- Grisilda Bakiasi
- Erin Bonner
- Fransheska Clara
- Anne DeHart
- Mian Horvath
- Mandy Levine
- Blanca Lopez
- Bridget Murray
- Mindy Reutter
- Lauren Sauers
- Emily Spiegel
- Maritza Vazquez-Trejo
In the G. Davis laboratory we study the evolution of development using the pea aphid, Acyrthosiphon pisum, as a model organism. The pea aphid is famous for its phenotypic plasticity. In one example, when exposed to short nights mothers produce embryos that will develop as asexual females, whereas when exposed to long nights mothers produce embryos that will develop as sexual females. A candidate for mediating this process is the insect hormone, Juvenile Hormone (JH). JH is best known for its role as a status quo hormone which determines the state of the aphid after each molt during development; if there are high levels of JH at molting the individual remains a nymph, and if there are low levels of JH the individual becomes an adult. In the context of reproductive fate JH is thought to induce the asexual fate: topically applied, JH can transform embryos that should have developed as sexual aphids into asexuals; additionally, JH titers inversely correlate with night length.Together this evidence suggests that JH mediates the induction of asexual fate under short night conditions. However, it remains unclear how JH titer is regulated by photoperiod. This summer, in order to gain knowledge of how JH is regulated, I will look at the expression of pea aphid orthologues of genes known to regulate juvenile hormone. Some of these genes are JHAMTs, which catalyze the last step in the synthesis of JH. Others are JHEs which are known to degrade JH. I will examine expression of these genes in both mothers and embryonic progeny, under photoperiods promoting both sexual and asexual fates (i.e., long and short nights). Comparing the expression of these genes under these different conditions should reveal the mechanism of regulation.
Evolution of the Photoperiod Response in Pea Aphid Reproductive Polyphenism
We are studying the role of photoperiod response in the evolution of Acyrthosiphon pisum (pea aphid) reproductive plasticity. Pea aphids exhibit a remarkable evolutionary response to the challenges posed by seasonal fluctuations in climate. During warm summer months, female aphids reproduce asexually, allowing them to quickly produce vast numbers of genetically identical offspring. However, as nights grow longer in the fall, aphids switch to sexual reproduction. In response to longer night conditions, they are induced to produce sexual females and males that will mate to lay fertilized eggs. These eggs are durable and frost-resistant, allowing the species to survive through the cold winter months. In the spring, the eggs hatch into asexually-reproducing females, and the cycle continues. By alternating their mode of reproduction in response to seasonal fluctuations in night length, pea aphids are able to maximize their reproductive fitness: they reproduce rapidly and efficiently throughout the spring and summer, and by maintaining the ability to reproduce sexually, are able to survive the cold winter and generate beneficial genetic diversity. Previous research in our lab has suggested that Juvenile Hormone (JH) signaling mediates the switch between asexual and sexual reproduction. It is thought that JH sends an asexual-promoting signal during the spring and summer (short night conditions), thereby specifying asexual reproduction during favorable environmental conditions. The goal of our research this summer is to more critically evaluate the conditions that control this switch in reproductive fate, and to further investigate the role of JH in inducing this switch.
We are first testing the photoperiod response of three strains of aphids in order to ascertain whether strains collected from different latitudinal regions have undergone changes in their response to increasing night length. We will do this by attempting to identify the Critical Night Length (CNL) of each strain, in the belief that different strains will have different CNLs. The CNL refers to the minimum hours of darkness that aphids must be exposed to in order to produce 50% sexual offspring over 5 days. We will test multiple different photoperiods in the different strains, using a newly developed and efficient method: we are building a staggered light box with ten compartments, each of which will simulate a different night length in a 24 hour cycle. The three strains being tested are from Tucson, Arizona; Atlanta, Georgia; and upstate New York. The strain from Tucson does not produce sexuals in its natural habitat, because the warmer temperatures and less severe winters mean that they do not need to produce frost-resistant eggs. It is unknown whether the Georgia strain will produce sexuals. As a result of the latitudinal, and thus climatic, dissimilarities between these locations, it is believed that the more southern strains have evolved over time to require more darkness to induce sexual reproduction, in comparison to the more northern New York strain.This is because it would be disadvantageous for the more southern strains to produce sexuals if the winters were not severe enough to require this mode of reproduction. To support this theory, we aim to show that the CNL for more southern strains is longer, i.e. it requires longer exposure to darkness to induce sexual reproduction.
The second part of our research will involve testing a possible mechanism behind the differences in photoperiod response between the strains. As discussed earlier, JH has been implicated as specifying asexual fate in aphids. We postulate that the probable difference in CNL between the strains may be explained by an increased sensitivity to JH in the southern strains. We are applying Kinoprene, a JH analog, in varying doses to adult aphids, and scoring their progeny for asexual or sexual fate. Previous research from this lab has found a difference in JH sensitivity between Tucson and the New York strain using kinoprene. The Tucson strain was found to be more sensitive to JH, producing a greater percentage of asexuals than the New York strain given the same dosage treatment. This experiment needs to be repeated, and we will also be narrowing the dosage treatments and applying these treatments to the Georgia strain as well.
RNA interference analysis of non-NMDA receptors in Leech Swimming
In order to understand the structure and function of the nervous system, the analysis of motor behavior is necessary and has provided some insight, particularly in regard to rhythmic motor patterns (e.g. breathing and swimming). Although research over the last 50 years has provided some understanding into the nervous system, information on the cellular and ionic mechanisms that underlie the production of rhythmic behaviors is limited and not tacit. To overcome this, alternating or removing ion channel function during the expression of the behavior through the use of RNA interference can lead to understanding the roles of these specific ion channels in relation to the expression of the behavior. Knocking down the expression of specific mRNA and proteins can create changes in behavior, which then provides an achievable approach to describing ion channel function in behavior.
Effects of Synaptotagmin-1 on Filopodia Formation During Neuronal Development
While neuronal networks are the basis for most functions of many organisms, we have a limited understanding of their mechanisms. Within each neuronal network are thousands of axons, each of which produces many branches. While the lengthening of axons is well characterized, much less is known about the mechanisms behind axonal branching. In Dr. Greif’s lab, we will investigate the potential role a protein called Synaptotagmin-1 (syt1) may play in axonal branching of developing eight-day-old embryonic chick forebrain neurons.
Syt1 is a widely characterized Ca2+-binding protein responsible for vesicular exocytosis at the synapses of neurons. At presynaptic terminals, syt1 works as a calcium sensor within a SNARE complex, which expedites the merging of vesicles to the plasma membrane, and subsequently the release in neurotransmitters, in response to elevated Ca2+ levels. Our working hypothesis is that, in a similar fashion, syt1 merges vesicles to the plasma membrane of a developing neuron’s axon, adding additional membrane to allow for branching. Previous research has provided evidence that a surplus of syt1 leads to increases in the number of axonal branches and filopodia formed during development, while deficits in the protein result in a decrease in such branching. This evidence suggests that the syt1 produced during early development regulates the formation of new branches and filopodia. Filopodia are small extensions along the axon from which branches develop; they are distinguishable by a purely actin cytoskeleton, while branches incorporate microtubules.
To confirm our hypothesis, our experiments will test how overexpression of wild-type syt1 affects axonal branching and filopodia development as compared with a mutant form of sty1, which has impaired Ca2+-binding abilities. We will replicate this experiment with in ovo electroporation to simulate conditions in vitro. After using electroporation to incorporate our plasmids, filopodia formation and syt1 expression will be analyzed using immunocytochemistry techniques and fluorescence microscopy. Depending on the results of the initial experiments, they may be followed by rescue experiments. If the expression of mutated syt1 negatively affects filopodia development, the rescue experiments will investigate whether and how function may be restored to the mutated syt1.
Creating expression vectors containing accessory proteins to leech glutamate receptors
The leech is an excellent model organism, widely utilized in the field of neuroscience because it exhibits a variety of distinct behaviors, including swimming, that are easily observed. Previous research has shown that glutamate receptors mediate all of the excitatory synaptic connections in the swim network. One approach to characterizing the role of glutamate receptors in swimming is through expression in Xenopus oocytes. In order to correctly express these glutamate receptors, however, several accessory proteins, including SOL-1 and STG, must also be expressed. The leech transcriptome database was searched for putative coding regions and primers were designed for these regions. Using PCR, postulated protein encoding sequences were amplified from an existing leech cDNA library. These regions were cloned into pGEM-T Easy for sequencing and compared to existing databases. The sequences were then cloned into expression vectors to be expressed in Xenopus oocytes so that glutamate receptors could be accurately studied. Sequences for STG1, STG2, and SOL-1B were isolated, sequenced, and cloned into expression vectors for later use. Based on BLAST searches, it is likely that these sequences do in fact code for the accessory proteins necessary for proper glutamate receptor activity and therefore, once cloned into expression vectors, will be useful in studying glutamate receptors in Xenopus oocytes.
Biophysical Properties of AMPA Receptor on Xenopus oocytes
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainite receptors are non-NMDA receptors which mediate all known excitatory synaptic connections in the leech central pattern generator (CPG). There is not much information about non-NMDA receptors, but by introducing mRNA of AMPA receptors into Xenopus oocytes their properties can be analyzed through two-electrode voltage clamp experiments. Then the distribution and location of AMPA receptors would be determined by in situ hybridization.
A Geographic & Geophysical Analysis of Mississippian Archaeology in the American Bottom
The archaeological region of the United States known as the American Bottom, located east of the Mississippi River in Illinois, was the first site of urbanization in North America and has been studied largely via government-mandated cultural resource management (CRM) surveys at proposed construction sites since the 1970s. Part I of this project contextualizes these extensive surveys (numbering 500-600 for the counties included) as I build from scratch a geographic information system (GIS) database compiling site information (use, time period, size) and survey report details obtained through an Illinois CRM database. This database will enable analysis of settlement patterns from the Late Woodland thru Mississippian periods (A.D. 600-1350) along attribute and spatial parameters, the latter of which is particularly significant for describing the composition of an archaeologically dense area such as the American Bottom.
Part II of this project focuses specifically on the site of Cahokia through a series of geophysical surveys studying effects of early urbanization on the landscape. Other American Bottom sites show a period of settlement aggregation followed by rapid decline in the centuries prior to the period of growth this study addresses. This period, concurrent with maize domestication, will likely be marked by population clustering evident in habitation sites and farmsteads. Geophysical surveys using magnetic readings and ground penetrating radar (GPR) detect soil anomalies, producing maps showing features such as houses, fences, and fire pits which we would expect to see in a growing settlement. This information may be incorporated into the system developed in the first part of this project and analyzed alongside other American Bottom sites.
Measuring Whole-body, Hemolymph and Embryonic Juvenile Hormone Titers in the Pea Aphid
The cyclically parthenogenetic life cycle of the pea aphid Acyrthosiphon pisum involves both sexual and asexual reproduction. The reproductive mode of offspring is determined during embryogenesis within the mother. Previous research correlates long photoperiod (short nights) to high Juvenile Hormone (JH) titers and implicates the level of JH titer experienced by the embryo as the signal that determines whether it becomes sexual or asexual. It is unknown whether it is embryonic or maternal JH that is ultimately responsible for determining the embryo's reproductive fate. Our project aims to quantify maternal JH titers when mothers are producing sexual and asexual progeny, and to quantify embryonic JH titers in sexual and asexual fated embryos. The goal is to identify which sources of JH (maternal or embryonic or both) are correlated with changes in reproductive fate. To do this we will collect JH samples from both mothers and embryos exposed to varying photoperiod conditions. We will send these samples to Dr. Fernando Noriega, our collaborator at Florida International University, for quantitative analysis. Knowing which source of JH is responsible for the shift between reproductive fates will help us to focus our later studies on evolved changes in the response to photoperiod, which we believe to be due to changes in JH sensitivity.
Juvenile Hormone Detection in the Pea Aphid
The cyclically parthenogenetic life cycle of the pea aphid Acyrthosiphon pisum involves both sexual and asexual reproduction. The reproductive mode of offspring is determined during embryogenesis within the mother. Previous research correlates long photoperiod (short nights) to high JH titers and implicates the level of Juvenile Hormone titer experienced by the embryo as the signal that determines whether it becomes sexual or asexual. It is unknown whether it is embryonic or maternal JH that is ultimately responsible for determining the embryo's reproductive fate. Our project aims to quantify maternal JH titers when mothers are producing sexual and asexual progeny, and to quantify embryonic JH titers in sexual and asexual fated embryos. The goal is to identify which sources of JH (mother or embryo or both) are correlated with changes in reproductive fate. To do this we will collect JH samples from both mothers and embryos exposed to varied photoperiodic conditions. We will send these samples to Dr. Fernando Noriega, our collaborator at Florida International University, for quantitative analysis. Knowing which source of JH is responsible for the shift between reproductive fates will help us to focus our later studies on evolved changes in the response to photoperiod which we believe to be due to changes in JH sensitivity.