Category Archives: 2021 Projects

Infection and Spore Yield of Daphnia Microsporidian

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Hannah O’Grady, a student at Mount Holyoke College, worked in the lab of Dr. Alex Strauss.

Abstract An important part of understanding how diseases spread and impact a community is understanding the tradeoffs that occur when a parasite generalizes. While sampling ponds in Whitehall Forest we discovered a potentially novel microsporidian that was able to infect at least five zooplankton in the Cladocera superorder at relatively high infection prevalence. We designed an experiment to investigate the potential costs of its generalism. We exposed isoclonal lines of two different species of Daphnia, each from two different lakes, to spores gathered from the dominant host in each lake to test whether the microsporidian was 1) more successful at infecting Daphnia of one species over the other or 2) whether it was more successful at infecting Daphnia from the same lake or 3) the same species from which the parasite spores were gathered. We also counted spore yield (a metric of parasite fitness) from the six infected species gathered from the field to test for differences across species, lakes or time. None of the Daphnia exposed to the spores in the lab became infected, leading us to hypothesize that there is an intermediate host for this parasite. Spore yields from field-collected hosts did differ significantly among host species, with higher spore yields in D. laevis (mean=446,415.55±412,977.53) and Diaphanosoma (mean=253,888.83±78,085.34) than in D. ambigua (mean=40729.12±41,396.68), D. parvula(mean=46250.06±49,432.78), and Simocephalus (mean=17083.17±13,025.14). There were no significant differences in spore yield across lakes or days. More research will be needed to find the intermediate host for the microsporidian as well as to determine its exact genus and species.

OGrady

Royally Split: Morphological divergence of parasites in milkweed butterflies

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Katie Yan, a student from Skidmore College, worked in Dr. Sonia Altizer‘s lab.

Abstract Ophryocystis elektroscirrha (OE) is a protozoan parasite found in Monarchs (Danaus plexippus). Same or similar OE-like parasites have been found in Queens (D. gilippus) and other Danaus butterflies. Experimental cross infection provided evidence of parasite specialization on natal host species via low infection rates on novel hosts, motivating in-depth analysis of parasite morphometric and genetic variation across host species from different locations. To examine OE and OE-like spore morphology across Danaus species, we looked at five host species from previously curated museum samples including: Jamaican Monarch (D. cleophile), Plain Tiger (D. chrysippus), Queen (D. gilippus), Common Monarch (D. plexippus), and Lesser Wanderer (D. petilia). Analysis of wild-collected museum spores showed that on average, Jamaican Monarchs and Common Monarchs had larger spores than other species. This relationship is consistent with the phylogenetic relationships of host similarity within the Danaus genus and further supports the hypothesis of parasite specialization on hosts. We then examined the influences of host and environmental factors on parasite morphology by analyzing spores from a cross-infection experiment involving Monarchs and Queens. Hosts in this experiment were fed either tropical milkweed (Asclepias curassavica) or swamp milkweed (A. incarnata). Monarchs and Queens were infected at a high rate by their natal parasites, some Monarchs were infected by Queen parasites, and no Queens were infected by Monarch parasites. Morphometric analysis of spores showed that Monarch parasites from Monarch hosts were largest, and that Queen parasites from Queen hosts were smallest; Queen parasites from Monarch hosts were intermediate in spore size. Other spore traits (shape, hue, density) were similar across treatments. Additionally, we found that spore size positively correlated with host wing area, suggesting that larger spores are found on larger butterflies. Milkweed species, sex, and final spore load did not predict variation in spore morphology. In summary, we found strong evidence for parasite specialization on different host species based on differences in spore size; further work should ask whether molecular genetic divergence of OE parasites across host groups matches differences in spore morphology and host phylogeny.

Yan

Scared stiff: Effects of a nematode parasite on fearfulness in bess beetles

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Anna Shattuck, a student from Tulane University, worked in the lab of Dr. Andy Davis.

Abstract Freezing is a defensive behavior seen in many different animals as a response to extreme threats such as predators. While research on freezing behavior is widespread, there is little known about how parasites may influence it. A non-lethal nematode parasite, Chondronema passali, can be found in the hemocoel cavity of bess beetles, Odontotaenius disjunctus. A single beetle can carry the burden of hundreds of these nematodes. Previous research has shown that parasitized beetles eat more and are more active, suggesting they are bolder and may be less prone to freezing. This experiment tests whether the nematode parasite influences fearfulness in beetles by exposing a population of bess beetles to different stressors and observing their freezing behavior in response to each. Following the trial period, beetles were dissected, and parasite load and sex were assessed. Out of 161 beetles, 14% were unparasitized by C. passali. We found that heavier parasite loads increase freezing durations in males and decrease freezing durations in females in response to a stressor. We suspected that female beetles experienced greater energy loss from being parasitized and laying eggs, making them more inclined to move and forage. To test this idea, we deprived 22 beetles of food for 48 hours in hopes they would display freezing behavior less when responding to a stressor. After being starved, only 4 beetles displayed freezing behavior, indicating fearfulness is tied to hunger.  Further investigation is needed in order to elucidate this relationship.

Shattuck

The Community Effects of Trematode Parasites on Species Interactions

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Kailah Massey from the University of Georgia worked with Dr. Emlyn Resetarits.

Abstract Trematode parasites have a complex life cycle that infects and castrates snails as their initial host. (Wood et al., 2007). The snails our team observed were Elimia type snails. These snails have top-down control over algae in aquatic ecosystems. Snails have total control over algae and influence lower trophic organisms that feed on algae. Changes done by top-down organisms have an inverse effect on the lower trophic level organisms. High levels of parasite infections can alter the resilience of an ecosystem. Furthermore, research has shown that trematode parasites can influence host consumption, potentially creating more of a strain and impact on ecosystems (Rosemond et al., 1993). To assess this claim, we conducted location surveys to quantify infection prevalence within snails at each site. We then constructed a chlorophyll consumption trial consisting of a blind experiment to determine if infected snails consumed more algae on average than uninfected snails. Our results indicate that a trematode infection can increase the consumption of chlorophyll in their snail hosts. Trematode parasites were responsible for up to twenty percent of chlorophyll consumption across our sites. Further research will include the differences between visceral and gonadal infections on the consumption of Elimia snails. 

REU-Poster-Kailah-Massey-1


The Tradeoff of Nutrition in Malaria Transmission

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Nathan Garcia-Diaz, a student from Willamette University, worked in the lab of Dr. Ash Pathak.

Abstract The effects of nutrition on malaria transmission was studied by collecting the most influential components of Vectorial Capacity. Vectorial Capacity (C) measures the Anopheline mosquito’s efficacy at transmitting the Plasmodium berghei parasites, and the largest factors impacting C are Extrinsic Incubation Period (EIP) and Vectoral Survival Probability (VSP). In order to force the mosquito to decide where to allocate nutrients, four conditions were created by combining two different treatments: Low Nutrient Treatment (1% Dextrose) and High Nutrient Treatment (10% Dextrose); Gravid (No Oviposition Site) and Not Gravid (Oviposition Site). Data was collected for EIP by examining sporozoite prevalence, and VSP was measured by mosquito mortality at fixed intervals after the infectious blood meal. To gain a more comprehensive notion on mosquitoes’ infectiousness, sporozoite density was measured alongside the other variables. The results from VSP data indicate that mosquitoes were most likely to survive if given the high nutrient treatment, and less likely to survive if the mosquitoes had the not gravid status. Additionally, when comparing EIP data between Gravid and Not Gravid statuses in the low nutrient treatment, gravid mosquitoes were infected sooner and at a higher rate than the not gravid counterparts. This pattern was seen again in the parasite density, gravid mosquitoes being more infectious than not gravid mosquitoes. It can be concluded that when infected mosquitoes are in a nutrient deficient state, gravid mosquitoes prioritize caring for its progeny rather than assembling an immunological response.

ngarciadiaz-REU-1

Assessing the impacts of Hyalophysa lynni infection on oxygen consumption of commercial shrimp

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Roland Berg, from Lewis & Smith College, worked with Megan Tomamichel and others in the lab of Dr. Jeb Byers.

Abstract Shrimp black gill disease (sBG), caused by the parasite Hyalophysa lynni, may be contributing to the recent declines in commercial shrimp populations off the Southeastern US coast. H. lynni attaches to shrimp gill tissue, triggering an immune response that causes gill melanization characteristic of sBG. While effective at killing the parasite, this immune response also deteriorates surrounding gill tissue; thus, sBG is speculated to alter host respiration. To address the impact of H. lynni infection on host oxygen uptake, we isolated individual shrimp in containers filled with artificial seawater and monitored the changing dissolved oxygen (DO) concentrations of their water over the course of five days. Afterwards, we diagnosed shrimp with sBG by pulling their gill tissue and performing a DNA extraction and PCR assay to identify the presence of H. lynni DNA. Although our results suggested that H. lynni infection did not impact host oxygen consumption, several other factors were significant predictors of a system’s DO concentration (most significantly time, shrimp length, water temperature, and shrimp gill color). Further research is needed to determine shrimp black gill’s effects on other gill functions, such as acid-base balance and ammonia excretion, as well as H. lynni’s impact on host respiration outside of restful experimental conditions.

Roland_REU_Poster_Scaled

OrNet: Spatiotemporal Analysis of Organelle Morphology

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Walter Avila, a student from Emory University, worked in the lab of Dr. Shannon Quinn.

Abstract Modeling changes in organelle morphology in response to infections is pivotal in studying pathogenic behaviors. Mitochondria are the most meaningful organelles to study because their structure changes dramatically in the presence of potentially fatal infections. Accurately modeling mitochondria could provide crucial information about severe infections. The project addresses this modeling concern by continuing development on OrNet (Organellar Networks). This Python framework constructs dynamic social network graphs of fluorescently tagged cells in microscopy videos to analyze the spatiotemporal relationships of mitochondria. These graphs are leveraged into eigenvalue arrays to describe organelle morphology over time quantitatively. We sought to improve the Temporal Anomaly Detection (TAD) technique in OrNet, which utilizes the arrays and detects when abnormal events occur in a video, indicative of organellar changes. The number of arrays rows and columns corresponds to the number of frames and eigenvalues, respectively. Currently, TAD takes the average eigenvalue at each frame and computes the distance between the average and the mean of a few preceding averages. If that distance exceeds a threshold, the frame is an outlier. This parameter-sensitive technique gives disproportionate weight to trailing eigenvalues. We assess how differently this model behaves when introducing a weighted average eigenvalue per frame. Using live videos of HeLa cells in different mitochondrial conditions, on average, the weighted average TAD declares a higher proportion of anomalous frames in cell videos from each mitochondrial condition than the simple average TAD code. This information about our model will guide how we implement OrNet in the future. Comparing how videos are analyzed differently by both TAD versions and other comparisons are necessary. We ultimately aim to use OrNet in large-scale genomic screenings of Mycobacterium tuberculosis mutants to understand better how these pathogens invade cells and induce cell death at the genetic level.

Avila

Frugivory Richness Predicts Ebola Spillover in Africa

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Mireya Dorado, a student at Northeastern University, worked in Dr. Patrick Stephen’s lab studying pathogen spillover

Ebola is a deadly filovirus that infects a variety of mammals including humans. Since the first documented outbreak i n 1976, there have been numerous field studies searching for the source of the spillover of Ebola. Only a few studies have directly investigated the effect of mammalian host biodiversity. These studies have been limited to the diversity of known Ebola hosts and bats. However due to Ebola’s broad host range, there has not been a systematic approach to which hosts may be important for spillover. Therefore, our goal was to determine whether and what aspects of mammalian diversity play a significant role in predicting Ebola spillover events. We calculated species richness of mammals in 50 kilometer by 50 kilometer grid cells across Africa. Statistical analyses were based on a presence absence approach, which compared species richness at sites of spillover t o pseudo-absence background locations. We used bagged logistic regression, a machine learning method, to create statistical models testing how well species richness of different mammal subgroups predicted spillover. Overall, we found that Cercopithecidae and Pteropodidae were the strongest taxonomic predictors of spillover (mean AUC=0.943 and 0.936 respectively), but diversity of frugivorous species was the best overall predictor (mean AUC=0.956). This strongly implicates a role of fruit in Ebola transmission and the significance of fruiting and masting seasons as ideal times for spread of infection.

Dorado

Investigating the Clinical Relevance of Patient-Reported Symptoms for Influenza Triage

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Jacqueline Dworaczyk, a student at Arizona State University, worked in the lab of Dr. Andreas Handel.

Telemedicine has become increasingly popular during the age of Covid-19. During a public health crisis, telemedicine could be used as a tool to triage patients and prevent burden on the health care system. In an exploratory data analysis, we investigated whether a symptom questionnaire could be used to predict influenza diagnosis. A symptom questionnaire containing 19 upper respiratory symptoms was administered to patients and clinicians (n = 2475 patients) at University of Georgia’s Health Center during the 2016-2017 flu season. Five clinical decision rules were applied to the symptoms reported by the patients and clinicians. The clinical decision rules’ performance when predicting influenza diagnosis was assessed using AUC, F1, MCC, sensitivity and specificity. A 7-11% drop in AUC was observed across all clinical decision rules when using the patient-reported symptoms as opposed to the clinician-reported symptoms.

In a sensitivity analysis, we evaluated the clinical decision rules’ ability to predict true, lab-confirmed influenza in a subset of our population who received PCR testing. While clinician-reported symptoms still performed better than patient-reported symptoms, the difference in AUCs when predicting PCR was significantly smaller. These differences in performance may be partially explained by a lack of agreement between patients and clinicians on the presence of signs and symptoms. Agreement between the patients and clinician’s questionnaire responses (n = 2475) was quantified using Cohen’s Kappa statistic and found that at best, patient’s and clinician’s had moderate agreement on three of the nineteen symptoms assessed.  Overall, it was found that using a patient symptom questionnaire to predict physician diagnosis led to a reduction in accuracy. Further studies need to be done to assess the clinical relevance of this reduction.

Dworaczyk

Phylogenetics of Dracunculus Nematodes in North America

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Madeline Giner, from the University of Texas at San Antonio, studied the phylogenetics of Dracunculus in the lab of Dr. Christopher Cleveland.

The Dracunculus genus contains parasitic nematodes that infect a variety of hosts, including reptiles and mammals. Dracunculus medinensis, the Guinea worm, has gained much attention due to its history of infecting humans. Less studied are other dracunculids, including Dracunculus insignis and D. lutrae which are native to North America. D. insignis can infect a variety of mammalian hosts, whereas D. lutrae specifically infects North American river otters (Lontra canadensis). The goal of this project was to investigate the genetic diversity of Dracunculus in wildlife species from the Eastern USA and investigate spatial and host patterns of infection. Phylogenetic relationships were examined using the cytochrome c oxidase I (COI) gene targets. We hypothesized otters would predominantly have D. lutrae, and D. insignis would be identified from other hosts. Our experimental methods included DNA extraction, gene-specific amplification (PCR), Sanger sequencing, and phylogenetic analysis using the software Geneious. Our results indicate that a majority of worms from otters (19/65), raccoons (Procyon lotor, 22/22), and Virginia opossums (Didelphis virginiana, 2/2) were D. insignis. However, a worm from an otter from Florida had 100% identity to a novel Dracunculus sp. previously detected in Georgia, and a Georgia otter worm is closely related to another novel dracunculid species from Florida. In conclusion, D. insignis was present in most locations and hosts, D. lutrae is absent, and an additional host is now known for two novel Dracunculus species. These data provide new information about Dracunculus diversity in US wildlife, but additional investigation is required.

GIner

Infectious disease professionals need better training in modeling

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Salil Goyal, a student at the University of California Berkeley, worked with Dr. Andreas Handel.

Models have become increasingly important in the field of infectious disease epidemiology, and broadly in the field of public health, in recent years because the ability of scientists and officials to make educated decisions based on data is important. However, many authors have stated recently (and especially during COVID) that programs that train people working with infectious diseases generally do not impart adequate training in understanding models. While many resources aimed at teaching infectious disease modeling do exist, they are aimed at different audiences and there currently exists no synthesis of these resources. As a potential solution to the problem, we offer here a comprehensive review of resources relevant to the pedagogy of infectious disease epidemiology, with a focus on modeling.

Goyal

What’s The Buzz Around Hydrogen Peroxide? An analysis of honey bee preference and mortality to differing hydrogen peroxide concentrations

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Carlos Martinez-Mejia from New York University worked with Dr. Lewis Bartlett.

Abstract Hydrogen peroxide has proven antimicrobial benefits and is created in honey when honeybees add glucose oxidase. This behavior makes honey bees another self-medicating animal and gives honey the longevity and antiseptic properties that we see. Hydrogen peroxide has been tested for toxicity in honeybees as well as parasites found in colonies. Honeybees have previously been seen to withstand higher concentrations of hydrogen peroxide than their insect counterparts. However, the exact toxicological limit of hydrogen peroxide that honeybees can withstand has not been officially established. Likewise, understanding if honeybees can identify these different concentrations and if this changes their preferences is still under investigation. Here we show that honeybees avoided hydrogen peroxide solutions in both sucrose and glucose when compared to the control sugar solution- differences in concentration and sugar were found to be significant in preference behavior. In addition, as hydrogen peroxide concentration increases- as does the proportional death of honey bees. At 4% H2O2 less than 40% of honey bees died. Knowing that hydrogen peroxide production for honey bees is a very metabolically taxing process- the hypothesis was that honey bees would rather have higher concentrations than go through the tiring process themselves. The results instead showed that on average honey bees avoided hydrogen peroxide when compared to the control sugar solution. Similarly, although honey bees have a higher tolerance toward hydrogen peroxide than other insects- 10% H2O2 was believed to be fully lethal but in some cases as many as 30% of honey bees survived at this dose. These results shed light on the relationship that honey bees have with hydrogen peroxide both regarding possible preference and toxicity threshold. We anticipate these trials to be a starting point for future pollinator health and pest control studies. Understanding the robustness of honey bees to such high concentrations of hydrogen peroxide opens the door for pesticide research that can effectively terminate pests while leaving the mass majority of honey bees unscathed.

Martinez_Mejia