Rabid Wolf Spider (Rabidosa rabida) Ecological Research at GBRS

 

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Fecundity in R. rabida

One of the ongoing R. rabida projects at the GBRS is describing the fecundity and life history of this spider.  Despite being abundant and easy to capture in large numbers nobody has characterized the numbers of offspring that were produced by each successfully breeding female in various habitats and across long periods of time.  Dr. Stork and a Harding undergraduate student researcher described the fecundity in this spider, in an Arkansas Academy of Science publication in 2017 (Hogland et al 2017).  Continuing this research, Dr. Stork et al. have been collecting fecundity measures for the past 3 years and will continue for the foreseeable future with the intention of correlating spider fecundity with GBRS weather station data and fluctuations in the overall insect community.

 

 

 

 

 

 

 

 

 

 

 

 

 

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Spider Microbiome

In recent years, the interactions of microbes in the gut to the health, activity, and behavior of animals has been of great interest in the scientific community.  Due to increased student interest in this topic, we decided to explore the microbiome of R. rabida to determine if this spider could potentially play a role as a microbiome model.  Harding undergraduate student, Patty Rivera, presented and published the seminal description of the microbial community on the surface of R. rabida in the Arkansas Academy of Science (Rivera et al 2017).  The following summer, a second student sampled the gut of the spiders collected at GBRS using ribosomal DNA identification to determine to gut microbiota of this spider. Following our success in exploring the microbial community, it was noticed that microbes were not equally distributed on or in the spider, and we hypothesized that proteins in the digestive fluid and or venom might be responsible for this.  So, Brandon Hogland, Amber Hug, and Dr. Stork ran mass spectroscopy on spider venom collected at GBRS. The results were very interesting!  With a trypsin lysis, over 10,000 peptides were found in the venom of R. rabida and the vast majority were like nothing seen in other animals.  More than 8,500 were “new” and of those several thousand showed strong potential for antimicrobial activity.  Previous description of venom proteins in wolf spiders had found 1 family of proteins the Lycotoxins which showed antibiotic activity (Yan & Adams 1998).  We found a few new variants of lycotoxins as well as several other promising peptides including one that most closely matched a human liver protein with antimicrobial activity.  These results spurred new collaborations with the UAMS Bioinformatics department to run more predictive analysis on the de novo peptides.  Currently, Brandon Hogland and Amber Hug are running antimicrobial assays on 19 of the de novo peptides and are finding promising inhibition of gram-negative bacteria.  In the summer of 2019 we began testing the susceptibility of R. rabida to its own venom.  If this spider is consuming prey that contains active venom proteins, then it might have developed some resistance which would lead to other interesting physiological studies.  We plan to collect venom from spiders captured at GBRS and inject prey items (either crickets or roaches) to determine an LD50 per unit mass.  We will then inject the calculated LD50 volume, half that volume, and double the volume into a spider and see if it survives or dies.  The LD50 data for prey items will provide useful background information for future venom work looking at peptides and the ecology of what causes peptide development.

 

 

Diurnal Activity and Sexual Dimorphism of Thermal Preferences

At GBRS we are also assessing the previously undescribed ecological data related to the diurnal activity of R. rabida. Previous descriptions of the thermal biology of R. rabida suggest that male and female wolf spiders have different thermal preferences, maximum thermal limits, and thermal sensitivities, especially at the warmer temperatures (Stork 2012). The preferred temperature for female spiders is ~ 32°C, which is warmer than would likely be encountered during typical nocturnal behaviors (Stork 2012). We hypothesize that diurnal activity, potentially related to reproductive activity in the female, likely accounts for the thermal, sexual dimorphism in this spider. Unfortunately, due to the lack of published data on this species, we cannot say with confidence where these spiders go during the day. To address this, we will capture male and female spiders at GBRS in July and early August (2019) using spotlighting methods developed by Wallace (1937), immobilize them in the field by placing them in a cooler of dry ice until they stop moving, and track them with either a string or an 8mm Biomark RFID tag (optimal method to be determined by preliminary lab testing). After the spider is tagged, it will be placed in a warm recovery container for ~30 minutes, before it will be released at the capture site. We will follow threaded spiders 24 h later to locate diurnal locations and take measurements of body temperature using a FLIR camera. The tagged spiders will be tracked using a Biomark HPR Lite reader and they will also have body temperature data taken with the FLIR camera. Body temperature readings will then be compared to published data on lab thermal preferences and tolerances of the spiders. This project field-testing the perceived thermal preference sexual dimorphism will be publishable in several journals including the top 20 entomology journal Environmental Entomology where the previous reports for this species are published (Stork 2012). The journal of Arachnology and the Proceedings of the Arkansas Academy of Science would also be likely publication opportunities for portions of this work. Two Harding University undergraduate students have agreed to work on this project. Payton Smith and Hayley Ford, both pre-med students, are working on methods, background information, and introduction write ups for the two approaches to tracking and the thermal applications. 

 

 

Microhabitat Preference of Rabidosa rabida

Another study in the pipeline involves microhabitat preference for R. rabida related to associations with specific plants at GBRS.  Rabidosa rabida climbs to the top of low vegetation at night to capture flying insects.  If spiders exhibit a plant preference for nocturnal foraging, this information would directly influence the future research direction of our microbial research.  In a preliminary study using 3X2m sample areas at GBRS, we captured spiders and recorded the species of plants on which they were captured.  At this time, preliminary

results do not show a plant preference, but we intend to

replicate this study with a larger sample size before we can

publish results. After sufficient survey work is complete, we will publish results in the Journal of Arachnology, Environmental Entomology, or the Proceedings of the Arkansas Academy.

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Literature Cited

Biomark, Inc. 705 S. 8th Street Boise, ID 83702. www.biomark.com

Brady, Allen R., and Kelly S. McKinley. "Nearctic species of the wolf spider genus Rabidosa (Araneae:Lycosidae)." Journal of Arachnology (1994): 138-160.

Corzo, G., and P. Escoubas. "Pharmacologically active spider peptide toxins." Cellular and MolecularLife Sciences CMLS 60.11 (2003): 2409-2426.

Foelix, R., 2011. Biology of spiders. OUP USA

Hogland, B., Stork, R., Hug, A. 2017. A description of variation in fecundity between two populations of wolf spider Rabidosa rabida in Searcy Arkansas using brood size measurements. Journal of the Arkansas Academy of Science 71(1): 47-50

King, Glenn F. "The wonderful world of spiders: preface to the special Toxicon issue on spider venoms." (2004): 471-475.

Nyffeler, Martin, and Klaus Birkhofer. "An estimated 400–800 million tons of prey are annually killed by the global spider community." The Science of Nature 104.3-4 (2017): 30.

Rivera, P., Stork, R., and Hug, A. 2017. A first look at the microbial community of Rabidosa rabida, a wolf spider in Searcy, Arkansas. Journal of the Arkansas Academy of Science, 71(1): 51-55.

Saez, Natalie J., et al. "Spider-venom peptides as therapeutics." Toxins 2.12 (2010): 2851-2871.

Solomon, Susan, et al., eds. Climate change 2007-the physical science basis: Working group I contribution to the fourth assessment report of the IPCC. Vol. 4. Cambridge university press, 2007

Stork, Ryan. "Intraspecific Variation in the Thermal Biology of Rabidosa rabida (Araneae: Lycosidae)(Walckenaer) from the Mountains of Arkansas." Environmental entomology 41.6 (2012): 1631-1637.

Vassilevski, A. A., S. A. Kozlov, and E. V. Grishin. "Molecular diversity of spider venom." Biochemistry (moscow) 74.13 (2009): 1505-1534.

Yan, L. and Adams , M. E. 1998. Lycotoxins, antimicrobial peptides from the venom of the wolf spider Lycosa carolinensis. Journal of biological chemistry 273(4): 2059-2066

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Bird Research at GBRS

 

Long-term Population Demography of Bird Species Captured at Gilliam Biological Research Station

 

Dr. Ruhl holds federal and state permits to capture and band all birds (excluding waterfowl, eagles, and endangered species) for research and educational purposes at the GBRS in White County, AR, USA. Birds are a fantastic study organism because they can be sampled year-round, sampling methods are low-risk for injury or mortality, and high levels of bird biodiversity at a single research site can provide numerous research opportunities.

 

The primary objective for the ongoing bird banding research is to use bird banding as a means to provide undergraduate students with research opportunities, and to aid in teaching efforts at Harding University. We are interested in the local distribution, habitat-use, survival, reproductive success, and movements of individual birds at the Gilliam Biological Research Station. By banding birds, we can identify individuals of a known history when they are recaptured. Several consecutive years of banding data can provide insight into long-term population trends. By banding birds on a consistent basis at the Gilliam Biological Research Station (GBRS), we are compiling a detailed long-term dataset that will be available to be utilized by other research institutions conducting research on population demography. In addition, these data can also be used for future student projects in the Advanced Ecology Lab curriculum.

 

On a weekly basis throughout the academic year, this banding station provides undergraduates (and interested faculty members) the opportunity to learn the basics of bird banding. Further applications of this program involve lab activities for a semester-long Ornithology course for biology majors as well as a 2-week bird identification course offered during the May intercession. We have already received a request from another institution asking to use GBRS banding data to help assess range-wide population trends. I was notified by the Federal Bird Banding Laboratory that a graduate student at Arkansas State University needed morphometric data for American Goldfinch, American Robin, Northern Cardinal, Ruby-crowned Kinglet, Song Sparrow, Tufted Titmouse, and White-throated Sparrow. Even though GBRS has only been collecting banding data for ~ 7 months, we were able to provide morphometric data for 90 birds!

 

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Using Radio Frequency Identification (RFID) to Test Assumptions of Optimal Foraging Theory on Wintering Birds at GBRS

 

Optimal foraging theory predicts that organisms should exhibit foraging behaviors that maximize net energetic gain. Therefore, when presented with a choice between prey items that vary in energetic currency (E) and handling time (h), organisms should select prey items that maximize the E/h ratio. The semester project for Animal Behavior Lab (BIOL 409) this spring (2019) used radio frequency identification (RFID) technology at the Gilliam Biological Research Station (GBRS) to determine whether the foraging preferences of Tufted Titmice (Baeolophus bicolor) reflected the predictions of optimal foraging models in situ. Dr. Ruhl and 7 Harding University students equipped two bird feeders with RFID readers. Biomedical engineering students helped with the design of the electronic components, and professor John White assisted the class with the electrical engineering aspects of this collaborative research process.

 

During the study, whenever a bird with an RFID leg band landed on a feeder, the RFID reader recorded the bird’s unique ID#, the date, and the time of the feeder visit. Feeders were placed ~ 1m apart from one another to represent a simultaneous foraging decision. For a 5-day period, the Animal Behavior Lab recorded baseline foraging preference data at both feeders using whole sunflower seeds (in the shell). They then replaced the whole sunflower seeds in one of the feeders with sunflower kernels (without the outer shell), and collected data for another 5 days. These two “prey items” (whole sunflower seeds and sunflower kernels) were used to test the predictions of optimal foraging cost-benefit models in an experiment in which the energetic currency of food was kept constant, but handling time differed. Chi-Square goodness of fit tests were used to determine if the foraging behavior of Tufted Titmice reflected a priori predictions of optimal foraging cost-benefit models in a situation when energetic currency is kept constant and handling time is reduced. With the exception of one individual, Tufted Titmice (TUTI) exhibited a preference for whole seeds over kernels, suggesting that prey choice in this context is likely influenced by additional factors.

 

Results from this preliminary study are depicted below:

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Quantifying Seasonal Fluctuations in Blood Profile Relative to Singing Ability in Avian Species

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Among Passerines, oscines sing complex songs during the breeding season, and exhibit a limited song repertoire during the winter. Suboscines, which produce much simpler innate songs/calls do not exhibit this seasonal variation in song repertoire. For song-learning oscines, the decrease in singing investment during the winter results in the disuse of the neurons that hold information and are responsible for song learning. Unlike most mammals, neurons that fall into such disuse are not rerouted to other parts of the brain, but instead die. This suggests that physiological changes across seasons would differ between oscines and suboscines. Suboscines, who sing the same simple, innate songs and calls throughout their lives, hypothetically would not experience this level of neuronal death. Neurofilament is a neuronal protein that is released into the bloodstream upon damage or death. In addition, there are changes in white blood cells across seasons that correlate with the timing of brain changes. However, it is currently unknown how much difference exists in neurofilament levels and white blood cell populations among passerines. The hypothesis is that seasonal changes in neurofilament proteins and white cell populations in blood will differ between oscines and suboscines.

 

We aim to optimize a method to quantify neurofilament proteins in peripheral blood of wild birds. Neurofilament proteins are heteropolymers that include a light, medium, and heavy chain subunit. Upon neuronal death, neurofilament proteins are released into the peripheral bloodstream. Neurofilament light chain (NEFL) levels tightly correlate with neuronal death. Therefore, measuring relative change in NEFL is a surrogate for neuronal atrophy and death. In wild birds, relative NEFL will be measured using dot blot analysis on blood plasma. Levels will be compared across seasons and bird species.

 

In addition, we aim to characterize the white blood cell profile of wild birds across seasons. Hematological profile of avian blood has been associated with stress, nutrition, and seasonal changes in studies involving captive birds. However, seasonal fluctuations in hematological profile and changes in specific cell populations of birds in situ is not known. We will characterize white blood cell populations in wild birds using flow cytometry. This technique sorts cells based on size and cell composition. Identified cells populations will be compared across seasons and among different bird species.

 

Successful completion of the proposed project will develop a foundation for studying seasonal changes in birds using peripheral blood. This is a sustainable practice that will allow future projects to develop additional questions. The proposed objectives will support student involvement in a cross-disciplinary project and promote collaborative research at a Harding, which is a primarily undergraduate institution.

 

 

Comparison of Avian Blood Parasite Load Among Species with Different Nesting Life Histories

 

In conjunction with the avian neurofilament research project described above, we will also make blood-smear slides for each individual from which a blood sample is taken. These slides will be used by a Harding undergraduate student to address questions related to bloodborne pathogens at GBRS. Previous research indicates that different nesting life histories associated with different nest substrate or nest type (e.g., cavity nest or open cup nest), and nest location (e.g., forest canopy, shrubs, grasses), may have an impact on the blood parasite load. We began compiling data to address this question in May 2019. 

 

 

Property-Wide Breeding Bird Density Surveys

(estimated start date: May 20, 2020)

 

This spring we will begin a multi-year effort of conducting breeding bird surveys at GBRS. Not only will this research provide necessary pre-treatment data for any future forest management scheduled to occur on the property (e.g., Short-leaf pine timber harvests), but by mapping the breeding bird density across different habitat types on the property, we will be better equipped to develop future research projects and accommodate collaborative projects with other universities and research institutions. Data from this point count survey (see geographic representation below) will help determine important habitat associations of breeding birds at GBRS and inform any future habitat management actions.

 

 

 

 

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Cooperative Breeding Biology of Tufted Titmice

 

Although Tufted Titmice (Baeolophus bicolor) are a relatively common bird species in North America, there is a paucity of detailed data describing their basic breeding biology. This species is not sexually dimorphic, so until recently, documenting differences in the allocation of parental care between males and females required color banding and direct observation. In addition, some observational studies from the 1970s and 1980s suggest that Tufted Titmouse breeding biology may be somewhat complex, and potentially may involve cooperative breeding in which helper Titmice (i.e., not the mother or father) were observed helping feed nestlings. We aim to use RFID technology to collect detailed nesting behavior data for Tufted Titmice at GBRS. Because Titmice are a cavity-nesting species, we hope to encourage them to adopt nest boxes. After Titmice have established nests and laid eggs in nest boxes, we will equip active nests with RFID readers. By recording visits to the nest boxes during the nestling stage with RFID, we will be able to compile detailed datasets explaining the parental investment of both parents as well as any helpers that are also fitted with RFID leg bands. So far, we have banded 8 Tufted Titmice with RFID leg bands, and we have built and deployed 7 nest boxes within a 200 m radius of the Biology Department shop at GBRS.

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Aquatic Community Research at GBRS

 

 

Amphiuma/Siren Life History Research

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In conjunction with the GBRS mud snake research project, we are also examining the distribution of the preferred prey of the mud snakes. At this time, the home ranges of all of the mud snakes (excluding one snake making two unexpected movements to the same location) are confined to the primary basin of Gilliam Pond.  Mud snakes are considered to specialize on feeding on large salamanders from both the Amphiuma and Siren genera. 

 

To determine whether prey availability is a factor in the home range size and usage, Dr. Steve Cooper and Harding undergraduate student Aric McKinney are conducting a tangential project surveying Gilliam Pond and all appropriate Amphiuma and Siren habitats within 100 meters of the pond’s borders.  The study perimeter was marked off using UTM coordinates relative to the basin boundary.  All suitable prey habitats were noted and UTM coordinates were recorded. 

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Water Quality Research at GBRS

 

In January 2019, three small (approximately 6-7m wide, and 1.5-3m deep) depressions were created in an effort to divert water off of a newly constructed GBRS road and allow access to the property from the biology shop on the NE border of the property. The depressions soon filled with water, both through percolation and rainfall creating three new ponds on the property. 

 

Ecological succession can be studied in terms of orderly development of communities resulting from changes in a physical environment and ultimately leading to a stable ecosystem in which maximum biomass and symbiotic function between organisms are maintained. As ecosystems develop in these ponds, total organic matter will increase and food chains will form. However, for any life to be sustained in the ponds, there must be the right nutrients and the right conditions. Conductivity and salinity, pH, temperature, turbidity, and ions present will fluctuate as new life forms. The chemical changes that occur during the succession of the pond are the focus of this project.

 

We intend to sample and analyze the three new ponds, and compare chemical levels with an established ("old") pond that will serve as a control. When it is not too wet, we are collecting water samples weekly; we are measuring temperature and PAR (photosynthetically active radiation) at the site using a Vernier LabQuest. The turbidity, conductivity, pH, ammonium ion concentration, and nitrate ion concentration of water samples are being analyzed in the analytical chemistry lab at Harding University. In the near future, we also intend to measure dissolved Oxygen content in the ponds.

 

Life observed in the ponds at this point includes water fleas and various insects, an unknown minnow species, algae, and spotted salamander (Ambystoma maculatum) larvae. As the ponds continue to develop, more life will appear in the ponds and the chemical values will shift. We hypothesize that turbidity will decrease as the concentrations of ions and conductivity increase.

 

This undergraduate research project is still very much in its infancy; thus, it is difficult to map any trends at this point. We plan to collect data continuously over multiple seasons to see how chemical values fluctuate with the ecological development of the ponds in the future.