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Alternative roles for Pseudomonas aeruginosa bacteriocins

Devin Hunt
Autumn Robinson
Lia Michaels
Mary Ellenbecker
Alison Coluccio
Patrick R. Secor
Laura K. Jennings

Bacteriocins are multi-protein assemblies that bear striking resemblance to bacteriophage (virus) tails. Bacteriocins are an extracellular contractile injection system that kill closely related bacteria by puncturing their cell membrane. Mounting evidence suggests that besides interbacterial competition, bacteriocins also mediate interactions between bacteria and diverse eukaryotic hosts by assembling extracellular hexagonal-bacteriocin arrays composed of numerous bacteriocin particles. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that produces bacteriocins called R2 pyocins which lyse susceptible bacteria. Based on homology to other contractile injection systems, we hypothesize that P. aeruginosa produces bacteriocin arrays that modulate host responses during infection. We have developed a method to quantify R2 pyocins utilizing the lysis of susceptible strains of P. aeruginosa. We are currently applying this quantification method to optimize the production and purification of pyocins to test in host-pathogen models. We have also generated a fluorescently labelled R2 pyocin for the detection of bacteriocin arrays using fluorescence microscopy. These experiments are essential to enable future evaluation of the effect of R2 pyocins on host pathogen interactions.

Analyzing Predator Impacts on Biomass in a Grassland System

Trevor Weeks

Predator-prey effects that alter the abundance, biomass, or productivity of a population community across more than one link in a food web are referred to as trophic cascades. Trophic cascades often specifically refer to “top-down” effects of predators on the rest of the ecosystem. These effects have been extensively studied in aquatic environments, however very few studies have examined trophic cascades in terrestrial ecosystems. The posterchild for terrestrial trophic cascades studies in recent years has been Yellowstone’s wolves. Their influence on aspen and willow communities via the regulation of elk has been well established, however no research has yet been done to understand the effect that wolves may have on a grassland system. To that end, I sought to answer the question of whether top-down effects regulate grassland biomass in a wolf-elk system. To answer my question, I utilized the extensive dataset collected over the last two decades during the Ya Ha Tinda Long-term Elk Monitoring Project. The Ya Ha Tinda is a mountain prairie ecosystem located on the eastern boundary of Banff Park in Alberta, Canada. The Elk Monitoring Project there is one of the longest continuous elk and predator research projects in the world. The sheer variety and volume of data collected over the course of this project provided me with the unique opportunity to address the complexity of terrestrial trophic cascades that has so often precluded studies in such systems. My study serves not only to help fill an egregious hole in this field of ecological research, but is also especially relevant as predators in North America are recolonizing much of their historic range. Understanding what impacts these reappearing predators may have on the ecosystem beyond their prey populations is therefore vital for management, conservation, and reintroduction efforts across North America.

Behavioral Impacts of the Gut Microbiome on Drosophila melanogaster

Ashley May Bielawski

Recent studies indicate alterations in the gut microbiome impact behavior including increasing aggressive behavior. The gut of an organism is home to a complex community of bacteria, fungi and viruses that collectively make up the gut microbiome. The prevailing hypothesis is that changes in the microbiome are communicated to the brain and subsequently impact behavior. In the Certel lab, we are identifying factors that regulate aggressive behavior and have been specifically examining the role of octopamine using the model system Drosophila. Octopamine (OA) is a neurotransmitter and neuromodulator that is required to promote aggression. Males without OA exhibit decreased levels of aggression and increased levels of inter-male courtship. Here we ask how changes in the gut microbiome impact male aggressive behavior and how is OA involved? These questions are relevant as we recently determined OA neurons located in the brain also innervate the gut. We generated germfree males and our initial results suggest germfree males fight longer and at higher levels than control males. I will present additional results quantifying the aggression of germfree, OA null mutants, and appropriate controls. Our results describe the impact of the microbiome on animal behavior which may be broadly conserved.

Development of a System for Transposon Mutagenesis of Bartonella bacilliformis

Finley J. Andrew

Bartonella bacilliformis is a tropical bacterial pathogen responsible for Carrión’s disease in humans. The lack of a system for random mutagenesis has greatly hindered our ability to efficiently study the agent’s molecular biology. Here, we report the first transposon (Tn) mutagenesis of B. bacilliformis, generation of a mutant library, and confirmation of five mutant strains by arbitrarily-primed PCR coupled with nucleotide sequencing. To accomplish this, B. bacilliformis strain JB584 was transformed by electroporation with the plasmid pSAM-R1; a vector initially intended for use in Rhizobium species. pSAM-R1 contains a transposase and a Tn encoding a kanamycin-resistance gene, allowing for selection and maintenance of the integrated Tn in the B. bacilliformisgenome. One mutant, designated JB584-4B2, was identified as having its flgI gene disrupted by the Tn. The flgIgene encodes the FlgI protein, an essential component in the P-ring used in the flagellar motor of bacteria. Thus, the motility phenotype of JB584-4B2 was subsequently examined on a novel motility medium. Results conclusively demonstrated that interruption of flgI gene created a non-motile mutant of B. bacilliformis. Taken as a whole, our results show that: 1) pSAM-R1 is a viable transposon vector for B. bacilliformis, 2) the plasmid can be employed to create a Tn library, and 3) arbitrarily-primed PCR is a suitable method for identifying and locating mutations generated by this procedure. When used in conjunction with B. bacilliformis strain JB584, this system of transposon mutagenesis and mutation identification allows for a new and expanded way to investigate the molecular biology of this emerging human pathogen.

Does Relative Condition Predict Probability of Spawning and Post-Spawning Mortality for a Migratory Inland Trout?

Maggie Wallace, University of Montana, Missoula

Fluvial Westslope Cutthroat Trout (Oncorhynchus clarkii lewisi) occupy a variety of river habitats over their life, moving long distances in order to complete their life history. Despite being iteroparous, individual Westslope Cutthroat Trout have high mortality associate with spawning and may skip spawning some years. We hypothesized that initial body condition at tagging is positively related to probability of spawning, migration distance, and post-spawning mortality. Additionally, we expect distance moved upstream may explain variation in post-spawning survival. We used radio telemetry to track movements of 70 non-hybridized Westslope Cutthroat Trout greater than 350mm in Rock Creek, Montana from 2018 to 2019. Sixty tagged individuals moved into tributaries during spawning season across both years and migrated up river between 0.2 km and 69 km to reach putative spawning sites. We observed 34 mortalities during and after the spawning season. We explored whether probability of spawning and post-spawning mortality was related to relative condition, tagging location in the drainage (low, middle, high), and upstream distance using a generalized linear model. Contrary to our expectations, initial results suggest that individuals in poorer condition were more likely to spawn.

Genetic analysis of changes in body length in Caenorhabditis elegans

Mikaya Terzo

The regulation of the cell cycle is an essential process that contributes to the normal growth and division as well as the prevention of tumor formation. A cell’s progress through mitosis is regulated by a network of proteins that affect the cyclin B–Cdk1 complex. In C. elegans, there are 4 different cyclin B genes that partner with Cdk1: cyb-1, cyb-2.1, cyb-2.2, and cyb-3. Although all are required for completion of embryonic cell divisions, the detailed functions of some of these cyclins, including any contribution to the regulation of animal size, are still unknown. After noticing a potential increase in length of worms that combined a mutation in fbf-2 RNA regulator with a hyperactive Cyclin B (cyb-2.1(h)), we investigated the relationship between hyperactive cyb-2.1, worm length, and regulatory pathways affecting animal size. Documenting animal growth over time in wild type and mutant strains allowed us to identify a significant increase of worm length in fbf-2; cyb-2.1(h). To further determine CYB-2.1 role in length regulation, RNA interference assays are being conducted to inhibit protein expression of numerous genes known to affect C. elegans length, through the TGFβ signaling pathway. A knockdown of each protein will be performed in both wild-type and fbf-2; cyb-2.1(h) double mutant. Wild-type worms are expected to show short body size after a knockdown. No decrease in the length of double mutants will indicate that CYB-2.1 genetically functions downstream of the knocked-down protein. The implications of findings will be discussed to place cyb-2.1 regulation in an established signaling pathway regulating worm length. Understanding cell cycle regulators such as cyb-2.1 and how they contribute to the regulation of animal size are crucial because loss of strict control over cell division can lead to developmental abnormalities as well as cancerous growths.

Investigation of Novel CYP26 Inhibitor for Treatment of Traumatic Brain Injury

Jacob M. Leatherwood, University of Montana, Missoula

Over 5.4 million Americans are living with traumatic brain injury (TBI) related symptoms today, with 10 million occurring annually on a global level. While once considered an isolated event, current research is redefining TBIs as the initiation of a disease process that can last a lifetime and impact multiple organ systems. Within the nervous system, injury occurs in two phases: the initial insult, and a post-traumatic inflammatory response. Retinoic acid (RA) signal transduction is activated when contusions, compressions, and lacerations occur in the nervous system, and recent studies have shown that endogenous RA in the brain supports neuronal protection, axonal growth, inflammatory signal modulation, and glial differentiation. RA biosynthesis is a highly regulated pathway where metabolism is controlled by retinoic acid hydroxylases, CYP26. Thus, inhibition of CYP26 is a promising mechanism to increase endogenous RA and promote healing from TBI. We hypothesize that inhibition of CYP26 with synthetic compounds will reduce TBI pathology through the modulation of neurotrophic and anti-inflammatory factors. To test this, in vivo experiments were performed with a small molecule CYP26 inhibitor, DX308. We found that DX308 significantly reduces TBI behavioral symptoms in rodent TBI models, including protection from paretic circling. Additionally, limb placement and sensory motor function significantly recover in the three days following treatment, suggestive of reduced inflammation and wound healing with DX308 treatment. Collectively, these studies suggest that CYP26 inhibition may rescue TBI pathology through increased levels of endogenous RA.

Leaf Choice of Pteronarcys californica in Rock Creek, Montana

Emily L. Hamant
Art Woods
James Frakes

The Giant Salmonfly (Pteronarcys californica), the largest species of stonefly in the world, are vital to streams in western North America. Understanding their diet may shed light on interactions between invertebrates and common riparian trees such as alder, cottonwood, chokecherry, dogwood, and willow. P. californica consume primarily fungal and microbial colonies on decomposing ‘conditioned’ leaves in streams. The rate and timing of leaf decomposition varies due to the toughness and chemistry of the leaves and the communities of microbes that colonize them. There are multiple species of leaves in the stream at any time, but they decompose with different dynamics. Leaves that quickly decompose disappear from the streams, leaving only tougher leaves during winter and the following spring and summer seasons. Because of this variability, P. californica may prefer leaves that decompose faster, such as chokecherry and alder, under short conditioning periods, but may prefer tough leaves, such as cottonwoods, under longer conditioning periods. We first asked whether stoneflies choose strongly among leaf type (based on either leaf species or conditioning time). We then assessed the consequences of those choices by measuring stonefly growth in no-choice assays (individuals given single leaf types). I also quantified leaf mass area for each leaf species as a proxy for toughness. This study showcases the complex interactions between riparian species and invertebrate growth. With the results of this study, restoration efforts on western streams may include planting a variety of riparian tree species to improve growth and winter survival of P. californica, and thus improve fisheries of western streams.

Rapid Evolution of the Rhopalodia gibba Mitochondrion in the Presence of an Emerging Nitrogen Fixing Organelle

Heidi E. Abresch, University of Montana, Missoula

The evolution of the mitochondrion from an endosymbiotic bacterium was the defining moment in the origin of eukaryotes. This understanding is based on multiple evolutionary studies that show that some organelles, such as mitochondria and chloroplasts, evolved from endosymbionts. Yet, understanding the process of organelle evolution remains one of the grand challenges in biology. Although several host-microbe relationships have been studied as key steps in organelle evolution, these examples typically focus on later stages of this process. The diatoms in the genera Rhopalodia, Epithemia, and Denticula have nitrogen fixing cyanobacterial endosymbionts called spheroid bodies, which are proposed to be emerging organelles. Understanding this diatom-spheroid body relationship provides an important model of that can provide insight into the early stages of how organelles evolve.

To understand this system, we obtained genome data for the spheroid body as well as the first genome data for the host, Rhopalodia gibba. We are using these data to investigate evolutionary changes in spheroid body and host genomes during the development of the symbiosis as well as to serve as references for mapping gene expression data. In this study, we compared the genome of the R. gibba organelles to other closely related diatom species that don’t have spheroid bodies. Analyses show that the chloroplast genome is changing at a similar rate compared to related diatoms. However, selection on the R. gibba mitochondrion appears to be relaxed. Translational genes appear to be changing at an increased rate compared to energy production genes. Additionally, horizontal gene transfer also contributes to mitochondrial divergence. This diatom-spheroid body relationship provides a rare opportunity to quantify these benefits and costs and could help elucidate the mechanisms of organelle evolution.

Sex-related Differences in Patellofemoral Joint Stress with Fighting and Approach Load Carriage

Marin Plemmons

Female soldiers are more than twice as likely than male soldiers to experience knee pain. In the military, carrying moderate and heavy loads is expected of soldiers, regardless of sex or physical capability, and is likely to contribute to the high rate of knee injuries in the military. Because of their lower body mass and leg strength, females may be more vulnerable to experiencing larger increases in knee stress, versus males while carrying heavy loads. Thus, we assessed sex-related differences in knee joint stress with the addition of moderate and heavy loads. Via 3-D motion capture, 34 healthy, well-trained individuals with load carriage experience completed instrumented treadmill trials with and without moderate (20-kg) and heavy (35-kg) load carriage. Peak knee stress was estimated with a musculoskeletal model and analyzed with repeated measure analysis of variance (ANOVA). Our data found that knee stress increased similarly with the addition of moderate load in both males and females; however, females experienced a disproportionately greater increase in knee stress with the heavy load compared to males. While these data suggest that females may need additional physical conditioning to improve their ability to carry heavy loads, females carried moderate loads as well as their male counterparts. This research is impactful to the field of exercise science and society (especially regarding the military) as it sheds light on sex-differences in load carrying capabilities. Furthermore, these findings can serve as an influential tool in developing additional research in the field of sex-dependent load carriage capabilities as it pertains to injury prevention.

The endosymbiont Wolbachia modifies temperature preference in insect host species

Chelsey N. Caldwell

Maternally transmitted Wolbachia bacteria infect most insect species and other arthropods. Wolbachia that naturally infect the fly Drosophila melanogaster (wMel) have the ability to block transmission of deadly viruses like Zika and dengue when transinfected into Aedes aegypti mosquitoes. Researchers have transformed Ae. aegypti populations with wMel on several continents, reducing human disease transmission. Despite the global prevalence of Wolbachia and its potential as a tool for biocontrol of human disease, we have limited understanding of how Wolbachia affect physiology and behavior of their natural Drosophila hosts. Research suggests the wMel strain modifies the temperature preference of D. melanogaster, but it remains unknown if Wolbachia generally influence host temperature preference. Using a thermal gradient apparatus, I determined the temperature preference of six Drosophila-host species in the presence and absence of their naturally co-occurring Wolbachia infections. Wolbachia infection significantly altered temperature preference for hosts infected with wRi (infecting D. simulans), wHa (D. simulans), wTei (D. teissieri), wSn and wSh (doubly infecting D. sechellia), and wMau (D. mauritiana). On average, infected flies preferred about a 0.5°C cooler temperature than uninfected flies. These results suggest Wolbachia effects on host temperature preference are not limited to wMel in D. melanogaster. Other data in our laboratory suggest that cool temperatures reduce Wolbachia titer in host bodies. We hypothesize that infected hosts choose lower temperatures to reduce Wolbachia abundance in their tissues. To test this, our lab is currently assessing Wolbachia titer in hosts reared in cool versus warm temperatures. Our work contributes to gaps in knowledge about Wolbachia effects on host behavior and motivates future analysis of additional systems, including transinfected mosquito vectors.

The Evolution of Dragons

Laura Janelle Mayfield, University of Montana, Missoula

Dragons have been depicted in human art as early as 4500 BCE. For centuries, these fantasy creatures have inspired countless folk and fantasy tales, as well as appearing in the art of different cultures around the world. Now there are thousands of different depictions of these huge, flying, fire-breathing lizards, but are any of them plausible? In this study, I referenced peer-reviewed scientific articles, phylogenetic analysis, and paleoart studies to create biologically-sound dragons. Basing the dragon lineage on a real branch of webbed-winged scansoriopterygids—an extinct family of climbing and gliding maniraptoran dinosaurs—I explored the possible wing-structure, fire-breathing abilities, and effects of adaptive radiation on these hypothetical dragons. My research, compiled in an illustrated and easy to read book, will be both entertaining and educational. Catching the attention of readers with the idea of scientifically correct dragons, I will then take them on an enlightening journey through evolutionary biology concepts such as homology, convergent evolution, and alternate evolution. It is important to educate the public on basic biology so that they better understand the scientific (or unscientific) research they are shown on media, helping to bridge the communication gap between scientists and the public.

The smell of attraction: cuticular hydrocarbon (CHC) profiles in a horned beetle

Chelsey N. Caldwell

The Asian Rhinoceros beetle, Trypoxylus dichotomus, is characterized by its large “pitchfork” horns on the heads of males. They use these horns to engage in male competition; battling each other for access to limited females. However, recent field studies suggest that females are choosy, selecting male mates based on traits other than horn size. In many insects, cuticular hydrocarbons are used by females to choose attractive, high quality males as mating partners. CHC’s are the molecules present in the waxy outer layer of the cuticle, functioning in waterproofing but also acting as pheromones. Individual CHC profiles can be indicative of physiological condition, as well as overall body size. In order to test whether CHC’s function this way in Rhino beetles, I conducted the first ever quantification of CHC profiles in this species. I collected CHC samples from both males and females and used Gas Chromatography-Mass Spectrometry (GC-MS) to test for correlations between CHC profiles, static condition (body size), and sex. I found that males and females have very different blends of cuticular hydrocarbons, that males are more variable from individual to individual than are females, and that some of this variation correlates with male body size. Thus, CHCs in rhinoceros beetles have all of the prerequisites of a reliable signal of sex and body size. Future studies will be needed to test whether CHC profiles also signal male physiological condition, and to explore whether females use these signals when they select mates.

Westslope Cutthroat Trout summer movement in western MT headwater streams

Hayden Cody, University of Montana, Missoula

In western Montana, Westslope Cutthroat Trout (WCT) have become increasingly confined to small headwater tributaries. In these systems, habitat degradation and Eastern Brook Trout (EBT) invasion pose major threats to WCT. Even though movement is a well-established key process enabling fishes to access spawning, foraging, and overwintering habitats and meet their resource needs in a heterogeneous stream environment, little is known about resident WCT movement in these headwater systems. As we work to conserve these populations, describing the scales of movement and understanding how they differ among streams is useful. The objective of this project was to describe summer WCT movement in seven Western MT streams of varying habitat characteristics (and differing Eastern Brook Trout presence) to contribute to limited existing knowledge on the topic. We captured 478 WCT and 250 Eastern Brook Trout across seven different headwater streams in June 2019 and implanted PIT tags in fish >70 mm in length. From July-September 2019, all streams were mobile read using a backpack RFID antenna three separate times and fish migration/emigration was monitored in three streams with fixed RFID antenna arrays. To reduce issues associated with potential shed tags, we only consider movements greater than 2m in the analyses. Approximately 82 percent of WCT moved more than 2m over the course of the season, although fish didn’t move long distances as median movement was less than 100m within each tributary. We found no correlation between WCT movement and fish length or EBT presence. Results indicate that study streams with high pool volume possessed limited fish movement when compared to streams with lower pool volume.

Winterization techniques for populations of Apis mellifera ligustica and Apis mellifera carnica in western Montana

Jessica Raty, University of Montana

Winterization techniques for populations of Apis mellifera ligustica and Apis mellifera carnica in western Montana


Abstract: The relationship between domestic honey bees (Apis mellifera ligustica and Apis mellifera carnica) and winter in a northern climate was studied to determine an effective, economical, and environmentally-sustainable method to keep the University of Montana Gardens’ colonies alive over the winter. Successfully winterizing bees would kickstart UM Gardens’ pollination and reduce the carbon impact from implementing new colonies every season, ultimately contributing to earlier, healthier and fuller plants and produce for the Food Zoo. The University currently owns two hives containing colonies of Apis mellifera ligustica and Apis mellifera carnica and has failed, thus far, to keep either colony strong enough to survive the winter. Keeping the honey bees alive over the winter will spare UM Gardens from having to buy new packages of bees every year, saving both money and start-up time. Established colonies lower the risk of implementing new colonies every season while reducing any footprint that shipping new bees across the country to Montana may have on the environment, which furthers UM Gardens’ mission of sustainability. I researched different types of winterization techniques from across the world by beekeepers in different climates, ultimately utilizing a combination of successful winterization techniques used by beekeepers in Alberta and Saskatchewan. These methods include using recycled wood chips and burlap for moisture absorption and reclaimed wood for the creation of candy boards. By applying sustainable and effective winterization techniques used in regions with similarly harsh winter climates, I designed and built my own system of winterization to keep the University of Montana’s bees alive over the winter. I applied my design to two hives by feeding the hives two different sugar mixes and building a windbreak. The results of this process will be fully revealed in spring 2020.