BIOLOGY, ECOLOGY & MANAGEMENT OF EMERGING DISEASE VECTORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1015010
• Multistate No.: NE-1443
• Project Start Date: Dec 10, 2017
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881

Performing Department
Biological Sciences

Non Technical Summary
Ticks and mosquitoes and the pathogens they carry are well studied organisms and can be models to improve our understanding of wildlife and human diseases.Exploring the Lyme disease system, this first project in the proposed work aims to understand why we observe high rates of infection of deer ticks with the Lyme disease agent, a bacterial spirochete callled Borrelia burgdorferi. We hypothesize there may be some, as of yet unknown, benefit for ticks of being infected with B. burgdorferi. Through controlled infection experiments in the laboratory, we will determine what benefits infection may offer to ticks under a variety of environmental conditions.The second project aims to improve our understanding of an emerging threat, the invasive mosquito, Aedes albopictus that is currently expanding its territory into the Northeastern United States. The proposed work is to monitor the population of Ae. albopictus in Rhode Island, and identify factors of the landscape that could facilitate its spread.The third project explores the understudied relationships between mosquito vectors and plants. Mosquitoes develop in standing water, often in habitats with plants of many different species. One such species is the carnivorous aquatic plant known as the common bladderwort. This project aims to explore the impact of this plant predator on the survival, development, and behavior of mosquito vectors. In addition, the project aims to develop the plant as means of controlling these mosquitoes in natural and artificial environments, such as aquatic gardens and backyard bird baths. Another example of how plants interact with mosquitoes is through nectar feeding. This project also explores the role of flowering plants to attract or repel mosquitoes as they search for places to lay eggs.

Animal Health Component

40%

Research Effort Categories

Basic

50%

Applied

40%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
721	3110	1070	25%
722	2499	1060	50%
721	3120	1070	25%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3120 - Spiders, mites, ticks, and other arthropods; 3110 - Insects; 2499 - Plant research, general;

Field Of Science
1060 - Biology (whole systems); 1070 - Ecology;

Keywords

evolution

ecology

entomology

invasive species

biocontrol

carnivorous plants

host-pathogen interactions

Goals / Objectives
Integrated tick management and community-centered approaches, including understanding the biology and ecology of novel and emerging tick-borne pathogens Ae. albopictus and Ae. aegypti, with a focus on surveillance, invasion ecology, genetics New Control Tools, including socio-ecological approaches

Project Methods
An often unstated assumption of species interactions in the Lyme disease system is that pathogens negatively impact vertebrate hosts (such as humans) but have little to no negative impact on ticks. However, recent evidence suggests that tick-borne pathogens, while negative for hosts, may have either negative or beneficial effects on ticks. There is evidence in the larger protozoal parasites like Babesia, which causes tick-borne disease Babesiosis, that infection and infection intensity (i.e. number of pathogens) can change tick traits of survival and fecundity. The presence of Babesia has been observed to reduce tick fecundity (Davey 1981) even while improving feeding success and survival of ticks when feeding on hosts with higher pathogen loads of Babesia microti (Randoph 1991). In contrast, both fecundity and feeding success were impaired in ticks infected with Theileria parva (Watt and Walker 2000). For bacterial and viral pathogens, this question of the impact of pathogens on the ticks remains open. This project focuses on demonstrating the impact of acquiring the bacterial spirochete, B. burgdorferi, on larval tick survival under different environmental conditions in the species I. scapularis.Experiment 1: Based on the hypothesis that B. burgdorferi impacts vector and host transcription to increase likelihood of transmission, the proposed experiments measures rates of engorgement of larval and nymphal ticks compared across infected and uninfected ticks. Ticks feeding on different body parts of mammalian hosts (white footed mice) will be compared. In addition to engorgement, RNA transcription in uninfected vs infected ticks will be compared to begin to elucidate the molecular mechanisms of impacts to engorgement.Experiment 2: Based on the hypothesis that B. burgdorferi - I. scapularis interactions (i.e. pathogen acquisition) increases tick fitness, the proposed experiment examines the differential survival of larval and nymphal ticks that are infected or uninfected with the spirochete under schemes of environmental stress gradients. The environmental stress will be induced via humidity conditions to mimic desiccation stress as well as via temperature conditions. Finally, freezing injury stress events will be assessed.Experiment 3: Based on the results of experiments 1 and 2, an observational experiment testing proportion of infection in I. scapularis in natural environments will b econducted to assess, across life stages, tick recruitment based on infection status. The methods for this experiment are to be determined based on the results of the previous experiments.Invasion Ecology of Aedes albopictus in Rhode IslandField based methods include mosquito collections via traditional methods (light traps, gravid traps, larval dipping), and with oviposition traps and BG sentinel traps that target container breeding mosquitoes, namely Ae. albopictus. It is important to note that in previous years surveillance traps have been those targeting Culicine mosquito species capable of transmitting WNV and eastern equine encephalitis (EEE). These traps are not efficient for trapping Ae. albopictus. Therefore, current routine sampling is not adequate for detecting the points of entry, persistence, or expansion of Ae. albopictus. We will design and implement a sampling program at three types of sites: 1) Import pathways, 2) tire dumps and 3) areas with warmer winter temperatures. Sampling at these sites will be implemented for 5 years.Weather monitoring: Currently there are 4 weather monitor stations in the state of Rhode Island. We will place HOBO data loggers that monitor temperature, humidity, and light level at sampling sites throughout Rhode Island that correspond to mosquito sampling sites. These data will be gathered continuously for the study period of 5 years.Laboratory Component: We will test survival to maturation of Ae. albopictus when subjected at each life stage to extreme cold temperatures similar to fluctuating day and night temperatures for each season. Specifically, we will test the impact of a cold temperature events and duration of cold temperatures that have been observed in sampling sites. Each life stage will be evaluated (egg, larva, pupa, adult). Eggs will be collected via ovitraps and transported to the USDA Biocontainment Facility at the University of Rhode Island. Insects will be reared in environmental chambers at controlled temperature, humidity, and light-dark schedule. The impact of low temperatures and extreme cold events will be evaluated on survival and development.Modeling of Invasion of Ae. albopictus: We will use a combination of statistical modeling and geographic information systems to create predictive models of the invasion potential of Ae. albopictus for RI based on the sampling data collected, experimentally estimated cold tolerance of the introduced Ae. albopictus eggs found in sampling, weather monitoring data, and spatial data on points of entry and calculated rates of spread for Ae. albopictus. We will develop a predictive spatio-temporal model of the invasion of the Zika vector for Rhode Island. This model will have broad applications for use in areas in the United States that are along the northern edge of the distribution of Ae. albopictus.Novel plant-based methods for biocontrol of larval mosquitoesUsing an experimental approach that incorporates field and laboratory based studies we propose two aims. Aim 1: To cultivate predatory plants for biocontrol of larval stages of Zika vectors. Aim 2: To develop novel botanical attractants for control and surveillance of Zika vectors. The proposed work directly relates to public health and disease prevention through Zika vector control. Note that each experiment will likely consist of a series of experiments but are grouped by general aim and hypothesis.Experiment 1: Laboratory evaluation of the bladderwort plant species (both invasive and native Utricularia species) as a predator of early and late instar Zika vector larvae and pupae. Cultivation of bladderwort will be in the Couret lab at the University of Rhode Island. No-choice experiments of bladdewort predation of mosquito larvae will be conducted in the laboratory as well as experiments with two or more species of mosquitoes.Experiment 2: Laboratory evaluation of the efficacy of Utricularia species in the control of Zika vector larvae with variations in environmental conditions of temperature, varying volume, water acidity, and nitrogen levels. Conducted at the University of Rhode Island in the USDA Biocontainment Laboratory.Experiment 3: Field study of the use of Utricularia species (identified in Experiment 1 and 2) in the control of Zika vectors Ae. aegypti and Ae. albopictus. Collaborators will be determined for the field study to evaluate the applied use of bladderwort as a control measure incorporated into an integrated vector management plan.Experiment 4: Controlled laboratory study of oviposition site preference with the presence/absence of alternative plants. Mosquito eggs will be provided by BEI Resources MR4 program through the CDC Entomology Branch. Methodology: Choice experiments for oviposition site preference conducted in experimental tents (BugDorm Insect Rearing Tents) for each mosquito species and for multiple plant species (identified in Experiment 1).Experiment 5: Laboratory study of importance of nectar components on adult longevity and male mating competitiveness in Ae. aegypti and Ae. albopictus. (Nectar sources identified in Experiment 4)Experiment 6: Depending on the results of Experiments 1-5, field studies of the use of botanical-baited oviposition traps as part of an integrated vector management program.

Progress 12/10/17 to 09/30/19

Outputs
Target Audience:Target Audiences: This work is interdisciplinary and therefore aimed at multiple audiences which included vector control/management professionals, public health/entomologists, biological science/vector-borne disease researchers, and the general public. Efforts: We conducted experiments testing the utility of the carnivorous aquatic plants in the genus Utricularia in lab and field trials to determine their impacts on local and invasive mosquito vectors. The findings from this research were presented at the regional Northeastern Mosquito Control Association meeting, the national American Mosquito Control Association meeting, and submitted to the journal Parasites and Vectors (currently under review). In addition, the process of completing this work was shared on social media accounts with the general public as well as in a talk at the RI Natural History Survey meeting. We conducted experiments to study the ecological interactions between black legged ticks, Ixodes scapularis, the pathogens they transmit, and their hosts. These experiments demosntrated that the acquisition of pathogens during the larval stage alters feeding behavior and survival of ticks. These findings were presented at the national joint meeting of the Society for the Study of Evolution (Evolution2019), the Evolution in Philadephia Conference (EPIC 2019), and will be presented at the international congress of entomology in 2020 (ICE2020). In addition, these findings were shared at the URI Open Farms event with the general public. We continued a field sampling project monitoring Aedes populations to identify the factors that facilitate or constrain the distribution of this invasive species. We presented a retrospective analysis of Aedes japonicus at the Northeastern Mosquito Control Association Meeting. Changes/Problems:There was a major delay in the construction of the environmental chambers needed to complete laboratory experiments of mosquito development to develop biological controls and study tick biology. These chambers were not completed until July 2019. The data collected in 2019 is currently being prepared for publication. What opportunities for training and professional development has the project provided?Professional Development for PI: Participation in the NE Center for Excellence in VBD Tick Workshop to learn tick biology, identificaiton, and control techniques. Graduate students: The project supported 1 year of training and research support for Samantha Schofield in pursuit of a PhD. Undergraduates: The project has supported 2 undergraduates for summer research in 2019. How have the results been disseminated to communities of interest?There is one publication in review in Parasites and Vectors and another in prep that will be submitted to Nature Scientific Reports. Dr. Couret has presented findings at local, regional, and national conferences and will present at the international congress of entomology in 2020. What do you plan to do during the next reporting period to accomplish the goals?This is the final report for multi-state project NE1443. We have joined a new multi-state project NE1943 on which we will report in the next reporting period.

Impacts
What was accomplished under these goals? (1) In 2019 through a laboratory experiment and a pilot semi-natural field experiments we have expanded the foundational understanding of the biology and ecology of Borrelia burgdorferi and Ixodes scapularis. We discovered that the acquisition of the pathogen by larval ticks a) alters their feeding/engorgement behavior, b) increases their survival under an array of humidity conditions, and c) as nymphs infection with B. burgdorferi increases their horizontal and vertical movement within the leaf litter and on questing dowels. (2) We have surveilled Aedes albopictus in RI and have discovered that while is range is wider than previously expected based on public health mosquito surviellance data, its expansion is associated with economically distressed neighborhoods. (3) We explored through controlled laboratory experimentation a natural predator as a biological control of mosquito larvae. Freshwater carnivorous plants in the genus Utricularia demonstrate they are a highly capable predator of mosquito larvae in the Aedes and Culex genera. Our research demonstrates these plants can be cultivated in small containers for control of container-breeding mosquitoes. These plants are widely distributed throughout the US and the genus has over 200 species distributed worldwide.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Couret J, Notarangelo M, Veera S, Le-Claire Conway N, Ginsberg HS, and Lebrun RL. under review. Biological Control of Aedes Mosquito Larvae with Carnivorous, Aquatic Plant, Utricularia macrorhiza. Parasites and Vectors
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Couret J, Lavoie N, Gettman A, Lebrun R, Ginsberg HS. 2019. A Retrospective Geospatial Analysis of the Environmental and Socio-economic Determinants of Aedes japonicus in Rhode Island, Northeastern Mosquito Control Association Meeting.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Couret J 2019 Lyme disease agent, Borrelia burgdorferi sensu stricto directly affects larval bloodmeal engorgement and survival of Ixodes scapularis. Evolution 2019 Meeting
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Predation efficiency of carnivorous aquatic plants: A novel biocontrol method for container breeding mosquito vectors. American Mosquito Control Association Meeting 2019. Couret J, Notarangelo M, Lebrun RL, Ginsberg HS.

Progress 12/10/17 to 09/30/18

Outputs
Target Audience:We have communicated findings to the general public, students of biological sciences, and researchers in the field of vector biology and medical entomology during this reporting period. We participated in the URI Open Farms outreach event at East Farm on August 25th, which had an estimated 600 visitors during the course of the day. During this event we shared our research results on the use of carnivorous plants for biocontrol of mosquitoes and on Lyme vector-pathogen interactions, as well as sharing basic science information on entomology, vectors, and vector-borne disease. Our portion of this event was facilitated the three undergraduate students, the technician, and PI Couret. Three undergraduate students have contributed to this project during the reported period, and have communicated their research through oral presentations and postersby participating in the annual Undergraduate Research Symposium in the College of the Environment and Life Sciences. PI Couret has communicated this work duiring the reporting period through the URI Alumni periodical Quadrangles, at the Evolution in Philadelphia Conference (EPiC 2018), at the International Conference on Lyme/Borreliosis in Atlanta Georgia (ICLB 2018). Three manuscripts are in preparation to be submitted to Nature: Scientific Reports, Journal of Applied Ecology, and Parasites and Vectors. During the reporting period a portion of this work has been published in the Journal of Medical Entomology (Couret et al. 2017, p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 11.0px Helvetica} 54(4): 1055-1060). Changes/Problems:Due to delays in purchasing and installing environmental chambers to study mosquito and tick vector biology in the laboratory setting, we have focused primarily on field based and modeling projects to this point. In the next reporting period we expect to shift to include addiitonal laboratory based rearing experiments to evaluate control methods rigorously under controlled environmental conditions. What opportunities for training and professional development has the project provided?During the reporting period this project has provided research opportunities and paid research time for 3 undergraduates, two from historically underrepresented backgrounds and one with visualdisability. How have the results been disseminated to communities of interest?We communicate to communities of interest primarily through social media, primarily through Instagram and Twitter. Over the reporting period our followers doubled and we now have approximately 630 followers on instagram and 300 on Twitter. Social media allows for rapid bites (pun intended) of information on our latest research on vectors to be shared with interested communities. We have connected with not-for-profit vector groups including patient advocates, museums, and science communicators. What do you plan to do during the next reporting period to accomplish the goals?We aim to publish additional manuscripts, present at conferences and invited talks, and continue to encourage and engage undergraduates in vector ecology and biology research.

Impacts
What was accomplished under these goals? (1) We developed a predictive model for the xenodiagnosis of the presence of Borrelia burgdorferi in reservoir hosts, white-footed mice, that is a useful tool for the surveillance of zoonotic Lyme (Couret et al. 2017). Further we discovered evidence that the spirochete Borrelia burgdorferi may be manipulating the tick vector in order to provide a transmission advantage, expandingour understanding of the biology, ecology, and evolution of tick-borne pathogens. (2) We have continued to survey Rhode Island for the invasive vector Aedes albopictus. Through the use of targeted surveillance techniques that are more sensitive to the presence of Aedini mosquitoes, we have shown the invasion of Ae. albopictus is much wider than previously thought or acknowledged through the RI State surveillance program. We are currently testing several hypotheses to explain the presence/absence of Ae. albopictus. Our preliminary results indicate that presence is correlated with economically distressed neighborhoods. (3) We are investigating a novel biocontrol method for Aedes mosquitoes using the aquatic plant Utricularia vulgaris. This herb is a predatory plant that mechanically traps small invertebrate prey. While it is widely distributed in freshwater ponds throughout the United States, it's use for biocontrol in small containers, backyard bird baths and ponds, has not been investigated. Our laboratory experiments have established a high predator efficiency for Aedes aegypti and Aedes albopictus, results we are preparing for publication and presenting at the 2019 AMCA meeting.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Couret, J*., Dyer, M.C., Mather, T.N., Han, S., Tsao, J.I., Lebrun, R., Ginsberg, H.S. 2017. Using Tick Scutal Indices of Larval Ixodes Scapularis (Acari: Ixodidae) As Method of Determining Pathogen Acquisition. Journal of Medical Entomology. 54(4): 1055-1060
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: International Conference on Lyme/Borreliosis (Atlanta, GA 2018) Effect of Infection Status on the Rate of Engorgement and Survival of Larval Ixodes Scapularis (Couret, J.*, Tsao, J., Han, S., Dyer, M., Stallworth, J.J.!, Mather, T.M., and Ginsberg, H.)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Evolution in Philadelphia Conference (EPiC 2018): Evidence of adaptive parasite manipulation by Lyme disease agent, Borrelia burgdorferi in black-legged ticks (Ixodes scapularis) (Couret, J.*, Tsao, J., Han, S., Dyer, M., Stallworth, J.J.!, Mather, T.M., and Ginsberg, H.)