Insect endosymbionts have been found in more than half of insect species. The roles these microorganisms play in their host could vary from parasitism to mutualism, but have not been well explored due to the high diversity of insects and their extremely heterogeneous life histories. In general, common endosymbionts, such as Wolbachia and Spiroplasma, could be beneficial to their host by increasing their longevity and the number of offspring, or by increasing the resistance of their hosts against other parasites such as nematodes. On the other hand, these endosymbionts could also cause male killing processes or post-mating cytoplasmic incompatibility in offspring, which could negatively Impact the host population. Alpine environments are thought to be relatively harsh to resident species, with low temperatures and oxygen concentrations, limited resources, and high solar radiation. The adaptations of alpine insect species, and the nature of their ecological interactions with microbes, is therefore quite interesting. In this study, we aim to understand the role of Spiroplasma sp. NR, in their host, the Nebria ingens species complex, by scanning the prevalence of infection, genetic diversity, and associating the infection rate with environmental factors and physiological, morphological, and molecular characteristics of the beetles. The preliminary results show a relatively high (57%) and geographically widespread infection rate of Spiroplasma throughout the Nebria ingens species complex.

Our lab focuses on evolution of aquatic organisms in fragmented habitats/landscapes. We are interested in anthropogenic and climatic effects on population-genetic structures of fishes. The Longnose dace, Rhinichthys cataractae, is a common inhabitant of riffle communities in Northeastern US streams. We require an aligned sequence of R. cataractae genome. Recent advances have dramatically expanded genome sequencing and analysis. Oxford Nanopore MinION, a 3rd generation technology, pulls DNA bases through nanoscopic pores, reading bases as they pass through from spectral reflectance of each nucleotide. After successful control experiments, we: extracted the DNA (checking its length, quantity and purity), prepared the library (repairing DNA ends), ligated motor proteins to DNA (SQK-LSK109), and loaded the DNA library into R9.4 and R10.1 flow cells. Genome assembly and alignment used Zebrafish, Danio Rerio, as the reference genome downloaded from the NCBI database. The D. Rerio genome is 1.42Gb arranged on 26 chromosomes. Sequencing used MinKNOW software. We utilized Guppy for high accuracy base calling after sequencing. Flye genome assembler, Medaka sequence polisher, and Guppy aligned the sequence up against the reference genome. We have three sequencing runs that yielded >14Gb of reads with N50>4Kb, and reads up to 50,000kb in length. The genome alignment will be discussed. The ability to sequence genomes of non-model organisms will elucidate the genetic basis of human and climatic impacts on the fishes in the wild. These findings will aid in future experiments about the genomic history of R. cataractae.

Lignin is an organic polymer that makes up a large portion of the world’s biowaste. It is found linked to cellulose in plant cell walls and is a waste product of the pulp/paper industry. Although very abundant in the world, it is primarily used as a furnace fuel and converted directly into carbon dioxide. However, lignin can be broken down by fungal peroxidases, but the process is too slow to be considered a commercially feasible choice. Termites and wood-eating organisms have been shown to depolymerize lignin through the use of bacteria and enzymes in their digestive tract in a fast and efficient manner in comparison to fungal peroxidases. Since bioinformatic and other investigations have to date failed to identify which enzymes are responsible for the breakdown, further investigations are needed to identify these depolymerizing enzymes. We propose to synthesize an organic probe molecule containing a fluorophore and a quencher to detect the exact location of these lignin degradation enzymes within these organisms’ digestive tract. In this 13-step synthesis, the creation of this lignin depolymerization detection probe will allow researchers to conduct in vivo experiments, which is anticipated to allow the development of lignin as alternative biomass for biofuel and fine chemical production.

The nonsense-mediated mRNA decay pathway (NMD) is an mRNA degradation pathway in most eukaryotic organisms which allows for the rapid recognition and degradation of mRNAs that prematurely terminate translation. This includes mRNAs containing premature termination codons alongside natural mRNAs. Natural mRNAs, when regulated by NMD, encode fully functional proteins which carry out a variety of extensive cellular processes. One of these processes includes bio-metal homeostasis. To explore the significance of NMD regulation on bio-metal homeostasis, mRNAs involved in copper homeostasis and cadmium detoxification were examined, which are known to be sensitive to the NMD process. Although copper serves an essential physiological role as a coenzyme, excessive intracellular copper concentrations can be directly linked to cell toxicity. Additionally, although cadmium ions are known carcinogens, pollutants, and teratogens, little is known surrounding cadmium’s underlying molecular mechanisms. Thus, to further understand the roles of both heavy metal ions on the NMD process, and therefore, on mRNA decay and gene expression, the growth rates and RNA expression rates of different strains of Saccharomyces cerevisiae were analyzed as they were cultured in a variety of environmental conditions. Through this, the effect of the presence or absence of these metal ions in relation to a functional NMD pathway was determined. It has been discovered that Saccharomyces cerevisiae strains from diverse genetic backgrounds respond differentially to environmental stimuli, and furthermore, that copper ions play a nutritional role, while cadmium ions play a toxic role, in the growth and expression of wild-type and NMD decay mutants.

Macrophages are specialized white blood cells, with roles in pathogen destruction. One of the key activators and regulators of this pro-inflammatory response is the src-family kinase (SFK) Lyn. Lyn exists in macrophages and other cells as two splice forms, LynA and LynB. Our lab used CRISPR/Cas9 gene editing to generate isoform-specific LynA knockout (KO) mice and LynBKO mice. Surprisingly, macrophages from both LynAKO and LynBKO mice showed higher levels of the remaining isoform. In addition, the levels of the other SFKs expressed in macrophages (Fyn, Fgr, or Hck) did not change, suggesting Lyn may have a unique regulatory mechanism that somehow senses the levels of the two isoforms. The goal of my summer research is to test whether this unknown regulatory system occurs in macrophages at the mRNA level or later at the protein level. Mouse cDNA and protein from LynAKO, LynBKO, and wild-type (WT) bone-marrow-derived macrophages (BMDMs) will be subjected to qRT-PCR and immunoblot analysis, respectively. qRT-PCR will be used to quantify the mRNA expression of LynA, total Lyn, and the E3 ubiquitin ligase c-Cbl. Results from these experiments will then be used to assess differences between mRNA and protein levels. Due to LynA being a selective target for protein degradation by c-Cbl, we expect there to be no differences in mRNA levels that would indicate this regulatory mechanism occurs post-transcriptionally, at the protein level. Because of Lyn’s role in macrophage activation, understanding Lyn’s regulatory mechanism will hopefully contribute to therapies aimed at decreasing inflammatory responses.