Progress 10/01/19 to 09/30/21
Outputs
Target Audience:The target audience for our project are communities that recreate, work, or live in forest lands as they are most at risk for tick bites and vector-borne disease transmission, as well as people that own animals associated with areas at risk for tickborne disease. This includes, in particular: Hunters, farmers, pet owners, parents, gardeners, Extension educators, field biologists, agricultural workers, recreational participants (hiking, biking, running, etc.), equestrians, camp educators, and campers. Changes/Problems:Objective 3: Develop tick surveillance and control tools that utilize candidate tick pheromones and host kairomones. Proposed aim 3.1. Test identified semiochemicals for Ix. scapularis attraction. Proposed aim 3.2. Design an attractive questing device. Proposed aim 3.3. Create an improved tick drag. The continuing COVID-19 pandemic was a major influence in the project. Field collections of animals was limited during 2020 due to institutional and State requirements to limit the spread of the pathogen. Given that the dominant and consistent semiochemicals from conspecific ticks and animal odors have either not been isolated or are just starting to be isolated, we were unable to complete objective 3. That being said, we switched to using live animals in our tick bioassays and our odor collection profiles look more comprehensive from live animals instead of the animal odor proxies such as cotton or excretory products. We also modified and streamlined our odor collection and tick bioassays and hope to identify and quantify these odors in the future so that we can test the chemical compounds for Ix. scapularis attraction or deterrence with the ultimate goal of incorporating these compounds into tick control or surveillance tools. What opportunities for training and professional development has the project provided?This project supported a graduate student and a post-doctoral scholar that were mentored in lab animal care, laboratory studies, and tick ecology. How have the results been disseminated to communities of interest?These results will be analyzed and summarized to be reported in peer-reviewed manuscripts and paper presentations at regional and national conferences. In addition, these results will serve as the baseline to continue to improve our understanding of tick ecology and host associations so that novel surveillance and control tools can be developed. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Assess nymphal Ix. scapularis responses to pheromones produced by conspecific ticks. Proposed aim 1.1: Assess attraction Ix. scapularis to odors produced by conspecific ticks. We tested groups of live ticks to determine if individual Ixodes scapularis and Ixodes ricinus are attracted to congregated conspecific ticks. For this objective, we established a protocol to test attraction between conspecific ticks in a 4-port.Thus far, we have replicates of I. scapularis and I. ricinus attraction to their respective conspecific ticks. For I. scapularis, we tested laboratory-reared (OSU Tick Laboratory, "southern lineage") and field-collected ticks (ticks collected from PA, northern lineage) and compared their responses. First, we tested attraction of the southern lineage (IsS) to other IsS ticks. Out of 44 ticks that were tested in different replicates, 26 ticks did not exhibit any response. Furthermore, out of 18 ticks that made a choice, 3 ticks made a choice towards the group of conspecific ticks and 15 ticks chose the other ports (controls with air flowing through empty glass chambers).Because we initially found low frequency of movement from IsS and nymphal IsN ticks were not yet in season, we also tested Ixodes ricinus (Ir) attraction to a group of Ir ticks. Out of 40 Ir ticks, 20 ticks chose the other ports in the arena, 10 ticks were found in the port leading to the group of conspecific ticks, and 10 did not exhibit any response. Proposed aim 1.2. Determine the chemical constituents of candidate pheromones produced by conspecific ticks. After testing the ticks in the bioassay, we took the tick groups in the glass chamber and extracted volatiles that ticks may release to attract conspecific ticks. We used two quantities to see if different numbers of ticks would produce varying amounts of volatiles. Tick groups of 25 nymphs and 50 nymphs were washed with n-hexane and then allowed to evaporate to make potential compounds more concentrated for direct injection into the GCMS machine.During the extraction process and GCMS analyses, we did not find any major odors from the tick species we tested. We hypothesize that the low frequency of ticks choosing conspecific groups of ticks could be related to tick feeding, such that individual ticks may be attracted to conspecific groups of ticks if the groups are actively feeding on a host. Objective 2: Evaluate attraction of Ix. scapularis to semiochemicals produced by white-footed mice. Proposed aim 2.1. Assess the attraction of Ix. scapularis to excretory products produced by mice. We established a protocol and collected preliminary data on different forms in which odors can be collected from animals. We tested used cotton collected from small mammal traps ("trap cotton"), cotton that was in the trap with white-footed miceor southern red-backed voles for 6 hours ("mouse hours" cotton), and fresh urine and fecal samples from white- footed mice and red-backed voles, when possible.Since the amount of time an animal spends in a trap can vary depending on when the animal first enters the trap, we standardized the amount of time a small mammal spent with cotton. We placed cotton into a clean trap with a white-footed mouse or red-backed vole for a designated period of time, approximately 6 hours.Out of 45 tested ticks, 41 ticks made a choice, with more ticks choosing the clean cotton.For both types of tests, ticks more often chose the control side, with the difference more pronounced for fecal samples. Proposed aim 2.2. Determine attraction of Ix. scapularis to hosts and hosts with conspecific tick burdens. We established a protocol to test Ix. scapularis host choice between white-footed mice and red- backed voles. We are using a round 4-port setup connected to an olfactometer. Animals were infested with ticks for a separate project and were then used in these trials.Thus far, our pre-infestation data show that ticks more often chose the control ports and not the ports connected to a mouse, regardless of the sex of the mouse. Of the ticks that made a choice, 14% and 18% of ticks chose the ports connected to a male or female mouse, respectively. We also found similar patterns when comparing tick choices between tick-infested and non-tick infested mice. Overall, when presented with live mice that are either infested or not infested and control ports with just air, Ix. scapularis more often chose the control ports, regardless of the sex of the mouse or tick infestation status. We were originally using laboratory-reared ticks from Oklahoma State University (OSU) in our behavioral assays and to infest the animals. In a separate experiment, we tested movement of ticks from laboratory-raised ticks from the southern US (OSU) and northeast US (CDC - colonies are replenished with ticks from the northeast US) and field-collected ticks from PA.Thus far, we found that field-collected "northern" ticks from PA on average move at a higher velocity, but not as far compared to the laboratory-reared southern ticks from OSU. On the other hand, laboratory-reared northern ticks from the CDC moved faster and farther than the southern counterparts. Proposed aim 2.3. Determine chemical constituents of candidate semiochemicals produced by white-footed mice. We developed a headspace method for analyzing trapped chemical components fromcotton with fecal and urine samples. A non-polar HP-5MS columnhoused in an Agilent 6890N (GC) interfaced with an Agilent 5973N mass spectrometer (MS) detector was used to identify different volatile components of eluted buffer. We identified a diverse set of compounds from white-footed mice and their excretory products. The identified compounds can be categorized into different chemical classes of pyrazines, sulfides, esters, ketones, acids and phenols and from small molecules with two carbons to larger molecules with fifteen carbons. Some of these compounds have been reported in previous studies about chemical ecology of animal odors and their function in terms of attraction or repellence of arthropod parasites. Urine compounds:Pyrazine 2,5 dimethyl is one of the common components of mice urine (Uchikawa et al., 2020; Vihani et al., 2020) and has been described for other rodents, mammals, and birds (Poldy, 2020). We found traces of this nitrogen-containing compound in most urine samples from field or laboratory- reared mice. This compound has been reported in headspace analyses of bont ticks Amblyomma variegatum, but the ticks do not produce a response when exposed to this compound (McMahon et al., 2001). Dimethyl sulfone is the smallest identified compound in our samples and has been previously described as a component in rodent urine (Novotny et al., 2007; Zhang and Zhang, 2011). Phenolic compounds, including phenol, 4-methyl phenol (p-cresol) and 4-ethyl phenol comprise another major part of urine volatile compounds. These compounds were reported from mice (Singer, Beauchamp and Yamazaki, 1997), rats (Díaz-Santiz et al., 2020).Recently, a study found that commercial odor from white-tailed deer containing a mixture of different phenolic compounds triggered an electrophysiological response in the Haller's organ of female Ix. scapularis ticks (Josek et al., 2021). Benzoic acid is another compound we found in our urine samples. Some animals, such as cattle, can produce this compound in the dermal gland, which evokes questing activity in Ix. ricinus ticks (Osterkamp et al., 1999). Fecal compounds: Benzaldehyde has been reported in rumen volatiles (Poldy, 2020) and hypothesized to be released during fecal excretion in our headspace assays. We also found different metabolites of butanoic acid but it remains to be revealed if these metabolites are released from feces or skin. Cyclohexane carboxylic acid is another compound that is possibly released from fecal pellets. This compound has been found to produce electroantennogram and behavioral responses in insects (Poldy, 2020).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Poh, K., Tiffin, M., Evans, J.R., and Machtinger, E.T. A needs assessment survey on vector-borne disease prevention methods used by Pennsylvania residents. Entomological Society of America Annual Meeting, Denver, CO. November 2021.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Poh, K. Gettin Ticky with the Veterinary Entomology Laboratory at Penn State University. Ohio State Tick Symposium, Columbus, OH. October 2021.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Poh, K. Parasite Hunters: Engaging the hunting community to uncover the status of ticks, deer keds, and their pathogens. The American Entomological Society, Virtual Meeting. February 2021.
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Poh, K., Machtinger, E. T. Behavioral differences in colony-reared and wild-collected ticks influence laboratory assessment of ecology. In preparation.
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Our target audiences for this project would be foresters and the general public that use forested habitat. Changes/Problems:Given the lack of and inconsistent responses from ticks in the Y-tube at different time intervals, heating, air/carbon dioxide levels, and humidity levels, we have switched to a petri dish method. This has yielded more activity from ticks and we are currently testing the petri dish method for consistent responses. In Objective 1, we were testing to see if ticks would be attracted to live ticks or if they produced pheromones to attract ticks, but we did not see consistent results. Therefore, we switched to the guanine, xanthine, and adenine mixture, which is similar to the byproduct of larval tick exuviae. This has been shown to produce responses by nymphal ticks in a previous study, so we will continue with this experiment. We also collected fresh excretion products (urine and fecal samples) from live mice to identify chemical constituents in addition to the trap cotton. During the live mammal trapping, we also housed mice for six hours with cotton to standardize the amount of time the mice spend with cotton. We will be testing all of these products in our experiments to fulfill Objective 2. Finally, we are currently using laboratory-reared ticks from Oklahoma State University, however, these ticks are considered the "southern" strain, which may behave differently from "northern" ticks (Arsnoe et al. 2015, 2019, Tietjen et al. 2020). In summary, southern ticks are known to quest at lower heights, quest less for hosts overall, and nymphal ticks may quest for reptiles instead of small mammals. Because of this, we have ordered a strain of northern ticks from BEI, but they will not be available until spring 2021. We will continue experiments with the southern ticks until they are available. What opportunities for training and professional development has the project provided?We have given extension and outreach talks at various in-person and online webinar events. These talks described tick biology, tick hosts, and tick prevention and safety. How have the results been disseminated to communities of interest?While results from this research have not been disseminated to communities of interest (foresters, general public), we plan on communicating our results in the future in the form of publications in scholarly journals, scientific conferences, and live talks/webinars with foresters and the general public. What do you plan to do during the next reporting period to accomplish the goals?We plan on finalizing our tick bioassay to measure attraction and finish processing our samples. As we are processing samples, the "most attractive" samples will go through the volatile compound extraction process. Finally, we will be developing our tick trap and improved tick drag using the most "attractive" chemical compound(s).
Impacts
What was accomplished under these goals?
Objective 1 (Machtinger & Poh): Proposed aim 1.1: We tested attraction of nymphal lx. scapularis to conspecific ticks, but this has not produced consistent results in tick response. We suspect that lx. scapularis may not emit any pheromones until they actively start to feed. Instead, we are now using a mixture of guanine, xanthine, and adenine as described by Allan and Sonenshine (2002). In the study, a 25:1:1 mixture produced similar responses to cask skins produced by blood-fed lx. scapularis larvae, so we predict that our results will follow a similar pattern. We are also testing different concentrations of guanine, xanthine, and adenine to see if nymphal lx. scapularis ticks will produce a similar or greater response. Proposed aim 1.2: Because we are changing our direction to look at different concentrations of guanine, xanthine, and adenine, we will not need to identify candidate pheromones produced by conspecific ticks. Objective 2 (Machtinger & Poh): Proposed aim 2.1: We have collected trap cotton containing excretory products for two field seasons (2019-2020). We have also collected fresh urine and fecal samples from mice in summer 2020. Finally, we collected used cotton using a standardized procedure in summer 2020, where mice were housed in traps for sixhours. This standardizes the amount of time mice spend with cotton as mice can be trapped in the wild at various intervals of time. We have developed and tested various tools to measure attraction. We have tested the Y-tube using a variety of heating, humidity, and air/carbon dioxide sources. This has produced inconsistent results, so we are now testing the petri dish method. The petri dish method seems to produce more consistent results, butwe will continue troubleshooting to ensure reliability. After we confirm consistency in our results, we will test our samples. Proposed aim 2.2: Due to Institutional Animal Care and Use Committee (IACUC) procedures, we will be starting colonies of white-footed mice (Peromyscus leucopus) by next spring to investigate nymphal tick behavior in response to infested and non infested mice. Proposed aim 2.3: For excretion products, we are still troubleshooting the extraction process;but we have identified some chemical compounds present in the cotton collected from traps. We are also planning to collect volatiles produced by live P. leucopus;but due to IACUC procedures, we will be extracting volatile headspaces of live infested and non-infested mice by next spring/summer. Objective 3 (Machtinger & Poh): Proposed aim 3.1: We are in the process of identifying the semiochemicals from Objective 2. Once chemical compounds have been identified, we will test them singularly or in combination for synergistic effects.
Publications
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