The Caenorhabditis elegans male and hermaphrodite nervous systems display sexually dimorphic development characterized, in part, by the presence of 8 hermaphrodite-specific neurons and 89 male-specific neurons. We are interested in identifying the genes and molecular mechanisms that govern sex-specific neural development in C. elegans. Through a mutagenesis screen using a pkd-2::GFP reporter to label male-specific neurons, we recovered several mutants that display defects in sex-specific neural development. Males carrying the sm129 mutation lack pkd-2::GFP expression in the male-specific CEM neurons that are involved in mate finding. Genetic epistasis experiments suggest that CEM neurons are improperly specified or differentiated. We cloned the sm129 mutation and determined that it is an allele daf-19 based on three pieces of evidence: (1) RNAi of daf-19 phenocopies sm129, (2) sm129 fails to complement a daf-19 null mutation, and (3) we found a mutation in daf-19 that likely affects splicing. We are also testing to see if sm129 mutants can be rescued by adding a wild type copy of daf-19. daf-19 encodes an RFX transcription factor that activates genes required for sensory cilia function in ciliated neurons such as the CEMs. daf-19 null mutants lack all sensory cilia, have sensory defects, and display a constitutive dauer phenotype (worms enter an alternative part of the lifecycle associated with starvation survival). We are currently investigating how this mutation affects ciliated neurons such as CEMs but does not affect dauer formation.
Aquatic ecosystems around the globe face an increasing threat of eutrophication from algal blooms caused by excess nutrients. On Cape Cod, the major route for delivery of the limiting nutrient, nitrogen, to coastal ponds and estuaries is groundwater seepage. In 2005, researchers at the Woods Hole Marine Biological Laboratory installed and began monitoring a wood-chip based NITREX™ permeable reactive barrier (PRB) designed to remove nitrates via denitrification at the shore of Waquoit Bay, MA. In this experiment, I took groundwater samples from an array of multi-depth sampling wells within and surrounding the PRB. From each well I measured a suite of physico-chemical parameters and in a subset of samples I analyzed metagenomic DNA for the presence and abundance of the nirS nitrite reductase gene and examined the structure of the microbial community using denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene. The PRB effectively reduced the nitrates from a maximum concentration of 109 μM in the core of the nutrient plume to below 10 μM inside and down-gradient of the PRB; however, a portion of the nitrate plume persisted at 3 meter depths underneath the PRB. Hydrogen sulfides present inside the PRB suggest seawater intrusion stimulates sulfate-reducing bacteria that may compete with denitrifiers for resources. Dissolved inorganic carbon (DIC) from respiration increased from 780 μM in the up-gradient groundwater to a maximum of 1985 μM inside the PRB. Dissolved organic carbon (DOC) increased from 8 μM in the inflowing up-gradient groundwater to a maximum of 130 μM in the PRB. High DOC concentrations extended down-gradient beyond the PRB. The presence and abundance of the nirS nitrite reductase gene, determined by PCR and qPCR, also increased inside and down-gradient of the PRB. DGGE results indicate the presence of distinct microbial communities among the sites. There was a high similarity between up-gradient PRB samples and control samples, with a different community emerging within the PRB, indicating that the PRB does have a measurable effect on microbial community composition.
Fewer than 2% of bats have the capacity to modify their environment to construct roosts. Tent-roosting bats cut and fold leaves to form roosts called “tents” and often use specific plant species for this purpose. Unlike the caves or hollow trees used by some bats, leaves possess an upper limit on their capacity to support weight. I tested the hypothesis that the maximum weight capacity that leaves can support limits the maximum social group size of bats that roost in them. I tested a secondary hypothesis that the bat Dermanura watsoni would preferentially use the plant species that can support more weight. I conducted research in the Tirimbina Biological Reserve (TBR), Sarapiquí, Costa Rica, between March and April 2012. To test the first hypothesis, I added weight incrementally to new leaves of three plant species until the angle of the leaves fell below that which bats naturally use. Philodendron fragrantissimum and Heliconia imbricata support one-third more weight than Asterogyne martiana. To address the second hypothesis, I determined plant abundance by systematic-random plot sampling along main paths in the reserve and systematically surveyed tents along the same paths. Patterns of leaf use by D. watsoni suggest a preference for A. martiana and complete avoidance of H. imbricata. Dermanura watsoni did not show a preference for plant species that support a greater maximum weight capacity. This study demonstrates that the maximum weight that the leaves can support is similar to the mean social group weight of D. watsoni and Ectophylla alba reported in the literature for these plant species and lower than the maximum reported social group weights. Therefore, it is possible that the maximum weight capacity of the leaves used to construct roosts limits the maximum social group size but may not be an important factor used for plant selection.
PAL-1 is a protein that regulates posterior development of Caenorhabditis elegans embryos. Although pal-1 mRNA is present throughout the entire embryo, the PAL-1 protein is only transcribed in the posterior end of the nematode worm. MEX-3, a RNA binding protein, binds to the pal-1 mRNA, preventing its translation in the anterior section of the embryo. The MEX-3 protein is essential to maintaining embryo polarity and ensuring that posterior features develop only in the posterior end of the worm. During development, MEX-3 is present throughout the 1-cell and 2-cell embryo stage. MEX-3 is then degraded in the posterior end during the 4-cell stage, allowing the expression of PAL-1 in the two posterior blastomeres. By the 8-cell stage, MEX-3 is depleted from the entire embryo with remnants remaining in germline cells. The ubiquitination pathway is hypothesized to mark MEX-3 for degradation, localizing the protein at various stages of embryo development. This study screened various E3 ubiquitin ligases to determine which ligases are specifically used to mark MEX-3 for degradation during embryo development. Double-stranded RNA was created for selected E3 ubiquitin ligases and then injected into adult worms. This invoked RNA interference (RNAi) of these ubiquitin ligases in the embryos of the adult worms. Knockout of genes D2089.2, F46A9.5, F59B2.6, and Y82E9BR.15 resulted in embryonic lethality. Fluorescence microscopy of GFP::MEX-3 (green fluorescent protein labeled MEX-3) revealed that only F59B2.6 (zif-1 gene) and Y82E9BR.15 (elc-1 gene) knockouts affected MEX-3 localization. Double knockouts of zif-1 and another developmental gene, mex-5, support the hypothesis that zif-1 acts after other regulatory events in MEX-3 localization.
Ten to fifteen minutes following death, a large release of CO2 is produced in many species when killed by high temperature. Studied in mosquitoes, hissing cockroaches, grasshoppers, and desert harvester ants, this post-mortal peak (PMP) appears to be temperature-dependent and, to our knowledge, does not occur in insects killed by means other than high temperature. Four effects were applied to common house crickets (Acheta domestica) to analyze the origin and properties of the PMP. First, it was shown that the PMP does not occur without oxygen. Second, post-mortal CO2 release was studied as a function of temperature-exposure following death and it was established that the phenomenon is dependent on extreme temperatures and runs to completion when exposed to temperatures above 60°C. Third, basic and buffered solutions were employed to assess the possible involvement of dissolved HCO3- (bicarbonate), the dissolved form of CO2, in production of the peak. Hemolymph factors like bicarbonate did not appear to have an effect on the PMP. Finally, exposure to hydrogen cyanide inhibited the PMP, demonstrating the involvement of mitochondria and cytochrome c oxidase in particular. Together, these results rule out any effect of hemolymph or possible CO2 stores in the body of an insect on the PMP. The PMP occurs as an aerobic mitochondrial reaction that requires high initiation temperatures. We believe that this underlying cause may be mitochondrial breakdown at high-temperatures. More specifically, fluidity of the mitochondrial membranes likely increases with high heat, disabling the established proton gradient and ATP production. The resultant accumulation of electron carriers allows for cyclic, but futile operation of the citric acid cycle and electron transport chain with remaining pyruvate stores.
The protein ubiquitination system is a targeted protein degradation pathway that is an essential component of cell cycle progression in mitosis and meiosis. Recent evidence indicates that the ubiquitin system is required for the degradation of zinc finger proteins that play important roles in embryogenesis. It is possible that the ubiquitin system regulates other proteins involved in early embryonic development by controlling which proteins are degraded, and thereby influencing cell fates. In Caenorhabditis elegans (C. elegans), there is a single ubiquitin activating enzyme, which has a well-understood function. The twenty-two ubiquitin conjugating enzymes in C. elegans have been researched to a moderate extent. Finally, there are believed to be about six hundred ubiquitin-protein ligases. Most of these ubiquitin ligases’ exact functions, the proteins they target, remain unknown. Ubiquitin ligases are perhaps the most interesting enzymes in the ubiquitin system because they determine which proteins are targeted for degradation. Two important proteins involved in the embryonic development of C. elegans are posterior alae defective 1 (PAL-1) and muscle excess 3 (MEX-3). PAL-1 is a homeodomain transcription factor protein that is required to specify posterior cell fates. MEX-3 is an RNA-binding protein that binds to pal-1 mRNA in the anterior cells and restricts the translation of PAL-1 to the posterior cells of the embryo, and thereby influences anterior cell fates. Both pal-1 mRNA and MEX-3 protein are present throughout newly fertilized embryos, but by the four-cell stage MEX-3 is depleted in posterior cells and can only bind to pal-1 mRNA in anterior cells, preventing the translation of PAL-1 in these cells. It is thought that MEX-3 depletion in the posterior cells is due to it being targeted by unknown ubiquitin ligases and degraded by the 26S proteasome. Research shows that two homologous mRNA binding proteins (MEX-5 and MEX-6) protect MEX-3 from inactivation and degradation in the anterior, allowing for the repression of PAL-1 translation. One major unanswered question is the identity of the ubiquitin ligase(s) that targets MEX-3 for degradation in the posterior of the embryo. This study attempts to answer that question. RNA interference screening of ubiquitin ligases that are expressed during embryonic development has permitted the identification of 20 ubiquitin ligases that do not target MEX-3 for degradation. Screening of additional ubiquitin ligases may lead to a better understanding of the regulation of many key proteins. By understanding more about the interactions between MEX-3 protein and pal-1 mRNA and how they are regulated, we will learn more about how embryonic development unfolds and what can potentially go wrong.
Competence for natural transformation is the physiological ability of bacteria to take up extracellular DNA. This ability is wielded by over 40 species of bacteria. We hypothesize that competence for natural transformation might be required for the GASP phenotype: a phenomenon by which cells grown to long term stationary phase can out-compete young cells co-incubated with them in stationary phase. In this experiment, we show for the first time that the soil bacteria Acinetobacter baylyi exhibits the GASP phenotype, and that knock out mutants of the gene comP which codes for a prepilin-like protein showed significantly reduced fitness levels during a GASP test when competed against young wild type cells. Knock out mutants of the gene comQ, which codes for a transmembrane protein, showed reduced fitness levels when competed against a young wild type strain in stationary phase but this difference was not significant. Defective GASP responses suggest that natural competence is important for cells to “age” (evolve) normally during long-term stationary phase.
Acinetobacter baylyi ADP1 has been studied in laboratories because of their competence for natural transformation and ability to adapt to different environmental conditions. A previous study has found 30 different genes in A. baylyi ADP1 that are induced by starvation during the long term stationary phase. ACIAD0167 is one of them, encoding a Vgr-like protein. The goal of this study was to test whether ACIAD0167 and other genes in its operon (ACIAD0166, ACIAD0168 and ACIAD0169) are required for twitching motility, or for surviving stressful conditions including heat shock, desiccation and DNA damage. Our study found that ACIAD0167 and the other three genes in the operon play a role in twitching motility in A. baylyi but apparently not in other phenotypes. The likely first gene in the operon, ACIAD0166, was cloned into wild-type ADP1 and over-expression of the gene caused a smaller twitching zone than one produced by wild type cells, further implicating the role of ACIAD0167-containing operon in twitching motility. These results indicate that ACIAD0166, ACIAD0167, ACIAD0168, and ACIAD0169 genes encode proteins, and should no longer be considered “hypothetical” genes. We also found a novel link among ACIAD0167, twitching motility and the type VI secretion system (T6SS). ACIAD0167 is found in the STRING network to be associated with genes involved the T6SS, whose structure resembles an inverted bacteriophage tail.
Neurons have highly asymmetric cellular morphologies and polarized cellular functions that are necessary for establishing neural circuitry and for proper functioning of the nervous system. Specialized processes, called dendrites, are used by neurons for reception of stimuli, while axons function in the transmission of signals. In neurons, mRNA localization and translational repression are used to change the protein composition of various regions of the cell, allowing for distinct axonal and dendritic morphologies and environments that are equipped for their various cellular tasks. A significant portion of the eukaryotic genome encodes for RNA-binding proteins (RBPs), which play important roles in localizing and translationally regulating RNAs. Since studies have shown that a large number of mRNAs are localized within dendrites, this suggests that the RBPs contribute broadly to neuronal development and function by localizing and regulating mRNAs. Based on a previous screen of RBP-encoding genes that affect dendrite morphogenesis in dendritic arborization neurons (da neurons) in Drosophila that identified 89 genes (Olesnicky, Killian, and Gavis; in preparation), I extended this screen to determine if any of these evolutionarily conserved RBP genes are important for dendrite morphogenesis in C. elegans PVD neurons as well. A significant decrease in dendritic arborization was found in dcr-1 mutants and preliminary results suggest that sup-26 and mtr-4 mutants may have decreased 3rd and 4th order dendritic branching. In addition, several other candidate genes are currently being investigated. Thus far, the results suggest that DCR-1/Dicer, an RBP involved in the microRNA pathway, SUP-26/Alan Shepard, an RBP implicated in translational control of mRNAs, and MTR-4/L(2)35Df, a component of the eukaryotic RNA exosome play an evolutionarily conserved role in dendrite development in flies and worms.
Aedes aegypti occupies several regions of the world and transmits well-known diseases such as yellow fever. The unique life cycle of this species displays its resilience with the initial laying of eggs, in short-lived puddles, that do not hatch until a subsequent period of flooding. Larvae accumulate resources while growing in the puddle but face the risk of habitat loss until they eventually pupate into an adult. While much is known about the ecology and molecular biology of this vector, there is an absence of information on the possible life history trade-offs that occur as a result of choosing this particular larval habitat. Growing faster and pupating sooner increase their probability of larval survival; however, this rapid growth rate results in smaller adult body size, which may impact survival as adults. Our study involved investigating the effect of age at pupation on fitness traits in the adult. Mosquitoes were tested for desiccation resistance as well as dry mass, water content, carbohydrate and lipid on the day of emergence, and their values were compared to each individual’s age at pupation. Results showed mosquitoes that pupated later displayed increased desiccation resistance as well as increased lipid content. This study demonstrates that time spent as a larva has positive effects on desiccation resistance, and this is not mediated by increased body size or body water stores. Larval development time also boosts lipid stores, which likely promote starvation resistance and may also be involved in enhanced desiccation resistance. Knowledge about life-history trade-offs affecting adult viability can be gained from this study. Thus, this information concerning pupation and emergence promotes a greater awareness of when to prepare for these mosquitoes such as after rainy seasons, as well as how to limit the available larval habitats.