Sperm competition theory predicts males should guard their mates with greater intensity when there is a higher risk of losing fertilizations. This relationship was tested in a spider, Nephila clavipes, first by evaluating factors (female age, mating status and duration of copulation with first male) that could affect the potential for sperm competition (likelihood of polyandry and sperm release by mating males) and second by relating this potential to intensity of post copulatory guarding. Female mating status influenced the potential for sperm competition (Study 1), but age did not when rematings were staged within a week after the female's normal two-day mating sequence with the first male (Study 2). Duration of copulation with the first male was an important influence on the potential for sperm competition (Study 3). When second males were presented four days after the female's final molt, longer durations with the first male reduced the likelihood of polyandry but did not influence sperm transfer. When second males were presented to females immediately after initial mating (Study 4), longer first male copulatory periods resulted in a reduced likelihood of polyandry and, when remating occurred, second males copulated for shorter durations, releasing fewer sperm. Hardening of the female reproductive tract, which occurs independent of mating, affected sperm release to only a minor degree (Study 5). Staged encounters revealed that postcopulatory guarding did vary, as predicted, with potential for sperm competition. Guarding intensity varied according to female status (Study 1) and, as expected, age of older, nonvirgin females did not influence intensity of defense (Study 2). When guarding trials were staged immediately after the first male had mated, intensity of defense was greater the shorter the first male's copulatory duration (Study 4). This coincided with the likelihood of polyandry and numbers of sperm released and consequently the potential for sperm competition. The nature of copulation and mate guarding in this species is consistent with sperm competition theory and appears not to result in conflict between the sexes / acase@tulane.edu

I investigated patterns of genetic variation in the North American gall-forming aphid, Pemphigus obesinymphae. In Chapters 2a and 2b, I developed and then implemented clone-specific molecular markers to investigate clonal mixing in P. obesinymphae . During its gall-forming phase, P. obesinymphae clones produce aggressive larval "soldiers", which altruistically defend their colonies from natural enemies. I showed that movement occurs between galls, indicating that P. obesinymphae colonies are not pure clones. I also showed that intruders behave selfishly, by not defending unrelated clones, and by accelerating development into reproductive adults. These results reveal a greater degree of complexity and conflict in aphid social groups than previously known. In Chapter 2c, I surveyed molecular variation in P. obesinymphae and its bacterial endosymbiont, Buchnera aphidicola. I found levels of variation at two Buchnera loci to be similar to those estimated from a previous study on a distantly-related aphid, Uroleucon ambrosiae. In the western US, P. obesinymphae and B. aphidicola were nearly monomorphic, and in the eastern US, estimates of synonymous divergence ranged from 0.08 to 0.16%. Most polymorphisms in sub-populations at low frequencies, indicating a recent purge of ancestral polymorphism. These results emphasize the importance of aphid population biology in shaping evolutionary patterns in B. aphidicola. In Chapter 2d, I explored the role of life cycle variation in speciation between Pemphigus aphids. P. obesinymphae and P. populi-transversus are closely-related and sympatric on the cottonwood, Populus deltoides (Salicaceae), but they have distinctly different life cycles. P. populi-transversus has a sexual stage that occurs in the fall, while P. obesinymphae produces sexuales in late spring. Field evidence indicates that intermediate phenotypes rarely occur, and mitochondrial and bacterial endosymbiont DNA sequences show no maternal gene flow between the two species. I considered the possibility of an initial allopatric phase in the divergence, and discuss the sequence of evolutionary changes that likely led to the sympatric divergence of P. populi-transversus and P. obesinymphae. The most plausible interpretation of available data is that a shift in timing of the life cycle in an ancestral population spurred divergence between the species pair.

Iron is essential for life and iron homeostasis is important for all species. Compared to the understanding of iron metabolisms in vertebrates, we know much less about insect intracellular iron homeostasis. The iron regulatory proteins (IRPs) play central roles in this process by interaction with iron responsive elements (IREs). Here, I report the cloning, sequencing, overexpression, purification and characterization of IRP1s from two insect species, Manduca sexta and Aedes aegypti. Electrophoretic mobility shift assays demonstrated that both IRP1s specifically bind IREs s not only from the same species, but also from human ferritin IRE. Another ferritin subunit also was cloned from Manduca sexta and an IRE was identified in the 5'-untranslated region of the mRNA, and the IRE reacted with Manduca IRP1 specifically. Transcription/translation assays demonstrated that both IRP1s repress ferritin synthesis in vitro, and the repression is IRE dependent. Iron administration to Manduca sexta increased hemolymph ferritin levels and decreased fat body IRP1/IRE binding activities without affecting either the IRP1 mRNA or protein levels. These data indicates that translational control of ferritin synthesis by IRP1/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. The different responses to reducing agent of Manduca sexta and mammalian IRP1s could provide a potential future strategy for designing pesticides in insect control.

The study of variation in arachnid genitalic structures has contributed to the fields of systematics and sexual selection. Simon (1892--1903) in his Histoire Naturelle des Araignees first divided the ecribellate higher spiders into two groups, the Haplogynae and Entelegynae, using reproductive morphology. Spider genitalia have been used as a taxonomic tool for distinguishing between taxa because of their species-specific morphological variation. Variation in spider genitalic morphology has inspired evolutionary biologists to test mechanisms of sexual selection by which the variation could evolve, ranging from Fisherian run-away selection, chase-away selection, and sperm competition. The Tetragnathidae are particularly interesting for a comparison between haplogyne and entelegyne reproductive morphologies. Within this entelegyne family, a reversal to haplogyny has occurred. Fifteen representative members of this family and four outgroup taxa were examined with scanning and transmission electron microscopy in order to describe the fine structure of spermathecae, including the distribution and density of spermathecal gland pores. While the function(s) of the glandular secretion are unknown, the distribution and density of spermathecal gland pores is discussed with regard to possible functions of the glandular secretion. The potential influence of spermathecal gland secretions on mating behavior and sperm competition is considered. Sperm release patterns have been examined in entelegynes, but previously were not available for haplogynes. The relationship between copulation duration and sperm release was determined in Tetragnatha versicolor, the first examination of sperm release patterns in any haplogyne spider. In this species, copulation duration is not proportional to sperm release. To examine the relative influence of spermathecal morphology and numerical sperm competition on paternity, sperm release and paternity was assessed in the entelegyne Nephila clavipes and the haplogyne Tetragnatha versicolor. The data clearly support differential sperm release between males as the cause of previously reported first-male advantage in Nephila clavipes and the mixed paternity found for N. clavipes and Tetragnatha versicolor in this study. The natural history, mating behavior, and sperm release were determined for a previously unstudied tetragnathid species, Glenognatha emertoni . This is the second examination of haplogyne sperm release behavior as well as the first description of an unusual escape behavior.

Although bees are herbivorous insects, they are seldom studied as such. My dissertation research characterizes the interactions between a guild of solitary cactus-specialist bees and the cacti they visit, examining these bees both as herbivores and as pollinators. I first examined the reproductive biology of two species of Ferocactus as background for the pollination study. Both F. cylindraceus and F. wislizeni were obligate outcrossers, and neither species was pollen-limited. Fruit set was high in both species, and reproductive output was limited by architectural constraints. Whereas most other herbivorous insects are antagonists of the plants they feed on, bees are usually regarded as mutualists (pollinators) of their host plants. However, not all floral visitors are pollinators, and pollen-foraging specialists have been postulated by some to be particularly antagonistic to the plants they visit. To test whether cactus-specialist bees are actually mutualists of the cacti they visit, I examined both the quantity and quality components of pollinator effectiveness of bees visiting F. cylindraceus and F. wislizeni. Despite the generalized morphology of the flowers of these plants, there were very few floral visitors other than three species of cactus-specialist bees. Flowers of both species were pollinated almost exclusively by cactus-specialist bees, primarily Diadasia rinconis. I also tested the pollen preferences of four species of cactus bees. Bees were offered flowers from their normal host cactus in which the stamens had been removed and replaced with novel pollens. Novel cactus pollens were accepted to some degree by all four species, but Diadasia rinconis and D. opuntiae accepted more novel non-cactus pollens than did either Lithurge apicalis or Idiomelissodes duplocincta. Both species of Diadasia showed significant acceptance of Sphaeralcea (Malvaceae) pollen, thought to be the ancestral host plant for the genus Diadasia. Caged, naive D. rinconis bees did not initiate nesting when presented with Sphaeralcea flowers alone; nesting began immediately when cactus flowers were offered. Once nesting was underway, most D. rinconis bees switched to Sphaeralcea if cactus flowers were removed. In both D. rinconis and D. opuntiae, there were striking differences in pollen preferences among individuals.

Food-for-protection mutualisms, interactions between two species in which one species provides protection from aspects of the biotic environment in exchange for a nutritional reward, show an exceptional degree of context dependency. The occurrence, strength, and outcome of these interactions often depend on the ecological context in which they take place. However, the causes and consequences of such context-dependent variation remain poorly understood. The protection mutualism involving lycaenid butterflies and ants provides an opportunity to explore many aspects of the ecology and evolution of interspecific interactions including the importance and predictability of ecological factors that produce context-dependent investment or outcomes in interspecific interactions. Ant-tended lycaenid larvae produce carbohydrate-rich secretions that are collected by attendant ants. In exchange for this food reward, ants may confer developmental benefits and protect larvae from predators and parasitoids. Both participants in this mutualism are capable of responding to changing ecological conditions and, thus, can quickly alter their level of investment or decision to participate in the interaction. In Appendix A, I present the results of field work that illustrate that ant tending provides the lycaenid butterfly, Hemiargus isola, with effective protection from parasitoid attack and enhanced larval survival. Lycaenids on plants from which ants were excluded were almost twice as likely to be parasitized as were lycaenids feeding on plants to which ants had access. In Appendix B, I present the results of laboratory experiments that show that the tentacular organ signal employed by H. isola is a generalized signal, conveyed by either a simple, tactile stimulus or a secretion of low volatility, which evokes an alarm response in attendant ants. Furthermore, I provide evidence to suggest that the function of the tentacular organ signal is context dependent and mediates lycaenid investment in the mutualism. In Appendix C, we present the results of laboratory experiments that demonstrate that altering the ratio of carbohydrate and protein resources available to ants influences their decision to participate in the mutualism with H. isola. Significantly more ants from colonies fed a low carbohydrate/high protein diet tended lycaenids relative to ants fed a high carbohydrate/low protein or high carbohydrate/high protein diet.

Symbiosis is a prevalent phenomenon among organisms throughout the tree of life, including the insects which often harbor maternally transmitted bacteria. Aphids engage in symbiotic interactions with several maternally transmitted bacteria, and many are known to associate with microbes known as secondary symbionts. These bacteria are typically not essential from the aphid's perspective, and until recently little was known about their roles in aphid biology or the coevolutionary histories of these symbioses. I have addressed these mysteries in this dissertation, through use of molecular and experimental analyses. My findings reveal that secondary symbionts are diverse and infect members of numerous aphid and insect taxa. Though they are maternally transmitted, their distributions can be attributed to occasional horizontal transmission among species. Consequences of symbiosis were observed at genomic levels, with "T-" and "U-type" symbionts exhibiting accelerated evolution in their 16S rRNA sequences. The "R-type" symbiont, in contrast, has not experienced accelerated substitution rates, though it does show a recent trend toward increased AT content, as observed for other symbiotic bacteria. Molecular and phylogenetic evidence presented in this dissertation suggest that secondary symbionts are generalists, capable of infecting numerous aphid hosts. Here, I also present experimental evidence in support of this hypothesis, demonstrating that two of three horizontally transferred symbionts are passed on maternally, at high efficiency, in a novel host, Acyrthosiphon pisum. However, not all efficiently transmitted symbionts would be expected to persist in populations of A. pisum, as some reduce aphid fitness. Finally, evidence obtained from my research and previous experimental and theoretical studies suggests that secondary symbionts should improve aphid fitness, though benefits may not accrue in all environments. Here, I examine the effects of temperature on the fitness effects induced by R-, T-, and U-type symibionts, finding that the R- and T-types confer benefits in aphids exposed to high temperatures, compared to slight and even non-existent effects on A. pisum reared under permissive temperatures. The U-type reduced fitness of aphids reared under high temperatures, revealing a potential cost to symbiont infection that could help to explain intermediate infection frequencies.

I utilize a variety of methods, from field studies of selection to phylogenetic systematics, to study the historical processes involved in the evolution of reproductive structures of milkweeds (Asclepias L. (Asclepiadaceae)). In an experimental study of a natural population of Asclepias tuberosa L., I explore the mechanisms by which pollinators select on the arrangement of flowers on a plant. I present an evolutionary model for the manner in which pollinators select on inflorescence design (the number of flowers in inflorescence units), considering reproductive success gained through both male and female function. In this model, the maximum fitness is achieved when proportional changes in male and female reproductive success with changes in inflorescence-unit size are balanced. The model is used to predict optimal inflorescence-unit size in Asclepias tuberosa, which roughly match inflorescence-unit sizes observed in nature. In studies of selection on plant reproductive characters in natural populations, the pollinators responsible for selection remain unidentified or are inferred from floral morphology. In the same experimental population of Asclepias tuberosa in which I studied selection on inflorescence design, I measured the effectiveness of all common pollinators. Although A. tuberosa possesses characteristics of butterfly-pollinated plants, and many butterfly species are found at its flowers, the most effective pollinators are bees. Furthermore, the most effective pollinators in the study population varied between years. These results suggest that inferences about the identity of effective pollinators should be based on careful measurements of effectiveness over several seasons. In order to study factors that affect the evolution of reproductive characters like inflorescence design in this large genus, it is desirable to employ a phylogenetic hypothesis of the relationships among Asclepias species. I present such an analysis based on morphological data. My results suggest that some of the North American species of Asclepias are more closely related to African species in other genera than to other North American species of Asclepias. Also, most infrageneric groups currently recognized are not monophyletic. Several novel infrageneric groupings are well-supported by my results. I also present analyses of the evolution of growth form and flower "horns" based on an explicit phylogenetic hypothesis.

In dipteran brains, a motion sensitive but color insensitive, pathway consists of large diameter neurons, organized as a precise retinotopic map. Several cell classes can be uniquely identified by their shapes, projections and relative positions in the neuropil. Morphological comparisons of small-field neurons in the second visual neuropil, the medulla, in 16 dipteran taxa reveal that those neurons that are involved in elementary motion computation are conserved in aspects that are expected to contribute to the functional pathway, but vary moderately in shape and cell decorations. On the other hand large-field neurons of the third visual neuropil, the lobula plate (LP), vary considerably in their numbers, shapes and positions within the mosaic. Those are neurons that integrate information from arrays of EMDs in a taxon-specific way. Because of the map-like organization of this neuropil, differences in cell size, architecture and cell number are of major functional consequence. Character evolution studies suggest that specific LP organizations are indeed tightly connected with specific functional properties; namely, differences that influence flight behavior. For example, the comparison of isomorphic vertical cells against hovering flight reveals multiple parallel origins of these two character traits. Other characters are closely associated with phylogenetic hypothesis, and no evolutionarily associated functional characteristics have been found. A parsimony analysis based on 32 neuroanatomical characters shows close similarities with conventional literature-derived hypotheses, suggesting the systematic value of neuroanatomical characters.

Relationships within Simuliidae, or black flies, are inferred from molecular sequences from four nuclear loci -the large ribosomal subunit (28S rDNA), elongation factor one alpha (EF-1α), dopa decarboxylase (DDC), and phosphoenolpyruvate carboxykinase (PEPCK) - and two mitochondrial loci - the small ribosomal subunit (12S rDNA) and subunit two of NADH dehydrogenase (ND2). Analyses of all genes provide varying degrees of support for subfamily and tribal limits within Simuliidae that are completely concordant with those inferred from the most intensive analysis of morphological data. Additionally, 28S and PEPCK suggest thaumaleids are more closely related to simuliids than are chironomids. Relationships within Simuliidae below the level of tribe were inferred from independent and simultaneous analyses of these data. Phylogenetic trees thought to represent best estimates of simuliid relationships were used to trace the evolution of several morphological characters and of natural partitions of these sequences, particularly codon positions in the four protein coding genes. The rather poor performance of these genes at intermediate taxonomic depths within Simuliidae is hypothesized to be the result of explosive diversification, properties of these genes, or a combination of the two. Potential hypotheses for the rather poor performance of these genes are proposed, and two types of genes hypothesized to be more informative in cases of explosive radiation are described.