Hox3 duplication and divergence in the Lepidoptera

Using the Speckled Wood Butterfly Pararge aegeria as the model species, this thesis presents the possible evolutionary significance of a set of duplications found in the Hox cluster of the Lepidoptera, called the Special Homeobox genes. An annotation of this duplicated cluster across a wide number of Lepidoptera was performed in order to assess patterns of duplication and loss across the order. The sequences recovered revealed a large amount of variation associated with the duplicate genes, indicating these are evolving very rapidly in different lineages. Patterns of sequence variation were examined to ascertain whether the observed variation was maintained due to selection at three separate levels of divergence: within the Ditrysia, within the more recently diverged Heliconius genus, and at the intraspecific level by quantifying nucleotide polymorphism within Pararge aegeria. Selective pressures were found to be operating between paralogous and orthologous genes, suggesting these have evolved, in part, under positive selection. The potential function of the duplicates was examined by means of CRISPR/Cas9 geneome editing, but revealed inconclusive results. Genome editing, however, was shown to be largely applicable to P. aegeria, and resulted in consistent mutations associated with wing patterning genes. The potential significance of the duplications for Lepidopeteran biology are discussed, as well as future applications for genome editing techniques in P. aegeria

Hormonal and transcriptional mechanisms underlying developmental plasticity of life histories in a seasonal butterfly

Temporal variation in abiotic and biotic variables such as temperature, rainfall, food availability or predation pressure profoundly affects the abilities of organisms to survive and reproduce successfully. Most organisms are remarkably flexible in the face of such heterogeneity in habitat quality, and display phenotypic plasticity in response to environmental variation, i.e. the production of alternative phenotypes from a single genotype, dependent on the experienced environment. The aftrotropical butterfly Bicyclus anynana expresses alternative adult life histories in its habitat's wet and dry seasons, including reproductive timing and lifespan. This thesis aims to increase insight into the hormonal and transcriptional patterns that underlie life history plasticity in B. anynana. The first question is how the environment experienced during development induces the two adult seasonal forms via conserved hormonal pathways. The second major question covered in this thesis is what transcriptional changes in the adult are associated with the seasonal forms, and how ageing differs between the seasons. Together, these data contribute to a better mechanistic understanding of plastic responses as adaptation to environmental variation, and provide starting points for research into mechanisms linking development and ageing in humans, and how events during early development can affect lifespan and human health.The research described in this thesis was supported by and carried out in the context of the EU-funded Network of Excellence LifeSpan (FP6 / 036894), with additional support by IDEAL (FP7/2007-2011/259679). The printing of this thesis was financially supported by the J.E. Jurriaanse Stichting, Rotterdam.UBL - phd migration 201

How does a butterfly embryo cope with environmental stress?

Butterfly abundance and species diversity have been in decline and several anthropogenic factors have been implicated in the observed downwards trend. This thesis focusses on environmental stress directed at butterfly eggs, using the model systems Pararge aegeria and Bicyclus anynana. Eggs are a vulnerable life stage in which environmental stressors can have a major impact on both survival and development. The majority of winged insects, including butterflies, develop a protective sheet of cells surrounding the embryo during early embryogenesis. This sheet of epithelial cells is called the serosa and appears to protect the embryo against toxins, desiccation and pathogens. I first developed a morphological staging including a description of the extra-embryonic serosa for P. aegeria. Such a description will aid in understanding the developmental perturbations environmental stressors may cause. One of the stressors butterflies are (inadvertently) exposed to are insecticides, which is reviewed in full here. A research programme is proposed to incorporate the developmental genetic mechanisms underlying the pesticide response in a conservation context. The effects of two hormone analogs (i.e. of juvenile hormone and 20E) to study their effect on endocrine disruption during embryogenesis; both a maternal effect and through direct exposure. Eggs displayed increased sensitivity early in development before serosa formation and were in general more significantly affected as a result of a maternal effect. Finally, using transcriptomics and in-situ hybridisations, the effects of a natural stressor in B. anynana eggs was investigated; bacterial exposure in combination with wounding. The serosa, rather than the embryo, was able to mount an immune response, evidenced by significant upregulation of immune-related genes, such as hemolin, and genes in the Toll, IMD, Mapk-Jnk-P38 and JAKSTAT pathways. In conclusion, the butterfly embryo copes with environmental stress, such as toxins and immune challenges, with the help of the serosa

Anti-predator adaptations and strategies in the Lepidoptera

This thesis examines visual anti-predator strategies employed by the Lepidoptera. I examine key aspects of pattern and behaviour and how they relate to the reduction of an individual’s predation risk. Symmetrical patterns have been found to be easier to remember and pick out, suggesting that symmetry is beneficial to aposematic displays. This suggests that symmetry may be maladaptive in cryptic patterning and asymmetry beneficial. In Chapter one, I report the results of a field experiment using artificial prey and wild birds to investigate how asymmetry and symmetry affect the efficiency of cryptic patterning to reduce predation. I found that asymmetry does not affect predation rate, in agreement with previous work. Yet, there is still the problem of how to mesh this with the potentially conflicting conclusions of symmetry studies. Chapter two examines aspects of the intimidation hypotheses of Lepidopteran eyespots. These address the generally larger and more centrally placed spots found on Lepidopteran wings and state that they startle or intimidate predators, providing time for escape. While it is agreed that eyespots intimidate or startle predators, the mechanism has not been agreed. There are two competing lines of thought 1) that ‘eyespots’ intimidate because they resemble the eyes of the predators’ own predators and 2) that it is the conspicuous colouration of the pattern that induces the startle or avoidance behaviour. The first experiment utilised artificial prey with differing ‘directions of gaze’ in a field setting. If purely conspicuous patterns direction of gaze should have no influence on prey survival. The results indicate that patterns imitating staring or upward gazes provide the greatest protection, suggesting that in some cases eyespots may be being perceived as eyes and not simply as conspicuous patterns. I wanted to see if it would be possible to find a way in which to measure or quantify the reaction of an animal to ‘real’ eyes, in order to compare it to the reaction to eyespots. Recent trials investigating human reactions to eye contact suggested a computer based method may be possible. In this second experiment we examined whether the direct gaze of a predator might produce a measurable effect in human subjects. I was not able find any effect, but it is unclear as to whether this is due to problems with the experimental set up. In Chapter three I investigate a factor often over looked in the study of crypsis, that of the behavioural adaptations that can enhance its efficiency. The larvae of the early thorn moth (Selenia dentaria) masquerade as twigs, using both colouration and behaviour adaptations. I compared the angle at which the larvae rested, to the angle at which real twigs deviate from the main stem. The results found that the larvae showed variation in their angle of rest and do not appear to match the angle of real twigs on the host tree. This result suggests that perfectly matching the angles of real twigs is not necessary to twig mimicry. While carrying out this experiment it was noticed that a breeze appeared to increase larval activity and induced a ‘swaying’ behaviour. This led me to examine whether mimic species may utilise the visual ‘noise’ produced by windy conditions to camouflage movement. Firstly, a small ‘proof of concept’ pilot was carried out, followed by a larger study using 2 different twig mimic species. The study involved measuring movement and swaying behaviour in 3 conditions (still air, wind setting 1 and 2). The results suggest that cryptic and mimetic lepidopteran species may use windy conditions to camouflage their movements and that some species may employ specialised ‘swaying’ behaviours. Cryptic species are limited in opportunities to move between foraging sites without increasing detection by predators, therefore, any adaptation that might reduce detection is extremely advantageous. In Chapter four I examine how conspicuousness and colouration are affected by living in a group, particularly in relation to other group members. A field experiment using groups of artificial prey, with differing densities and group sizes was used to explore the effect of group size and density on the predation risk and detectibility of cryptic prey. My results show that, as expected, larger groups are more likely to be detected, but that the increase is much slower than a linear increase. This suggests that groups must increase considerably in size before any individual group member will suffer increased predation risk. The second experiment examines the ‘oddity effect’ and how it affects predation. This hypothesises that when confronted by grouped prey, predators can increase their kill rate by concentrating their efforts on capturing unusual or ‘odd’ prey, a strategy that reduces the ‘confusion effect’. A field experiment was conducted with groups composed of differing proportions of two artificial cryptic prey types. Groups with odd individuals did not suffer an increase in conspicuousness and were not attacked more often. However, once located and attacked the groups did suffer a greater predation rate. Odd individuals were predated at a greater rate than normal individuals and the rate did not change as more or less odd individuals were added to the group. A computer based ‘game’ was used to further investigate the oddity effect. The results from the initial run of the game appeared to show strong evidence for the oddity effect, with a further significant increase in this effect when attention is split between foraging for prey and scanning for predators. To be confident of this result the experiment was repeated with the ‘odd’ and ‘normal’ seed patterns reversed. The new data set strongly suggested that much of the effect seen in the previous experiment was due to a difference in pattern visibility between the two seed patterns. Nevertheless, the results indicated that selecting odd seeds is quicker than selecting normal seeds. The results from both the field and computer trials suggest that preference for odd prey may improve predator foraging speed and efficiency. Chapter five investigates whether cryptic and non-defended prey could reduce their predation risk by grouping with aposematic and defended prey. This was tested using artificial prey in a field setting. My results show that undefended non-aposematic prey can benefit by grouping with aposematic prey with no evidence that predation rates for aposematic prey were adversely affected by this association. If confirmed this might illuminate the origins of Batesian mimicry. I have investigated a range of anti-predator adaptations and strategies in the Lepidoptera and in particular pattern elements and use of crypsis and aposematic displays. These anti-predator strategies are important in that they modify predation rate and so directly influence the evolution of species. While I have been able to provide evidence for some current hypotheses, in many respects my results demonstrate that there is still a lot to learn about visual anti-predatory strategies

To see and not be seen

Die vorliegende Arbeit behandelt Tarnung im Tierreich und der Menschheitsgeschichte. Um die Mechanismen zur Täuschung von Feinden und Vermeidung von Entdeckung und Attackierung besser zu verstehen, werden die Eigenschaften von visuellen Systemen unterschiedlicher Spezies und aus der Sicht verschiedener Wahrnehmer genauer betrachtet. Während der Beschäftigung damit, wie sich Feinde und Artgenossen wahrnehmen, wird ein Überblick über einige Testmaterialien gegeben, wie die Manipulation von Farbmustern oder Körperteilen eines Objektes um die Antwort eines Empfängers zu untersuchen, Manipulation des visuellen Hintergrunds, Computersimulationen und auch Szenarien zur visuellen Suche. Bereiche der visuellen Wahrnehmung welche “Camouflage breaking” beeinflussen, beinhalten unter anderem Prinzipien von Figur-Hintergrund-Segmentation, Objekterkennung und Kantenerkennung im menschlichen visuellen System, welche mit tierischen Sinnessystemen verglichen werden. Mechanismen von Tarnung und Täuschungsfärbung aus der Natur wurden auch im humanen Kontext angewendet. Beginnend mit den umfassendenen Gebieten von Kunst, Militär und “dazzle painted“ Schiffen, wird die Verbindung von Camouflage mit der menschlichen Kultur, und neuere Entwicklungen auf dem technologischen Sektor präsentiert. Trotz allen Erkenntnissen ist das Wissen um die genauen Wirkungsmechanismen von Tarnung spärlich. Durch weitere Forschung auf dem Gebiet der Interaktion von visuellen Systemen können diese jedoch genauer verstanden werden.This work focuses on camouflage in the animal kingdom and in human history. In order to gain a deeper knowledge of mechanisms for avoiding detection or attack and for deceiving predators, the properties of visual systems of different species are explored from the mind and eyes of various perceivers. While inspecting how predators and conspecifics see each other, an overview of several testing material is given, such as manipulations of color patterns and body parts of an object to examine the response of a receiver, manipulation of visual backgrounds, computer simulations and also visual search scenarios. Areas of visual perception that influence camouflage breaking include among others principles of target-background segmentation, object recognition and edge detection in the human visual system, which are compared with animal sensory systems. Mechanisms of camouflage and deceptive coloration from nature have been adopted to the human context. Starting with the broad area of art, military and dazzle painted ships, the connection of camouflage with human culture and recent developments on the technological sector is presented. Despite all that insight, knowledge of how camouflage works is spare but by further examing the interactions of visual systems we can understand perception more precisely

Program Abstracts, 99th Session, Iowa Academy of Science, April 24-25, 1987

Presentation abstracts from the annual meeting of the Iowa Academy of Sciencehttps://scholarworks.uni.edu/ias_docs/1037/thumbnail.jp

Human-carnivore conflict: livestock resource selection, predation, and signal-based mitigation

Human-carnivore conflicts occur globally and are a leading cause of carnivore population declines. Such conflicts usually occur when carnivores predate livestock and can include preemptive and retaliatory killing of carnivores by livestock farmers. In northern Botswana, livestock farming is a widespread and culturally important practice. Subsistence farming enterprises commonly abut protected areas, and human-carnivore conflicts are common. Understanding interactions between livestock and carnivores, and how livestock use resources and habitats generally, are important components to managing these conflicts. Throughout this thesis, I explore human-carnivore conflict in northern Botswana. I found that livestock resource selection and predation vary seasonally and spatially in relation to ecological and anthropogenic features in the landscape. Predation sites are subsequently avoided by cattle in the short-term, but not by goats. Contemporary mitigation to minimise livestock predation events commonlyincludes lethal control and broadscale exclusion by artificial barriers and aversive interventions, yet naturally occurring deterrent signals fine-tuned through evolution are rarely considered. Lions roar to deter conspecifics from territorial boundaries, which prey and subordinate carnivores eavesdrop on and modify their movement and behaviour in response. I used lion vocalisations to understand livestock (prey) responses to this apexcarnivore and to test how effective roars are in deterring lions and other carnivores. Using a high-tech experimental approach, I found that (1) cattle avoid lion vocalizations, while goats do not, and (2) lions are not deterred by lion roars played-back from Remotely Operated Acoustic Repellent stations (ROARs), nor are other human-carnivore conflict species occurring in the area. Finally, I used a commonly occurring anti-predator signal in nature,demonstrating that artificial eyespots painted on cattle rumps deter lions from attacking cattle. Collectively, the results from my thesis can be used to better manage livestock in a landscape of risk, and to promote human-carnivore coexistence by deterring predation. Applications derived from my thesis to promote human-carnivore coexistence can be used across Africa and the globe

A quantitative and qualitative approach to cuttlefish (Sepia officinalis) body patterning

Cuttlefish are renowned for their ability to quickly alter the colour and texture of their skin, for camouflage and communication. This is due to the presence of thousands of pigment-filled sacs, known as chromatophores, which are distributed across the skin. The chromatophores are innervated by motoneurons, which dilate the chromatophores to create the spots, stripes, and other markings, known as chromatic components. There are 34 recognized chromatic components, and it is an interesting question how cuttlefish coordinate the expression of these components to camouflage and communicate. The digital age has introduced new, powerful algorithms and methods to tease out subtle features in the coloration patterns, by means of image registration, segmentation, and identification, as well as methods for modeling the underlying control systems. These tools offer major new insights into the mechanisms of visual perception. In addition, powerful techniques have recently been developed that have yet to be applied to this complex visual motor control system. These methods have large potential in helping discover what features between the pattern and the environment are necessary to prevent detection. Here I present four laboratory experiments, that for the first time use machine learning models, to investigate cuttlefish pattern formation, implementation, and information. The first two experimental chapters investigate how cuttlefish orchestrate their chromatic components for camouflage patterns, and what strategies they employ on diverse backgrounds. I demonstrate that components are expressed more independently than previously believed, finding that the range of patterns expressed lie on a continuum, allowing us to suggest a revised classification scheme for cuttlefish body patterns. The diversity of patterns seem to imply that a cuttlefish could use its repertoire flexibly to display the maximally cryptic pattern for a given background, however I show that cuttlefish to not in fact select a single (possibly optimal) camouflage pattern, continually alter their appearance on a given background, and that the frequency of change increases in relation to the size of the objects in the environment. My third chapter investigated the language-like properties of cuttlefish communication using human speech recognition models. From our subset of cuttlefish patterns, I discovered cuttlefish utilize a lexicon of 10 patterns, with language-like properties such as: they obeyed Zipf’s law, contained around 1.6 bits per display, and interestingly, while 2 patterns were visually similar, they were displayed in separate contexts. By implementing a regression onto the patterns, I introduce a basic dictionary of cuttlefish terms and their meaning. From my investigations into cuttlefish intraspecific signaling, I discovered two previously undocumented patterns, used in agonistic encounters between cuttlefish. My final chapter describes these patterns and the contexts they are displayed