Microbial odors are essential for attracting mosquitoes to their hosts, but their role in attraction to other nutrient sources remains unknown. Sugar sources provide nutrients that lengthen the lifespan of both male and female mosquitoes and increase the vectorial capacity in host-seeking female mosquitoes. Microbial odors have been shown to act as proxy signals for the availability and composition of certain nutrients, such as amino acids and mineral ions, found in nectar and fruit sap. As microbes are abundant in flowers and fruits, we hypothesize that Microbial Volatile Organic Compounds (mVOCs) from these nutrient sources play an important role in the feeding behavior of mosquitos. To test this, we analyzed the microbial community of a known attractive fruit, Mangifera indica ‘Keitt.’ Microbial species were identified from our environmental samples through amplicon sequencing of the 16S and 26S regions. Isolates of the most abundant and relevant species were cultured for mVOC collection and chemical analysis via Gas Chromatography and Mass Spectroscopy (GC-MS). Behavioral assays were then performed with Aedes aegypti mosquitoes to observe the effect of microbial odors on the attractiveness of nutrient sources. Through the identification of behaviorally-relevant microbial compounds, we can gain a stronger understanding of the ecological dynamics of mosquito chemoreception and microbial community signaling, which can help inform future mosquito-control measures.

Malaria is one of the most prevalent diseases worldwide, with nearly half of the world residing in regions at risk of transmission. It is commonly spread to humans through the bite of a female Anopheles mosquito infected with Plasmodium spp. parasites. When feeding, mosquitoes ingest biogenic amines at concentrations found in the blood of the host. Adult patients with severe malaria have altered concentrations of serotonin and histamine in their blood compared to healthy individuals. Previous work showed the ingestion of serotonin and/or histamine concentrations associated with adult patients with severe malaria influenced key mosquito behaviors, such as the tendency to take a second blood meal, flight behavior, and visual object inspection—traits that are related to the transmission of malaria. However, the mechanisms by which serotonin and histamine modulate mosquito behavior remain unclear. Given the known involvement of these biogenic amines in physiological processes, we hypothesize that the ingestion of varying serotonin and histamine concentrations in Anopheles stephensi mosquitoes will alter their distribution across tissues. Mosquitoes are fed with deuterated serotonin and histamine at levels associated with patients with severe malaria versus healthy individuals. These deuterated compounds serve as tracers to distinguish endogenous (natural) from exogenous (ingested) serotonin and histamine in the tissue. The mosquitoes are dissected to retrieve the head as a proxy for the brain, the midgut, and sensory appendages including maxillary palps, antennae, legs, and proboscis. All tissue samples are extracted and analyzed via liquid chromatography-mass spectrometry (LC-MS) to distinguish and quantify deuterated and non-deuterated serotonin and histamine. By viewing all aforementioned tissue regions and comparing endogenous versus exogenous biogenic amine levels in the samples, we hope to understand the modulation of biogenic amine distribution in An. stephensi tissue and offer insights into possible connections between neuromodulators and behavior in the mosquitoes.

With an estimated billion people impacted by malaria, yellow fever, and zika every year, the mosquito has built its reputation as the "World's Deadliest Animal." Mosquitoes have an exquisite sense of smell, widely studied for its role in locating human hosts for blood meals. However, mosquitoes also use their olfactory systems to locate and access sources of plant sugar- an essential food that contributes to longevity and fecundity. In fact, only female mosquitoes feed on human blood, whereas all mosquitoes rely on plant sugar as a source of energy. Despite plant sugar being an essential nutrient source, relatively little is known about the identity of plants preferred by mosquitoes for their sugar meals. Unlike prior studies, this research utilizes molecular techniques to uncover the mosquito plant diet as found directly within the mosquito gut. In my research, I use wild-caught Aedes aegypti and Aedes albopictus mosquitoes- the vectors for yellow fever, zika, dengue, and chikungunya. I first extract the DNA within its digestive tracks, then use PCR to amplify plant-specific sequences. Following, I use DNA sequencing to identify the plant species consumed based on barcoding markers. Each plant possesses a unique bouquet of scent chemicals that may influence mosquitoes' foraging behavior. To investigate the profile of chemicals emitted by mosquito-attractive plants, I analyze their volatile organic compounds (VOCs) using gas chromatography-mass spectrometry (GC-MS). I first separate the individual VOCs present in the plant species using gas chromatography, then identify and quantify the compounds using mass spectrometry. By linking these chemical profiles to mosquito feeding preferences, this research helps determine which plant volatiles attract mosquitoes, providing insight into how odor-based mosquito traps can be improved. This work contributes to a broader understanding of mosquito feeding ecology, with applications for public health and mosquito population management.

Manduca sexta is a model lepidopteran insect organism which has been widely used in the field of chemical ecology due to its impressive olfactory senses. Odorant reception plays an important role in locating nectar sources, mating, and ovipositioning. Insects detect volatile chemical compounds (VOCs) present in their complex environment primarily through their sensory organ antenna. Each antenna is made up of thousands of olfactory receptor neurons (ORNs) and each neuron detects specific odor molecules with specific odorant receptor proteins. The whole genome sequencing of Manduca sexta has identified the major chemosensory receptor proteins: odorant (ORs), ionotropic (IRs) and gustatory (GR) but the role of each receptor is still unclear. In this project, we are investigating the role of female-biased odorant receptors OR5 and OR6, which might be involved in detecting VOCs present in their environment and play an important role in mating and oviposition. To investigate the role of these ORs, we have generated mutant strains by using a CRISPR/Cas9 approach and we are checking their effect on odor detection and oviposition behavior by comparing them with wild type strains. We are also performing an RNA-FISH experiment to visualize the ORs and locate the olfactory sensory neurons in the female antennae. In addition to this, we are also working on developing a neurogenetic tool which will allow us to measure the neuronal activity in response to different olfactory stimuli by generating a pan-neuronal BRP-GCaMP6s transgenic line.

The Manduca sexta hawkmoth, a proficient pollinator, employs its antennae to efficiently navigate its surroundings. The antennae have highly sensitive olfactory receptor cells, allowing the moths to have an acute olfactory sense and odor recognition ability, making the moth antennae an ideal candidate tissue for developing reliable biosensors. In contrast, commonly used portable artificial sensors are inefficient and inaccurate in chemical detection. To evaluate the moth antenna's effectiveness as a biosensor model, I assessed the longevity in neural activity of the antenna via an electroantennogram over 24-hour durations. To do this, I attached an excised antenna to a circuit board to measure voltage variations across the antennal nerves during odor stimulation to understand the baseline antennal lifespan. To increase the longevity of electrical activity, I formed a hydrogel solution to enclose the antenna to protect it from drying out, and Leibovitz's L-15 Medium so the antenna has access to Amino Acids to build and repair its tissues, serving as an energy source. Preliminary findings show a tradeoff between longevity and electrical activity, where the antenna-only trials had high voltage readings over 4 hours while the hydrogel antenna had less intense electrical readings over 8 to 12 hours. The hydrogel proved to be a quality medium for the diffusion of the L-15 media over long periods of time, preserving the antenna from dying prematurely. These results demonstrate that moth antennae are a suitable model for  highly accurate and efficient long-duration biosensors and support the feasibility of implementing them in devices that detect and identify substances of interest with a longer life span. Future work will apply machine learning methods for enhanced chemical discrimination in disease diagnosis.

One of the most deadly creatures, the Aedes aegypti mosquito, flies under the radar of the everyday person far too often. However, as known carriers of vector-borne diseases such as dengue fever, Zika and yellow fever, these insects pose a significant risk for disease transmission. Our study aims will investigate the role of human-associated odors and colors in mosquito learning. I plan to test hexanoic acid and acetophenone, both odors which are commonly found on human skin. Acetophenone in particular is found in increased levels in patients with dengue fever, so by studying that particular odor more can be learned about the spread of disease. Using aversive training, mosquitoes are exposed to these odors paired with a shaking stimulus that simulates defensive swatting. A human-like color (orange) will additionally be paired with the odor to assess whether visual and olfactory stimuli presented together will strengthen the learning. After training we will place them in a two-way maze to test whether the scent and/or color was learned or not. This research will contribute to understanding how mosquitoes associate human-related  visual and olfactory cues, which may help inform future research on mosquito behavior and control strategies.

Initial work from our lab has demonstrated that decision-making in OCD patients varies along the approach-avoidance behavioral axis depending on their response to deep brain stimulation (DBS) treatment. Patients who do not respond to DBS exhibit heightened risk aversion and consistently make avoidant decisions, while responders balance risk and reward in their decision-making process. In contrast, patients who receive excessive stimulation display disinhibited behavior, making choices that maximize reward regardless of potential risk. While this 2D task has provided valuable insights into approach-avoidance behavior in OCD, it does not fully capture the naturalistic behavioral responses observed in daily life. To address this limitation, we are developing a virtual reality (VR) task designed to quantify approach-avoidance behavior in a dynamic 3D environment. Participants complete probabilistic decision-making trials while wearing a VR headset, allowing for precise tracking of eye movements, hand positioning, and body dynamics. This project aims to provide a naturalistic task to analyze movement velocity and behavioral trends to identify neural biomarkers associated with approach-avoidance tendencies. This work will act as a stepping stone, enabling deeper insights into how neuromodulation regulates these behaviors. We are currently finalizing the development of this VR task and will demonstrate the feasibility of using this task to collect unique behavioral data on approach-avoidance behaviors. In the future we hope to use this task to identify biomarkers of approach-avoidance behavior and use that information to further refine neuromodulation treatment for psychiatric disorders.

Periodontitis is a severe inflammatory disease that damages the gingiva and has been linked to systemic diseases, such as diabetes, heart disease, and respiratory disease. These serious health complications express the importance of studying oral microbiota and their interactions. This research investigates the growth dynamics of Fusobacterium nucleatum and Porphyromonas gingivalis, two gram-negative anaerobic bacteria that play a significant role in inducing the progression of periodontitis, under mono- and co-culture conditions. By collecting monoculture and co-culture growth curves along with cell counts, and LIVE/DEAD BacLight staining we are identifying their optimal growth phases and interactions. With this information, we seek to optimize these cultures for downstream experiments, including bulk and single-cell RNA sequencing, to identify unique genes signatures from cell-cell interactions implicated in periodontal disease progression. Understanding these dynamics will contribute to future studies on the persistence of periodontal infections and broader research on gene expression.

Individuals with Stage II/III severe periodontal disease were recruited from the Graduate Periodontics Clinic at the University of Washington School of Dentistry using an approved IRB (STUDY00016871). Subgingival plaque samples were collected from four tooth sites: an active inflamed site, two adjacent tooth sites, and a distant healthy tooth site. Sterile paper points were inserted into the gingival sulcus for 30 seconds. DNA was isolated and extracted using the Qiagen AllPrep DNA/RNA Mini kit (Cat. #80204), then purified and concentrated using the Zymo Clean & Concentrator (Cat. #D4014) kit. 16S rRNA libraries were generated and sequencing was performed on the MiSeq platform (Illumina, San Deigo, CA, USA) using 300bp paired-end chemistry. Raw reads were processed and analyzed using Qiime2 and the DADA2 algorithm to generate amplicon sequence variants (ASVs), which were then classified using the expanded Human Oral Microbiome Database (eHOMD). Differences between tooth sites were assessed within and across individuals. Correlation between taxonomic levels and clinical data was also assessed. Data analysis is still being performed at this time. Based on previous literature (Pawolski et al, 2005, Kerns et al. 2023), we expect that a subgingival community gradient radiates from tooth sites affected with periodontal disease toward distant healthy sites. We aim to resolve this within individuals using ASVs for the first time. Additionally, we anticipate an increase in disease-associated bacteria within actively inflamed tooth sites, such species within Porphyromonas, Tannerella, and Treponema genera. Furthermore, we anticipate a gradient of perio-pathic disease-associated bacteria will decrease in relative abundance the further away from active diseased sites. We expect that results from our study will highlight the presence of a subgingival microbiome composition and enrichment of specific gram negative perio-pathic disease-associated species within clinically healthy tooth sites in patients with active periodontal disease despite the absence of clinically observed inflammation.

This study investigates the mechanical properties of A500-C steel round hollow structural sections through tension testing of coupons cut from tubes with various radii and thicknesses. Testing was conducted following ASTM A370 standards to evaluate relationships between carbon equivalent, strength ratios, and ductility. The carbon equivalent was determined using the International Institute of Welding equation with chemical compositions obtained from mill certifications. Strength ratios that were analyzed include measured tensile-to-yield strength, measured-to-mill certification values, and measured-to-nominal values, with comparisons to the ratios prescribed for design in the American Institute of Steel Construction Seismic Provision. The results indicate that most strength ratios and ductility metrics showed no significant correlation with HSS thickness or carbon equivalent. However, some trends were observed. Negative correlations were found between the ratio of measured to nominal ultimate strength and thickness, the measured tensile-to-yield strength ratio and thickness, the ratio of measured to mill-certification yield strength and carbon equivalent, and the ratio of measured to nominal yield strength and carbon equivalent. A positive correlation was observed between the measured tensile-to-yield strength ratio and carbon equivalent. These findings help provide insight into the variability of A500-C steel properties and their dependence on chemical composition and wall thickness, with potential implications for design assumptions in the structural design code.

Individuals with Stage II/III severe periodontal disease were recruited from the Graduate Periodontics Clinic at the University of Washington School of Dentistry using an approved IRB (STUDY00016871). Subgingival plaque samples were collected from four tooth sites: an active inflamed site, two adjacent tooth sites, and a distant healthy tooth site. Sterile paper points were inserted into the gingival sulcus for 30 seconds. DNA was isolated and extracted using the Qiagen AllPrep DNA/RNA Mini kit (Cat. #80204), then purified and concentrated using the Zymo Clean & Concentrator (Cat. #D4014) kit. Whole genome libraries were generated and whole genome sequencing was performed on a NovaSeq X (Illumina, San Diego, CA, USA). Relative abundance of species-level assignments represented by clusters were determined, and best matched genomes were then used to generate species pangenomes for comprehensive multi-genome wide read mapping and gene-level analysis. Taxonomic and gene level functional analysis was also performed. Data analysis is still being performed at this time. Based on previous literature (Basic and Dahlén, 2023), we anticipate that the activity of certain microbial metabolic pathways associated with oral disease and their functions will be heightened in the active disease site and lower on the non-affected site within strain-level disease-associated bacteria, such as those of the Porphyromonas, Tannerella, and Treponema genera. Specifically, we expect amino acid fermentation and lipid metabolism activity to be increased in the active disease site. We expect that results from this study will highlight the presence of heightened activity of bacterial metabolic pathways and functions at the strain level associated with specific gram negative perio-pathic disease-associated species within clinically healthy tooth sites in patients with active periodontal disease despite the absence of clinical observed inflammation.

Periodontitis, the most severe form of periodontal disease, affects ~50% of Americans and is expected to continue increasing as a major public health concern globally. Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) have been identified as synergistic oral pathogens that play a key role in advancing periodontitis via immune subversion; however, bulk RNA sequencing fails to elucidate the genuine synergistic interactions among these populations due to culture heterogeneity. To capture the true cell-cell interactions within complex polymicrobial communities we are utilizing microbial split-pool ligation transcriptomics (microSPLiT), a cutting-edge high-resolution single-cell RNA sequencing approach to illuminate novel interactions between Fn and Pg. To optimize microSPLiT for oral bacteria, this study explores quantifying RNA and DNA within Fn and Pg mono-cultures to pinpoint ideal sample populations and library preparation conditions needed for accurate single-cell gene expression. Qubit fluorometric quantification was used to quantify RNA and DNA. Pg possess known mechanisms that confer resistance to assorted antimicrobial agents; therefore, an increased concentration of enzymatic reagent may be necessary for permeabilization steps within microSPLiT. These findings are expected to help optimize microSPLiT for Fn and Pg while directly advancing our understanding of in vitro interactions between two pervasive oral pathogens.

The glymphatic system is a brain-wide network of perivascular spaces that facilitates waste clearance by exchanging cerebrospinal fluid and interstitial fluid, promoting the clearance of solutes like amyloid β and tau from the brain. Impairment of the glymphatic system has been demonstrated in models of traumatic brain injury (TBI), which has emerged as a risk factor for neurodegenerative conditions like Alzheimer’s disease. These findings suggest that glymphatic impairment may contribute to the development of post-traumatic neurodegenerative conditions, highlighting the potential for therapeutic intervention. Post-TBI sleep disruption, headaches, cognitive deficits, and the brain's subsequent vulnerability to downstream neurodegeneration is a particular concern among veteran and athlete populations. Prazosin, an alpha-1 adrenergic receptor antagonist, has been used clinically in these groups to treat trauma-induced nightmares, where it has been shown to alleviate sleep disruption. However, little is known about its impact on the glymphatic system which is most active during sleep. Here, we hypothesized that glymphatic function is enhanced by blockade of central adrenergic tone, and that this modulation would improve deficits observed in mild blast and impact TBI models. To evaluate the effect of prazosin on glymphatic function in a murine model, we assessed glymphatic function in sham, blast TBI, and impact TBI mice following a 28-day treatment with prazosin. Post-injury behavioral tests were conducted to evaluate cognitive impairments across treatment groups. Using an intracisternal co-injection of infrared and conventional fixable fluorescent tracer, CSF distribution was evaluated through a validated in vivo dynamic imaging technique and paired with whole-slice fluorescent imaging. Our findings so far suggest enhancement of glymphatic function in sham-TBI prazosin treated groups compared to the control. Continued study may better elucidate the mechanisms that underlie post-TBI neurodegeneration, and provide insight into potential targets for treating neuropathological conditions linked to glymphatic system impairment.

The Aedes aegypti mosquito is the primary transmission vector for dangerous diseases, such as yellow fever and dengue viruses. Emerging vector control efforts use strategies based on mass release of sterile and transgenic male mosquitoes to suppress local populations. These strategies rely on modified males outcompeting wild-type males in securing mates. However, our knowledge of mosquito mating remains limited. Male mosquitoes locate mates by identifying the distinct flight tone (WBF) females produce by beating their wings, which ranges from 450-600 Hz. But auditory neurons in male antennae are tuned to lower frequencies (200-400 Hz). It is hypothesized that as males fly and encounter a female, nonlinear interactions between male and female WBFs occur in the antennae, producing additional tones called distortion products. These distortion products are what male auditory neurons believed to be tuned to. Distortion-based hearing predicts that only flying males can detect a female flight tone. We tested this prediction by conducting behavioral phono-taxis experiments to compare the acoustically-mediated behaviors of flying and walking wild-type Ae. aegypti. Fifteen to twenty male mosquitoes that were either free to fly or had their wings removed were placed in a cage that had a speaker on either side. Pure tones, ranging from 100-1000 Hz, were played randomly from one of the speakers. Positive phono-taxis responses were quantified by recording the number of male mosquitoes that moved towards the speaker while a sound played. In contrast to the prediction of distortion-based hearing in mosquitoes, we found that non-flying, walking mosquitoes responded to similar female-specific WBFs that free-flying mosquitoes did. This challenges current theories on distortion products being the main way male mosquitoes hear and locate mates and suggests there are more complexities to the mosquito hearing process than initially believed. Future work will focus on further characterization of mosquito hearing processes.