identification from antennal transcriptome data and caste/subcaste specific expression
The aim of chapter 2 was to identify receptor candidates of the different sensory modal- ities in addition to genes that might be differentially expressed between castes and/or
subcastes ofA. vollenweideri. By specifically analyzing the expression patterns of OR coding genes, I wanted to identify pheromone receptor candidates.
The antennal transcriptome data was blasted with sequences form other eusocial in- sects in order to identify the homologs inA. vollenweideri . Differential expression of antennal genes was analyzed based on caste and subcaste specific microarrays. It is tested if expression levels of members of specific gene families can be linked with task allocation in subcastes and life style of the sexuals. Beyond that it was assessed if the AL-phenotypes are reflected in the expression levels of OR coding genes and thus if pheromone receptor candidates can be identified. Based on the sequence informa- tion from the transcriptome data we established the phylogenetic relationships of OR coding genes across several ant species.
modality bias in eusocial insects
1.1 Introduction
The brain as an organ is very costly and consumes a high proportion of the energy in relation to its size. Already 20% of the basal metabolic rate in humans is consumed by the brain even if the brain size only makes up 2% of the total human body mass (for review see [195]). Single brain regions can make up large proportions of brains and hence being the main processing brain region consuming most of the energy allocated to the brain. The cost of a large brain or brain region is always balanced by benefits that come along with an increase in brain volume or brain regions. Usually benefits are an increased performance of the entire brain or specific brain regions like it was found in primates [196].
A huge variation in brain size is described for primates with more derived species show- ing an increased relative brain size compared to more basal primate species. Early research in this field found that brain size correlates with the ecological stimuli that need to be processed. Primates with larger brains were often found to feed on fruits instead of eating leaves. As fruits are not available all year long and more patchy in their distribution compared to leaves, it was suggested that primates eating fruit needed more cognitive ability for the location and memorization of the food sources and that therefore their brains were larger [197]. Primates not only differ in their diet, they also differ in the group sizes they live in, from very few individuals to groups of around hundred individuals [196]. It seems that primates living in larger groups have a social organization within the group whereas groups with few individuals are less stable and change in composition [198]. Primates of large groups communicate with each other in order to coordinate foraging and/or to maintain group cohesion [196]. In addition to the ecological hypothesis Dunbar et al suggested a different mechanism for the increase in brain size, called the social brain hypothesis [196, 198]. They found a correlation between group size and brain size and so it was suggested that the increase in brain size might be an adaptation to the social group living rather than to local- ization and memorization of food sources [196, 198]. An increase of a specific brain
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region, e.g. the neocortex, can influence the total brain size of a species especially if it not changing all or any other brain areas [199]. But a volumetric increase of a brain region does not necessarily lead to an increase in total brain volume as the total volume might constrain volumetric differences. If there is an increase in only a specific brain region, cost and benefit of the increase might be balanced and adaptive and other brain regions might be changed due to that. Closer examinations of the brains of primates revealed that it is the neocortex that scales with the group size [196, 199]. In this case the allometry of the brain regions to each other is shifted and the change is qualitative rather than quantitative [196] leading to an increased performance of the neocortex for social stimuli processing.
Comparing the brain body allometry of mammals with that of ants, it was shown that ants have comparatively smaller brains than expected from the allometry of mammals [200]. The same study showed that the body mass as a measure for the allometry is misleading because of differences in the skeleton, exoskeleton versus endoskeleton. The exoskeleton of insects contributes with a proportionally large part to the body mass [200]. Even if only soft body tissue is taken into account for brain body allometries of ants, brains of ants are still unexpectedly smaller in comparison to mammals [200].
This miniaturization of the brains might suggest that benefits leading to an increased brain or brain regions in primates might not necessarily lead to the same outcome in ant species. Beyond that, complex forms of social division of labor exist in more derived eusocial species where colony size can be enormous. In this case, workers exhibit a high degree of specialization and task allocation which is linked to size polymorphism [201].
Based on these findings there were two alternative hypothesis about how the brain size correlates with social complexity and social performance in eusocial insects. On the one hand, it was suggested that the brain size in more complex organized species is increased in line with the findings in primates that socially complex environments co-evolve with larger computational power of the brain. On the other hand it was supposed that brain size would decrease in more complex organized eusocial species because of the high degree of specialization in division of labor [187]. The second hypothesis is based on the finding that the collective behavior of a colony can be de- scribed as a self-organizing process where individuals perform rather simple behaviors repetitively but the outcome on colony level is a complex behavior as a summary of all individuals of the colony (for review see [68]).
Comparisons across desert ant species suggested that brain size scaled with colony size and that ant species exhibiting large colonies had indeed larger brains than species with small colonies [200]. Studies in the higher Attini, in contrast, showed that brain size scales with the degree of social complexity with a maximum meaning that brain
size increases with colony size up to a certain point where this relationship turns and brain size starts to decrease again [202]. Hence, brain size partially seems to scale with the social complexity, maybe because of the increased social stimuli that colony mem- bers have to process. But if the behavioral specialization further increases like in the polymorphic workers of attine ants, it seems that brain size decreases and this might be due to an increased specialization of colony members which might be accompanied by a reduced performance of the individual. Complex task allocation in polymorphic subcastes is thought to lead to a reduced behavioral repertoire of the workers where task switching is only possible to a certain degree [203, 204]. It is suggested that the reduced behavior in specialized species leads to a decrease in brain size in order to save energetic costs that large brains cause [195].
Volumetric changes of brains or brain regions can have multiple causes which might also co-occur. Differently sized brain regions might contain different number of neu- rons or their morphology might be altered, for example the number of synapses could be changed. Changes in synaptic contacts in insects are well described for experience related plasticity in adults [205, 206]. Volumetric changes across different species how- ever, might be due to the same processes but originate from altered developmental programs before adult emergence.
Individual brain regions can largely influence the total volume of the brain but can as well change their volumes without affecting total brain volume so that other brain regions decrease in size [196]. If volumetric changes in brain regions are independent from other brain regions, the allometry of brain regions varies for different species and is known as mosaic evolution [207, 208]. Instead of correlating the entire brain volume with the performance of the species, individual brain regions should be investigated similar to the example of the primates. Along this line of argument, the mushroom bodies (MBs) were believed to be more complex in eusocial insects compared to soli- tary insects due to their social lifestyle [209]. MBs are multimodal processing centers receiving input from olfactory and visual neurons [96, 210, 211] and are shown to be in- volved in learning and memory [212]. Strausfeld et al even suggested that the MBs are homologous structures to the neocortex of primates [210, 213] which is controversially dicussed. Recent studies with a moderate taxon sampling, discovered that the common ancestor of Hymenoptera already had complex MBs (90 Mya) and that the complex structure of the MBs is independent from a solitary or social lifestyle at least for bees, wasps and other basal phytophagous hymenopterans [214]. However, the study was not specifically testing whether MB size correlates with social performance and hence we can only assume that the MBs are equally complex in all eusocial Hymenoptera because of their important role in multimodal processing and memory formation.
