Thesis (M.Sc. (Zoology & Entomology)) - Rhodes University, 2008.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity. / Medicine, Faculty of / Graduate

There are three social dimensions within a family: parent-parent interactions, parent-offspring interactions, and offspring-offspring interactions. All of these interactions are subject to evolutionary conflict, which occurs whenever interacting individuals have divergent evolutionary interests. Family interactions and family conflict are often influenced by phenotypic and genotypic traits of the parents and the offspring. An important phenotypic trait is body size, which can affect fecundity, mating success, and fighting ability. An important genotypic trait is inbreeding status (i.e., whether an individual is outbred or inbred), which can influence its overall quality or condition. In this thesis, I investigate the independent and interactive effects of inbreeding and parental body size on family interactions in the burying beetle Nicrophorus vespilloides. I first show that the body size of the two parents influences the resolution of sexual conflict over the amount of parental care (Chapter 2) and over the consumption of a shared resource (Chapter 3). Here, the shared resource refers to the carcass from which both the parents and the offspring feed over the course of the breeding attempt. I then show that females that won or lost a fighting contest provide more care to their offspring compared to beetles with no fighting experience (Chapter 4). This indicates that female burying beetles make parental investment decisions based on their experience with a contest (which is independent of body size) rather than the outcome of that contest (which is dependent on body size):. In the second half of my thesis, I examine whether family interactions also influence and are influenced by inbreeding depression (Chapters 5–8). I find that a female's mating preference for an outbred versus an inbred male is conditional on her own inbreeding status: inbred females preferentially mate with outbred males, whereas outbred females are equally likely to mate with an outbred or an inbred male (Chapter 5). Even though sibling competition does not appear to have an effect on the offspring's inbreeding depression (Chapter 6), the presence of the mother during larval development can reduce the severity of inbreeding depression (Chapter 7), and this effect depends on the mother's body size (Chapter 8). In Chapter 9, I discuss the broader implications of these findings for evolutionary biology, ecology, and conservation biology.

Inbreeding is associated with reduced fitness, a phenomenon known as inbreeding depression. I investigated direct and indirect effects of inbreeding on social traits associated with parental care in the burying beetle, Nicrophorus vespilloides. This species breeds on small vertebrate carcasses and the parents provide care by maintaining the carcass and regurgitating food to begging larvae. I quantified the survival of outbred offspring produced by inbred and outbred parents. I found that inbred offspring had reduced survival compared to outbred offspring, and that outbred offspring produced by inbred parents survived less well. Such intergenerational effects of inbreeding suggests that inbreeding may affect the amount of parental care provided to offspring. I tested this by investigating the amount of care inbred and outbred male and female parents provided to outbred offspring. I found no reductions in the amount of care provided by inbred parents but found that parents provided more care when their partner was inbred. In addition, I investigated effects of inbreeding on parent-offspring communication, when either female parents or their offspring were inbred. I found that whilst inbred offspring begged less, parents provided inbred offspring with more care. The effects of inbreeding had significant consequences affecting biparental negotiation and parent-offspring communication. Next, I tested for the effects of inbreeding on the antimicrobial properties of secretions that both parents apply to the carcass during larval development. I found that the bactericidal activity of inbred male parents was reduced compared to outbred male parents during the dispersal stages and no evidence for the secretions of inbred and outbred female parents differing. Finally, to test whether the strong inbreeding depression found in this species influenced the mating decisions, I presented females with related or unrelated males, and found no evidence that females avoided inbreeding. These results show that to accurately estimate the fitness consequences of inbreeding the social effects on all individuals within a family must be accounted for.

Parent-offspring communication is widely regarded as having evolved to provide the parent with honest information about the hunger state of its offspring, thus enabling it to mediate conflict over resource allocation between parents and offspring. The conflict is caused by the offspring benefitting from receiving more care than the parents are selected to provide due to the costliness of care. I studied the role of parent-offspring communication as a mediator for the conflict in the burying beetle Nicrophorus vespilloides. The burying beetle is an excellent study system for this question, as the larvae, that are raised on carcasses of small vertebrates and cared for by both the male and the female beetle, beg for food from their parents with highly distinguishable begging displays. First, I examined whether offspring adjusted their begging to different classes, or individual adult beetles. I found that while the larvae did not discriminate between male and female beetles, they adjusted their care to cues indicating individual recognition of adults. Second, I tested whether begging was based on offspring size at egg stage, and found no indication that offspring adjusted their begging to improve their innate quality. Third, I examined whether parental response to begging exhibits behavioural plasticity when the internal clock for the timing of reproduction for the parent, and the demand from the larvae do not meet. I found that the parents adjusted their care based on the amount of begging exhibited by the larvae. Fourth, I investigated whether parental adjustment of care based on offspring begging incurs a reproductive cost to them. I found that the females paid a cost in fecundity, but not in the number of dispersing larvae or their own survival. My original contribution to knowledge is therefore to show through these four studies, that offspring begging is adjusted based on parental cues, and can directly affect proximate parental behaviours, and also incurs a reproductive cost to their future reproductive success, thus providing more experimental evidence for the importance of parent-offspring communication, and its implications to the evolution of parental care.

In this thesis, I investigated the role of parental care in evolution. Parents provide the environment in which offspring develop and therefore have a large influence on their offspring's phenotypes, and so are in prime position to influence evolutionary processes. I used an experimental approach, and focused on the burying beetle, Nicrophorus vespilloides. The burying beetle is a perfect system for this question: they exhibit elaborate biparental care which is correlated with rapid speciation in the Nicrophorus genus. I started with a thorough exploration of burying beetle ecology and how the guild structure and interspecific competition in local populations can shape phenotypic evolution of my focal species, N. vespilloides. Interspecific competition shapes how the carrion niche is partitioned, which feeds back onto the evolution of body size within Nicrophorus reducing competition. The evolution of parental care in this genus likely facilitated its adaptive radiation, as parental care is linked with body size, both within and across species. But to what extent does the ecology shape the production and maintenance of phenotypic and genetic variation? I then use a quantitative genetic approach to show that body size and development time of N. vespilloides shows no additive genetic variation. Evolution of these fitness related traits can only occur through maternal effects or sibling effects. I tested this prediction by mimicking the radiation of the burying beetles by imposing my own selection on body size when parents could care for their offspring and when they could not. The presence of post-hatching parental care dramatically changed how populations responded to selection, through a combination of cooperation between parents and offspring, and cooperation between offspring. As well as shaping the evolutionary potential of populations, an experimental change in parental care can induce new selective forces, favouring adaptive novelties for the new social environment. Larvae evolving without parental care evolved disproportionately larger mandibles when small to better adapt them to a life without care. Much is known about the evolution of parental care across the animal kingdom, but what happens next: are the burying beetles a "one-off"? I compiled data across the arthropods comparing clades that exhibit post-hatching parental care with their sister clades and show that clades with care are more species rich. While the mechanism may not be the same as with Nicrophorus, I discussed other potential mechanisms that may be at play in the role of parents in evolution.

The cost of reproduction hypothesis suggests that allocation into current reproduction constrains future reproduction. How organisms accrue reproductive costs may differ between species and with varying levels of resource quality. Burying beetles are model organisms for testing for the cost of reproduction because of their unique natural history; beetles utilize small vertebrate carcasses for reproduction and they and their offspring feed exclusively on these discrete resources. Burying beetles also can utilize a large range of carcass sizes for reproduction. We tested for the cost of reproduction in two species of burying beetles, Nicrophorus marginatus and Nicrophorus guttula found in Central Utah by breeding beetles on a range of carcass sizes (5g, 10g, 20g, 30g, 40g, and 50g carcasses). We also used a manipulation experiment to force beetles into over-allocating energy into reproduction to assess reproductive costs. For both species, reproduction was costly, with beetles suffering reduced lifespan and reduced lifetime fecundity with increased resource quality. Both species also showed clear signs of senescence, having reduced brood size and lower efficiency as individuals aged. Females did not show indications of terminal investment in terms of female mass change, unlike the previously studied Nicrophorus orbicollis, which gained less mass after each reproductive attempt as it aged. Nicrophorus marginatus consistently outperformed N. guttula in terms of total number of offspring produced for all carcass sizes. Nicrophorus guttula populations may continue to persist with N. marginatus by exploiting a less desirable but more abundant resource.

Body size is one of the most commonly studied traits of an organism, which is largely due to its direct correlation with fitness, life history strategy, and physiology of the organism. Patterns of body size distribution are also often studied. The distribution of body size within species is looked at for suggestions of differential mating strategies or niche variation among ontogenetic development. Patterns are also examined among species to determine the effects of competition, environmental factors, and phylogenetic inertia. Finally, the distribution of body size across the geographic range of a species or group of closely related is looked at for indications of the effects of climate and resource availability on body size at different latitudes and altitudes. In this collection of research, I address the evolution and importance of body size in burying beetles (genus Nicrophorus). Body size is important to several aspects of burying beetle natural history, including competitive ability, fitness, parental care, climate tolerance, and locomotor activity. In Chapter 1, I use a large data set of body size measurements for seventy of the seventy-three Nicrophorus species to make inferences about the distribution of body size within the genus, across its geographic range, and the importance of body size in speciation. I found that the range of body sizes is not normally distributed, with an overrepresentation of small-sized species. I also found that expansion of the burying beetle range has been restricted by their inability to tolerate warm, dry climates, and therefore the majority of burying beetle diversity occurs in the temperature mid-latitudes of the northern hemisphere. Body size also seems to be important in speciation, as almost all sister taxa are significantly different in body size. In Chapter 2 I use common garden experiments to assess the importance of body size for males and females in competition, reproductive output, and starvation resistance. Body size is equally important for both sexes in starvation resistance, but it is more important for males in competitions for carcasses and for females in reproductive output. In Chapter 3 I test for fitness consequences of multigenerational effects of body size in offspring. I found that the larger offspring that are produced by larger mothers and on larger carcasses had higher fitness than small offspring. In Chapter 4 I test for the possibility of brood parasitism in two species of burying beetles, N. guttula and N. marginatus, which co-occur over part of their geographic ranges. I found that both species are able to detect and remove parasitic larvae. Finally, in Chapter 5 I compiled parent and offspring body sizes from seven species of burying beetles and use them to compare the heritability of body size among species using comparative techniques and a meta-analysis. I found that body size heritability is different between species, but is low for the genus as a whole. Together, these projects provide valuable information on the evolutionary significance of body size in Nicrophorus, and indicate compelling questions for future research into the evolution of body size in burying beetles.