1. Dermacentor variabilis
Gut Microbiome Analyses of Lone Star, Black-Legged and Dog Ticks William Bennett, Biomedical Informatics Jeremy Seto Ph.D, Biology New York City College of Technology, CUNY
Abstract
The focus of much tick research is often on the detrimental effects caused by the pathogens found inside the ticks. However, some focus has been shifted towards researching the natural microbiota already present and trying to determine if host-pathogen interactions are influenced by the gut microbiota. Lone Star, Deer / Black-Legged, and Dog ticks at each life stage (larval, nymph, adult) had their guts collected and analyzed. Larvae were unfed, nymphs had fed once, and the adults have fed at least twice. Three samples of each species adult male and female were collected along with three samples of their nymph (not sexed) and larval stages. The Lone Star tick at adult, nymph, and larval samples were each comprised mostly of Anaerococcus Octavius while the adult, nymph, and larval samples of the Deer and Dog ticks were each comprised of a variety of different organisms. In the long run, this can imply that some microbiota are conserved between the different life stages of certain species of ticks and this information can be analyzed further for future research on the effects microbiota have on their host.
Introduction
Tick-borne diseases are an emanating public health issue due to ticks being vectors for multiple diseases. The three species mostly found in the NYC area are the Lone Star, American Dog, and Deer ticks. They are responsible for Tulameria, Rocky Mountain spotted fever, Lyme disease, and more.
Ticks have three developmental life stages - larva, nymph, and adult – with the main difference being that larvae only have six legs until they develop into a nymph with eight legs.
The microbiota living in each tick species varies due to their feeding routine and play an essential role in the species survival. However, it has been shown that when these ticks are invaded by different obligate parasites, their gut microbiome changes. According to Fuente et. al, through host – parasite interactions, the tick’s immune response can be altered to promote survival and transmission of the parasite and also insuring host death.
Methods Cont.
Dog Female Adult Dog Male Adult
Step 3: After dissection, the guts were scraped from inside the adult tick and stored in sterile 2 ml collection tubes.
Step 4: DNA was isolated from the adult samples using the PowerSoil® DNA Isolation kit protocol.
Deer Nymph Lone Star Nymph
Lone Star Larva
Larvae samples were incredibly small and almost impossible to dissect the guts out with the tools and magnification used. DNA isolation of larvae samples came from using whole larvae. No dissection done.
Step 5: After DNA isolation, PCR and gel electrophoresis were done to amplify and view any DNA found.
Step 6: Samples were sent to Molecular Research DNA in Shallowater, Texas for 16s sequencing using Ion Torrent PGm.
Results Cont.
The software QIIME (Quantitative Insights Into Microbial Ecology) was used to analyze the microbial communities found for each classification level and produced the following taxonomy figures (*Kingdom excluded):
Phylum Class
Order Family
Genus
Removal of scutum
(top shell)
This is the transmission cycle of Lyme disease. Deer ticks are the host vector for Borrelia burgdorferi.
Results
Amblyomma americanum
Ixodes scapularis
Dermacentor variabilis
* Pie charts were made using Microsoft Excel
Discussion
In the Lone Star samples, Anaerococcus octavius was found to be the most abundant species and more prevalent in the adult stages. A connection can be made where this species is needed more in the adult stage since the adults feed more often. They are found in normal intestinal flora but can be obligate parasites and this can possibly play a role in host infection.
In the Deer samples, Rickettsia monacenis, Roseomonas ludilueritiae, Symploca atlantica, and Dorea spp., were found in the adult female, adult male, nymph, and larvae samples respectively. R. monasenis is known to induce Spotted Fever-like symptoms. R. ludilueritiae has been shown to cause bacteremia. We can see in the adult stages, pathogenicity seems very likely. D. spp., was found in the larvae samples and has shown to contain selective inhibitors that act as antimicrobial agents. This microbe can possibly aid in the survival of the tick.
In the Dog samples, the females were most abundant with Nitrospina species and Hoeflea spp., in the males. Hoeflea species have been shown in studies to inhibit the growth of other bacteria. Knowing this, a possible bio-intervention could be developed.
A further study of the samples will be done with an analysis of metabolic functions found.
Methods
For this project, three larvae, nymphs, and three male and female adults of Deer and Lone Star species were used. For the Dog species, only three male and female adult samples were used. Each species were collected beforehand and retained in ethanol.
Step 1: Paraffin wax was melted and poured into a petri dish.
Step 2: Nymph and adult tick samples were imbedded into the paraffin before it solidified by using the tip of a hot glue gun to create a small dent in the paraffin. *Larvae samples were not dissected due to size*
Lone Star Female Adult
Deer Female Adult
Citations
Centers for Disease Control and Prevention (CDC), Lyme Disease. Retrieved November 28, 2017, from
Edwards, K. T., J. Goddard, and A.S. Varela-Stokes. (2009). Examination of the Internal Morphology of the Ixodid Tick, Amblyomma maculatum Koch, (Acari: Ixodidae); a “How-to” Pictorial Dissection Guide. Midsouth Entomologist, 2, Retrieved from
Tick identification [Digital image]. (2017). Retrieved November 28, 2017, from
Removal of scutum
(top shell)
Video of dissection
Acknowledgements
I would like to thank CUNY, New York City College of Technology, and the Emerging Scholars Program for giving me this opportunity to do research this semester. I would also like to thank Professor Seto for mentoring me during this semester and guiding me throughout the research. We also would like to thank Waheed Bajwa from the Department of Health and Mental Hygiene for providing us with the tick samples.
Removal of scutum
(top shell)