Scott Pitnick - US grants

A research group at Arizona State University proposes to purchase an element/isotope analysis system for use in environmental biology. Environmental biology is a critical area for development because of a rapidly expanding population and associated environmental stress in the Southwest. The proposed instrumentation will support studies of subjects such as: stream- riparian and lake-ecosystems, focusing on nutrient cycling and transformation; comparative physiology research on insect nitrogen excretion; and host nutritional quality and reproductive success in natural populations of cactophilic flies. The University will support this endeavor by providing a technician dedicated to the laboratory, space and necessary improvements in the space, maintenance costs, and cost-sharing on capital purchases. The projects are all characterized by substantial involvement of student researchers (graduates and undergraduate). This instrumentation will allow a significant improvement in training of these students.

Developmental instability (DI) is the failure of an organism to develop normally, in ways ranging from physical anomalies and disease to abnormal behavior. The causes of DI include genetic and environmental factors and their interactions. In man, DIs include cleft lip, congenital heart disease, schizophrenia and chromosomal abnormalities as well as exposure to teratogens such as alcohol and isoretinoin. However, despite extensive theoretical explanations, it is not known how developmental homeostasis breaks down to produce abnormal phenotypes. This award supports a workshop titled "Developmental Instability: Its Origins and Evolutionary Significance". This conference will discuss ways to measure developmental instability, genetic and developmental origins of instability, human health and phenotypic variation, and DI's relevance to population biology and evolution in general. The coonference breaks new ground and has a strong interdisciplinary approach, joining the fields of evolutionary and developmental biology. %%% DI has far-reaching implications for population biology, environmental biology and human health. DI is reported to increase in populations of organisms whose habitats are polluted or otherwise altered, as well as in those experiencing bottlenecks and subsequent inbreeding. Despite this lack of knowledge, DI is already employed as the basis for decisions regarding whether species are endangered or humans are at risk.

Males of a number of species produce more than one type of sperm, some types contain and some lack DNA. Males of Drosophila obscura produce sperm of various discrete lengths, a phenomenon known as polymegaly. While all types contain DNA, the potential biological benefit of producing different lengths is unknown. In fact, the evolutionary significance of this polymorphism is not understood in any species. The present study tests the biological and evolutionary significance of polymegaly in some members of the Drosophila obscura group. In addition to a better understanding of basic reproductive biology and evolutionary theory, this research may assist in the development of biological control programs. One current methodology for "natural" insect pest control is to release many sterile males in the hopes that females will mate with these males rather than fertile males currently in the population. Since a number of insect species exhibit sperm polymorphism, this research may provide information useful in pest management programs.

9402161 Pitnick This U.S.-Mexico award will support Prof. Scott Pitnick of Arizona State University in a research collaboration with Prof. Edward Pfeiler of the Instituto Tecnologico y de Estudios Superiores de Monterrey, Guaymas campus. The investigators intend to use allozyme variability of four cactophilic Drosophila species, endemic to the Sonoran Desert, to characterize the genetic structure of all four species and to compare the genetic structure of each species at different seasons in the context of resource availability. They will also examine the relationship between genotype and differential tolerance to thermal extremes. The four Drosophila species endemic to the Sonoran Desert have been an important model for studies of ecological genetics. Their usefulness, however, has been limited by the lack of knowledge of the genetic structure of these populations and how genetic structure varies with resource availability and with season. The proposed work, through complementary use of facilities and expertise, will increase our understanding of the field. ***

Pitnick 9806649 Among fruitfly species, the length of sperm and of the female's sperm-storage organs have evolved very rapidly, with some species producing relatively few, gigantic sperm, as much as 20 times their body length. Such unusual sex cells challenge our understanding of the fundamental nature of sex differences and of the evolution of reproductive strategy. Two goals of the proposed work are to determine (1) the evolutionary relationship between these male and female traits and (2) the functional significance of variation in sperm and female sperm-storage organ length. To this end, populations of Drosophila melanogaster will be artificially selected for longer and shorter sperm-storage organs. Comparison between males within the selection lines and in "associated male" lines will permit discrimination of the roles of pleiotropy and sexual selection in generating a correlated response in sperm length to female sperm-storage organ length evolution. Productivity and sperm competition experiments will quantify the relationships between sperm length, female reproductive tract morphology, and patterns of sperm use by females. A third goal of the proposed work is to determine the extent to which divergence in sperm length and female reproductive tract morphology contributes to reproductive failure among populations/species. Factors that prevent interbreeding among populations, or "reproductive isolating mechanisms," are critical to the speciation process. Reciprocal crosses among the selected lines will determine the extent to which divergence in sperm and female reproductive morphology can lead to reproductive isolation among populations. Experiments determining the fate of sperm in hybrid matings among five closely related species will extend this analysis of reproductive isolating mechanisms. Number of sperm transferred, eggs fertilized, and eggs hatching per mating will be determined and used to quantify levels of sperm/female and sperm/egg compatibility for all possible hybrid a nd control crosses. Results from these studies will enhance our presently limited understanding of postmating, prezygotic and postzygotic, extragenomic reproductive isolating mechanisms and broaden our perspective on Drosophila speciation.

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Pitnick

The crucial event for the origin of new species is the formation of reproductive isolation. How one species splits into two reproductively isolated groups of organisms, however, is poorly understood. Fruitflies of the genus Drosophila have been a model system for studies of speciation. These studies have focused on premating or postzygotic isolating mechanisms, or those processes that determine whether or not males and females mate and whether or not they produce viable and fertile offspring, respectively. The multitude of events occurring between insemination and egg fertilization has been largely ignored in studies of speciation. Recent work, however, indicates that sperm, seminal fluid, and female reproductive tract characteristics that are of central importance for successful reproduction are rapidly divergent.

The investigators postulate that this divergence will cause diminished reproductive success whenever members of genetically differentiated populations interbreed, and they propose a series of experiments to explicitly test this hypothesis using D. arizonae and its sibling species D. mojavensis, both endemic to North America. Because D. mojavensis from different geographic regions are considered to be incipient species, the investigators will take advantage of a naturally occurring continuum of reproductive isolation for testing their predictions. Preliminary data indicate that populations of D. mojavensis differ significantly in the traits to be measured. By conducting laboratory crosses between members of different populations and species with known reproductive characteristics, the investigators will determine how these characteristics lead to reproductive barriers central to the formation of new species. Results of these experiments may have important implications for the formation of new species.

Sperm-female coevolution: mechanisms, models and theoretical implications.

Recent work has demonstrated that the shapes of sperm and of the female
reproductive tract evolve in concert. This pattern is attributable to a
process known as female "sperm choice," with female reproductive
tract characteristics producing a bias in patterns of paternity when
females mate with multiple males, such as biasing fertilization in favor of
longer sperm. Research proposed here will investigate (1) physiological
mechanisms of female sperm choice, (2) possible benefits to
females of having the ability to "discriminate" among sperm variants, (3)
costs and benefits to males of variation in traits that contribute to
fertilization success, and (4) implications of producing few,
large sperm for sex difference theory.

Disparity between the sexes in the size of their sex cells is believed to
be the cornerstone of all sex differences (e.g., body size, aggressiveness,
ornamentation). However, contrary to popular belief, males of all species
do not produce many tiny sperm. In fact, sperm cells are the most diverse
cell type: in nearly all animal groups, they evolve in size, shape, and
structure so rapidly that even closely related species can usually be
discriminated by their sperm. Because having the correct species-specific
sperm structure is pivotal for successful reproduction, sperm divergence
may function as an "engine of speciation." Understanding the causes of
sperm evolution is thus important to our understanding of the biological
basis of sex differences and of the process of speciation.

Because of the small size of sperm and the technical challenges of observing events taking place inside the complex reproductive tract of females, there are numerous, fundamental aspects of reproduction that remain poorly understood in internally fertilizing species, including humans. This project is a detailed investigation of genetic, physiological and behavioral aspects of events taking place between insemination and fertilization in a model system: the fruit fly Drosophila melanogaster and closely related species. For the first time, these events will be directly observed in real time using unique transgenic fly strains producing sperm with heads that express green (GFP) or red fluorescent protein (RFP). Experiments will explore mechanisms of sperm migration, sperm storage, egg fertilization, the mechanisms by which sperm from different males compete to fertilize the eggs of twice-mated females, and mechanisms of ejaculate-female incompatibility contributing to reproductive failure in hybrid matings between closely related species.

The proposed research has the potential to generate watershed advances in the fields of reproductive physiology and genetics, sexual selection and speciation. Resulting progress in our understanding of sperm behavior within females and the identification of candidate genes contributing to sperm-female incompatibility are further likely to lead to advances in our understanding and treatment of human infertility.

Sperm have tremendous diversity, but the evolutionary significance of variation in sperm form is poorly understood. One of the more unusual kinds of variation is conjugation, where two or more sperm join at the head to form a single functional unit. Conjugation is commonly interpreted as an adaptation to sperm competition and has independently arisen in groups as diverse as opossums, snails, and diving beetles. This study examines the evolutionary pathways and fitness consequences of sperm conjugation. The researchers will use behavioral observations, biochemistry, and cytology to (i) compare the intensity of sperm competition in species with and without sperm conjugation, (ii) test the hypothesis that conjugation provides protection from spermicidal environments, and (iii) quantify the effects of sperm length and conjugate size on motility, an important correlate of fertilization success.

The intellectual merit of this research program includes its potential to reveal the relationship between sperm form and function and to improve understanding of the selective environment of sperm, a greatly neglected aspect of sexual selection. This project will provide training for a PhD student and valuable laboratory and field-based research opportunities for undergraduates. Resultant data will be submitted to public databases to facilitate research, and specimens collected during the course of the project will be deposited in museum collections (e.g. NMNH).

Spermatozoa are the most diverse cell type, exhibiting rapid and dramatic evolutionary changes in form. However, the genetics and adaptive significance of sperm form are poorly understood. Investigations are proposed (1) to examine the relationship between sperm length and motility/behavior within the female reproductive tract, and (2) to identify genes of major influence on sperm length. These studies will be conducted with populations of the fruit fly Drosophila melanogaster that have been experimentally evolved to have unusually short or long sperm, and several closely related species differing substantially in sperm length. All populations have been genetically engineered to produce sperm whose heads glow green or red under fluorescent light. This material will enable, in the first investigation, unambiguous discrimination among sperm from different males within twice-mated females, as well as direct observation in vivo of real-time sperm motility and sperm-sperm and sperm-female interactions. The second investigation will use breakthrough gene sequencing technology (i.e., restriction-site associated DNA mapping) to identify genes associated with sperm length variation.

The proposed research has the potential to generate watershed advances in the fields of reproductive physiology and genetics, sexual selection and speciation. Many undergraduate and graduate students will be trained in the course of this work. Resulting progress in our understanding of sperm behavior within females and the identification of candidate genes contributing to sperm form are also likely to lead to advances in our understanding and treatment of human infertility and in the further development of assisted reproductive technologies used in the preservation of threatened and endangered species.

Name a species in nature, and chances are, its females use sperm from more than one male, resulting in multiple paternity of offspring. This sperm competition generates strong selection that drives rapid evolution of both male and female reproductive traits and may contribute to the maintenance of species boundaries. Despite its evolutionary importance, sperm competition has been difficult to study within its natural environment, the female reproductive tract, because sperm from different males are generally indistinguishable, and the female reproductive tract is often poorly suited for microscopy. The proposed research exploits the experimental and genetic tractability of the fruit fly, Drosophila melanogaster, to generate 200 genetically differentiated populations whose sperm heads express a green or red fluorescent marker. Thus, the research will investigate how sperm variation contributes to sperm competitive success, how sperm traits genetically vary and covary, and ask how the female's genome contributes to her sons' sperm traits. The research will also quantify male×male×female genetic interactions and examine how rearing environment impacts sperm traits.

The proposed research will greatly advance understanding of the complex genetics of sperm traits and of sperm-female interactions underlying variation in reproductive success. Human infertility diagnoses of unknown cause may be a consequence of interactions between partners, but distinguishing potential sources of incompatibility necessitates a detailed understanding of reproductive processes and the genetics of reproductive success. Also, fluorescent-sperm populations generated for this research are proving valuable in laboratories worldwide studying genetics, development and fertilization and in educating the public about reproductive biology.

Male gametes are the most morphologically diverse cells in the animal kingdom, ranging from very simple cells to sperm with missing or multiple flagella and conjugated sperm that form cooperative units. To understand how this diversity evolved, it is necessary to investigate gamete morphology from an evolutionary developmental perspective, an approach widely applied to questions of embryonic development in whole organisms but that has never been applied to cellular morphogenesis. The proposed research will exploit the uniquely long spermatozoa in fruit flies of the genus Drosophila (up to 5.8 cm) to further investigate previously identified candidate genes for sperm length. Drosophila also present an especially elegant system due to their experimental and genetic tractability. These genes will be validated as potentially important in spermatogenesis, and their molecular evolution across the Drosophila lineage will be investigated. The research will also explore how these genes function during spermatogenesis using in vitro culture methods that allow cell development to be observed and manipulated in the controlled environment of a culture dish.

The proposed research will greatly inform the scientific community's understanding of how gamete diversity has evolved at the developmental, molecular and cellular levels. Sperm are functionally important evolutionarily (affecting reproductive fitness and speciation), as well as biomedically and economically (informing fertility and in vitro fertilization practices in humans and domestic animals). Studies of the genetics of sperm form may also improve our understanding of general flagellar and ciliary development, including ciliopathies involved in human disease (e.g., cystic fibrosis, polycystic kidney disease).

Traits that influence reproductive success can be both remarkably elaborate and differ greatly between related species. Variation in such traits not only influences an individual?s success in populations, but can also help form new species when groups differ in characteristics important to choosing a mate. Measuring reproductive success becomes more complex when animals mate with multiple individuals within a single breeding cycle, a common occurrence. Reproductive success, a major influence on trait evolution, is then shaped not only by success in achieving matings, but also by competitively fertilizing eggs, and producing high quality offspring. Using lines of flour beetles whose males? produce sperm with red or green fluorescent tagged heads, this research will evaluate the outcome of these different selective episodes and identify their contribution to reproductive success. The use of the fluorescent sperm lines also enables identification of mechanisms underlying differences in competitive fertilization success within populations, as well as differences in fertilization capability in hybrid versus same species matings. Finally, this research will explore how repeated matings influence offspring production in this highly promiscuous species.

Because flour beetles are economically significant stored grain pests, insight to their reproductive system can inform management efforts. Additionally, the project will include involvement of multiple undergraduate researchers and outreach to the Syracuse City School District.

The process of evolution is expected to favor the single most successful behavioral tactic in a population and eliminate less successful options. Nevertheless, in some species, males adopt different behavioral tactics for acquiring mates, and it is unclear how males decide which tactic to adopt or what the reproductive consequences of this decision might be. In the yellow dung fly, some males compete with one another over access to females at breeding sites, whereas others search for feeding females in non-competitive environments. This study aims to understand the environmental and genetic factors that influence the choice of reproductive tactic in yellow dung fly males and how these alternative tactics are maintained within the same species. These results will help explain the wide diversity of animal traits observed within species throughout nature. Additionally, opportunities will be provided for high school and undergraduate students interested in biology to gain hands-on research experience by participating in data collection and analysis associated with this work.

The co-occurrence of status-dependent alternative mating tactics is an example of extreme phenotypic variation persisting within a population. In addition to differences in mate securing behaviors and morphology, males of alternative tactics are also expected to differ in reproductive investment strategies due to tactic-specific variation in the degree of gametic competition that males face. The appropriate genetic model for understanding the evolution, maintenance, and expression of such phenotypic variation has long been the subject of intense debate. The "environmentally-cued threshold" model has recently emerged as a powerful quantitative genetics-based approach for investigating these long-standing questions. However, the difficulty of accurately quantifying lifetime fitness associated with each tactic has restricted its use in empirical studies. This research will overcome these obstacles by using large, experimental enclosures that simulate field conditions, as well as molecular analyses for paternity assignment, to assess lifespan and reproductive success associated with the size-dependent expression of alternative mating tactics in the yellow dung fly, Scathophaga stercoraria, which is a model system for behavioral ecology. All behavioral, morphological, and genetic data obtained from this research will be deposited into a public repository such as Dryad.

This work will contribute to our understanding of factors affecting fertility and how differences in the interactions between sperm and the reproductive tracts contribute to the reproductive isolation of species. After leaving the testes, sperm undergo numerous changes as they move through the male and female reproductive tracts. These changes to the sperm cells, how they vary among species, and their importance for fertilization success are not well understood. This project will characterize these changes, at the molecular level, within and among closely-related species of fruit fly to understand their evolutionary history, the cause of specific changes, and the effects for sperm survival and fertilization success. This project will also result in the development of a "Genes, Genomes, and Society" (GGS) educational program to introduce high-school students to the field of genomics. It will sponsor a genomics workshop for New York High School teachers to encourage adoption of the GGS program. Finally, some high school students and teachers will also participate in laboratory research.

Using whole-cell proteomic analyses, sperm will be tracked, beginning with storage in the male seminal vesicle, through ejaculation and following protracted storage in the primary sperm-storage organ of females. Identical analyses will be conducted using two sibling Drosophila species (D. simulans and D. mauritiana) and for reciprocal hybrid matings between the species, where evolved ejaculate-female compatibility has potentially been compromised. Proteomic analyses will be similarly conducted on female reproductive tract secretions. The resulting datasets will provide, for closely related species, an exhaustive library of the sperm proteome throughout the life history of sperm, which will permit all alterations to sperm (protein gain, loss and modification) to be discerned. With regard to proteins added to sperm during storage, reciprocal sex-specific labeling will distinguish male- from female-contributed proteins. Molecular evolutionary and genomic analyses of proteins involved in sperm-female interactions, together with the data from hybrid inseminations, will address questions about rates of divergence, genetic architecture and the putative role of sperm-female interactions in speciation.

It is increasingly easy to sequence the genomes of species. The utility of genomic resources, however, is limited without knowledge of the complex developmental, physiological, morphological and behavioral traits that genes code for. Indeed, improving the ability to predict important traits (e.g., lifespan, disease) from knowledge of genotypes is one of the top priorities of modern biology. Much research uses the fruit fly, Drosophila melanogaster, to map phenotypes onto genotypes. This project will begin by characterizing a set of traits that capture core aspects of life history for ~250 related species of Drosophila. These analyses will be complemented by mapping genotype-phenotype relationships across the 30 species of Drosophila with fully-sequenced genomes. All data will be made available through a public database. This resource will enable strategic decisions about future genome sequencing priorities. Furthermore, the database will assist the integrated study of diverse life history traits in the context of both resource ecology and phylogeny.

Using standardized protocols, all ~250 species of Drosophila and related genera (Samoaia, Scaptomyza, Zaprionus and Gitona) maintained by the Drosophila Species Stock Center will be phenotyped for a set of 18 morphological, behavioral, physiological and developmental traits, Collectively, these traits capture core aspects of sexual selection and life-history syndromes, in addition to providing characters relevant to ecological selection. These data form the basis for a valuable public database on Drosophila "evolutionary phenomics," along with accompanying phylogenetic resources. The data will also be used for two kinds of state-of-the-art comparative analyses. First, Bayesian phylogenetic modeling, applied to data for all species, will be used to evaluate rates of diversification and co-evolutionary dynamics among character states and across the clade using a range of alternative trait diversification models. Second, two complementary analytical approaches will be applied to all Drosophila species with fully-sequenced genomes to explore genotype-phenotype relationships for all traits, with the goals of identifying candidate genes underlying trait diversification and setting the stage for studies of the molecular evolution and genomic architecture of Drosophila biodiversity.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.