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Honors Theses Honors College
5-2020
Molecular Characterization of Wolbachia and its Impact on the Microbiome of Exotic and United States Ticks
Cailyn G. Bobo
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Part of the Entomology Commons The University of Southern Mississippi
Molecular Characterization of Wolbachia and its Impact on the Microbiome of Exotic and United States Ticks
by
Cailyn Bobo
A Thesis Submitted to the Honors College of The University of Southern Mississippi In Partial Fulfillment of Honors Requirements
May 2020
ii Approved by:
______Shahid Karim, Ph.D., Thesis Adviser Professor of Biological Sciences
______Jake Schaefer, Ph.D., Director School of Biological, Environmental, and Earth Sciences
______Ellen Weinauer, Ph.D., Dean Honors College
iii Abstract
Wolbachia endosymbionts are obligate intracellular bacteria in the phylum α-Proteobacteria.
They infect approximately two-thirds of arthropods including insects and nematodes. These bacteria manipulate host reproductive biology through a series of mechanisms that include feminization of male progeny, parthenogenesis, male killing, and cytoplasmic incompatibility.
These features make Wolbachia an effective agent of controlling insect populations, as previously reported in different mosquito species. Likewise, the bacteria have also shown promising ability to interfere with the ability of mosquitoes to transmit several viral pathogens.
This study was conducted to fill an existing knowledge gap on the prevalence of Wolbachia in different tick species and also compare the microbiome of infected and uninfected ticks. A polymerase chain reaction (PCR) based assay was used to characterize Wolbachia endosymbionts from three United States tick species (Amblyomma americanum, Amblyomma maculatum, and Ixodes scapularis) and two exotic species (Hyalomma anatolicum anatolicum and Rhipicephalus microplus), by amplifying the 16S ribosomal RNA and heat shock (GroEL) genes of Wolbachia. This study employed multiple sequence alignment and phylogenetic trees to resolve evolutionary relationships between Wolbachia and other insects and arthropods.
Microbiome analysis was done by using Illumina sequencing of the V1-V3 variable region of the bacteria 16S ribosomal RNA. Overall, a low prevalence of Wolbachia was found, but the identified strains showed close similarities to other Wolbachia species from Aedes aegypti, Aedes albopictus, and Chrysoma megacephala. Uninfected ticks were all lab-reared ticks, while all ticks found to be positive for Wolbachia were ticks collected from the wild. I also observed a distinct difference in the bacterial species composition between infected and uninfected ticks.
The microbiome of Wolbachia infected ticks exhibited a low species richness and diversity as
iv compared to that of uninfected ticks. This study revealed that ticks share similar Wolbachia endosymbionts with mosquitoes. In addition, it provides research on interactions between
Wolbachia endosymbionts and tick species, which will subsequently direct targeted tick control.
Keywords: Wolbachia, cytoplasmic incompatibility, tick, 16S ribosomal RNA, heat shock GroEl gene, tick control
v Table of Contents
List of Figures…………………………………………………………………………………...viii
List of Abbreviations……………………………………………………………………………...ix
Chapter 1: Introduction……………………………………………………………………………1
Chapter 2: Literature Review………………………………………………………...... 3
Ticks and Their Significance……………………………………………………………...3
Control of Ticks and Tick-borne Diseases…………………………………...... 7
Wolbachia…………………………………………………………………………………9
Chapter 3: Methodology…………………………………………………………………………13
Tick Collection…………………………………………………………………………...13
Tick Identification…………………………………………………………...... 14
Genomic DNA Extraction…………………………………………………...... 14
Molecular Identification of Tick Species……………………………………………...…15
Molecular Detection of Wolbachia………………………………………………………15
Detection of Wolbachia 16S rRNA……………………………………………………...15
Detection of Wolbachia GroEL……………………………………………...... 16
PCR Sequencing and Phylogeny………………………………………………………...17
Microbial Profiling……………………………………………………………………….17
Data Analyses……………………………………………………………………………18
Chapter 4: Results………………………………………………………………………………..20
Tick Species Studied……………………………………………………………………..20
Wolbachia Prevalence……………………………………………………………………22
Microbiome Composition…………………………………………………...... 24
vi Bacteria Species Abundance………………………………………………...... 24
Diversity Analysis…………………………………………………………...... 25
Chapter 5: Discussion and Conclusion……………………………………………...... 27
References…………………………………………………………………………...... 33
vii List of Figures
Figure 1. Acquisition and roles of microbiome on tick biology…………………………………4
Figure 2. Application of Wolbachia in reducing mosquito population and pathogen transmission……………………………………………………………………….10
Figure 3. Illustration of methodology……………………………………………………………13
Figure 4. Morphological identification and molecular confirmation of tick species…………….20
Figure 5. Phylogenetic analysis of amplified COI sequences with tick species…………………22
Figure 6. PCR analysis of Wolbachia species…………………………………………………...23
Figure 7. Genetic relationship of identified Wolbachia………………………………………….23
Figure 8. Relative abundances of bacteria at the species taxonomic differences………………..25
Figure 9. Rarefaction analysis of Wolbachia infected and uninfected ticks……………………..26
viii
List of Abbreviations
CDC Centers for Disease Control
CCHVF Crimean-Congo hemorrhagic fever virus
CMM Candidatus Midichloria mitochondrii
COI gene Cytochrome oxidase I gene
DENV Dengue virus
Faith-pd Faith phylogenetic distance
FLE Francisella-like endosymbiont
Fstz gene Filamenting temperature-sensitive mutant Z gene
GroEl gene Chaperonin groEL [Escherichia coli] gene
HRTV Heartland virus
OUT Operational taxonomic units
PCR Polymerase chain reaction
POWV Poswassan virus
USNTC United States National Tick Collection
QIIME2 Quantitative Insights into Microbial Ecology
Wsp gene Wolbachia surface protein gene
ix Chapter 1: Introduction
Ticks have become a major public health concern due to the pathogens they carry and transmit to human hosts. These organisms depend on mammals, such as humans, for blood meals and, unfortunately, the pathogens they pass on during the feeding period can cause major infectious diseases in humans (Anderson and Magnarelli, 2008). Due to their medical significance, a large importance is placed on research dedicated to the control of ticks and tick- borne diseases (Bonnet et al., 2017). In previous years, chemical control methods were favored to combat the spread of ticks and the pathogens they carry, primarily through the use of acaricides (Valle and Guerrero, 2018). Ticks have developed a resistance to these chemicals, which has created a need for newer methods of control (Abbas et al., 2014). Researchers in recent years have directed their attention towards possible methods of biological control. One of these methods could include the genus of bacteria known as Wolbachia. Wolbachia is an endosymbiont identified in two-thirds of insects, including mosquitos (Hilgenboecker et al.,
2008). Research related to mosquitos and Wolbachia shows the endosymbiont capable of decreasing populations of mosquitos by rendering males sterile through a phenomenon known as cytoplasmic incompatibility (Stouthamer et al., 1999). This endosymbiont is also believed to interfere with the transmission of other harmful pathogens that reside in its host (Audsley et al.,
2017). Based on the effects of Wolbachia on mosquitos, there are questions of its presence in ticks and whether it can interfere with their reproduction and pathogen transmission.
Purpose and Hypothesis
The purpose of this study was to investigate the prevalence of Wolbachia in tick species found in the United States and in exotic ticks, and to compare the microbiome of Wolbachia infected and uninfected ticks. I hypothesized that the presence of Wolbachia would alter the
1 native microbiome. I expected to observe a difference in the microbial species richness and diversity between Wolbachia infected ticks and noninfected ticks.
2 Chapter 2: Literature Review
Ticks and their Significance
Ticks are obligate, blood feeding ectoparasites classified in the Acari subclass of arachnids (Anderson and Magnarelli, 2008). Recently, a larger emphasis in the scientific community is placed on the research of ticks and tick-borne pathogens. This sudden surge in research can be attributed to both the medical and veterinary significance of ticks (Bonnet et al.,
2017). Ticks are second only to mosquitos when it comes to serving as vectors of diseases affecting humans (de la Fuente et al., 2008), and are credited with transmitting the greatest diversity of both humans and animal pathogens, which include bacteria, protozoa, and viruses
(Jongejan and Uilenberg, 2004). The requirement of a prolonged blood meal and the need to feed on host blood at each life stage makes the tick a complex model organism. The number of hosts ticks have for each life stage varies between species. While on its host, the tick will undergo engorgement by taking full or partial meals of blood (Anderson and Magnarelli, 2008). During the feeding process, the tick may ingest blood contaminated with a pathogen, depending on the health of its current host. The tick also becomes capable of passing any pathogens it may be carrying to a susceptible host during this process (Anderson and Magnarelli, 2008). The possibility of pathogen transmission classifies ticks as an extremely important and significant vector (Anderson and Magnarelli, 2008).
In addition to transmitting pathogens, ticks also host a very rich and diverse community of microorganisms that range from bacteria to fungi and viruses. This community of organisms is collectively known as the microbiome (Fig. 1). Some members of the tick’s microbial community have been described to benefit the tick host. A Coxiella-like endosymbiont is thought to be important to the tick’s reproductive and nutritional fitness (Gotlib et al., 2015; Zhong et al.,
3 2007). For example, Coxiella-like endosymbionts are thought to synthesize important nutrients for the tick (Gotlib et al., 2015). A study conducted by Zhong et al. (2007) saw a decrease in egg hatching and a lower number of viable larvae when female ticks were treated with an antibiotic specific for killing Coxiella-like endosymbionts. Other members of the microbiome have also been reported to interact with tick-borne pathogens by facilitating or inhibiting pathogen colonization and subsequent transmission. Narasimhan and colleagues (2014) showed that perturbing the normal microbiome of the black-legged tick interferes with the ability of the Lyme disease pathogen to colonize within larval ticks. Abraham et al. (2017) also reported that
Anaplasma phagocytophilum, a bacterium transmitted by I. scapularis, modifies the tick’s expression of an antifreeze glycoprotein, which subsequently dysbiosed the microbiome, hence facilitating the bacteria’s colonization.
Figure 1. Acquisition and roles of microbiome on tick biology. The composition of the tick microbiome is influenced by the tick feeding on humans and animals, as well as by its environment. The microbiome can influence the tick’s life cycle, homeostasis, and feeding.
4 Tick-transmitted pathogens are responsible for multiple human diseases that continue to spread across the United States and the world; some of these include Lyme disease caused by the spirochete Borrelia burgdorferi, which is transmitted via the tick species Ixodes scapularis.
Consequences of untreated Lyme disease can include facial paralysis, arthritis, and rash (Centers for Disease Control (CDC), 2019). Rocky Mountain Spotted Fever is caused by Rickettsia rickettsi, a pathogen transmitted to humans through the species Dermacentor variabilis. If left untreated, Rocky Mountain Spotted Fever can lead to a compromised respiratory system, tissue necrosis, and damage to the central nervous system (CDC, 2019). Other severe illnesses caused by tick-borne pathogens in humans include Tularemia, Ehrlichiosis, and the alpha-gal syndrome
(CDC, 2019).
Ticks are also capable of transmitting harmful viruses to humans and other animals, which are known as tick-borne viruses (Parola and Raoult, 2001). One of these dangerous viruses to humans is known as the Crimean-Congo hemorrhagic fever virus (CCHFV) (Zivcec et al.,
2016). This virus is most often transmitted by tick species of the Hyalomma genus and was first discovered in 1945. It remains a major public health concern due to previous epidemics in parts of Asia (Shi et al., 2018). Another important virus harbored in ticks that poses a significant risk to the United States is the Heartland banyangvirus (HRTV). This virus causes Heartland virus disease in humans with symptoms such as fever, nausea, and muscle or joint pain (CDC, 2018).
This virus is primarily transmitted by Amblyomma americanum (Bosco-Lauth et al., 2015). The
Poswassan virus (POWV) is another tick-borne virus that was first discovered in 1962 in
Poswassan, Ontario (McLean and Larke, 1963). Infection by the Poswassan virus can lead to encephalitis or meningitis, and in worst cases, death (CDC, 2019). This virus is primarily
5 transmitted by ticks of the Ixodes genus, and since its isolation, cases have been documented in the United States (McLean and Larke, 1963; Smith et al., 1974).
A reported 84 species of ticks are known to inhabit the United States and only ten of these species have been documented to bite humans (Merten and Durden, 2000). Tick species that have been documented as common vectors for disease in the United States include
Amblyomma americanum, I. scapularis, and A. maculatum. These species can all be found in varying distributions across the United States. The blacklegged tick, I. scapularis, is known for its transmission of B. burgdorferi and Ehrlichia muris euclairensis, which are the causative agents of Lyme disease and Ehrlichiosis, respectively (Eisen et al., 2017). The Lone Star tick, A. americanum, with distribution in the Southeastern, Atlantic coast, and the Midwest United
States, transmits Francisella tularensis, Ehrlichia chaffeensis, and Rickettsia rickettsi, and has also been shown to be involved in causing the alpha-gal syndrome (Crispell et al., 2019).
Another member of the Amblyomma genus is the species A. maculatum, more commonly known as the Gulf Coast tick. This tick species is the vector for the pathogen Rickettsia parkeri, which is the causative agent of spotted fever and is found along the Gulf coast and Atlantic coast, as well as in Kansas, Oklahoma, Arkansas, and Kentucky (Budachetri et al., 2014; Eisen et al.,
2017).
Outside of the United States, ticks also contribute to the spread of the tick-borne pathogens. Exotic tick species include the cattle ticks, Hyalomma anatolicum anatolicum and
Rhipicephalus microplus, which are known to be found in large populations in Pakistan (Karim et al., 2017). Rhipicephalus microplus is a significant livestock pest, causing a reduction in milk production, damage to hides, and in some cases death, to livestock in the cattle industry (Karim et al., 2017; Rodriguez-Vivas et al., 2018). Many of these consequences are related to the tick-
6 transmitted haemoparasites associated with R. microplus, such as Babesia bovis and Anaplasma marginale (Rodriguez-Vivas et al., 2018). Hyalomma anatolicum anatolicum is also an important cattle endosymbiont known for its transmission of pathogens from the Theileira genus
(Singh et al., 2015).
Although exotic tick species are not geographically distributed within the United States, some of the species have been found in the country because they may have been imported to the
United States by migratory birds (Mukherjee et al., 2014; Budachetri et al., 2017). Tick-infested migratory birds will carry the attached tick endosymbionts with them as they travel to different geographical locations, such as the United States, therefore establishing populations of new species to the country (Budachetri et al., 2017).
Control of Ticks and Tick-borne Diseases
The use of chemical acaricides has become the most common practice to control and eradicate ticks, especially in the cattle industry (Valle and Guerrero, 2018). These acaricides target ticks in their parasitic life stage, which is the point during their life cycle when they feed on a host (Graf et al., 2004). Unfortunately, several unwanted side effects have occurred due to the widespread heavy use of acaricides. One drawback of this method is the risk of chemical contamination of cattle products (Valle and Guerrero, 2018). Also, reports of increasing resistance of many species of ticks to acaricides have become common (Abbas et al., 2014).
With the failures of chemical control in some instances, the need for new methods of control has become even more crucial. In the past several decades, researchers have turned to the development of vaccines against tick-borne diseases for humans and other animals. A breakthrough in the area of tick and tick-borne disease control came in 1986 when the Bm86 glycoprotein was discovered on the membrane of cells within the tick’s gut (Willadsen et al.,
7 1989). Researchers were able to utilize this glycoprotein as an antigen to develop a vaccine that would target the tick gut when they fed on a vaccinated host (Willadsen et al., 1989). Vaccines developed from the Bm86 glycoprotein affect the tick’s ability to digest blood and to take up nutrients. Therefore, female ticks are unable to use the blood and its nutrients to lay their eggs, which leads to a decrease in the population. Because the Bm86 glycoprotein was originally isolated in the R. microplus species of ticks, the vaccine was found to be an effective method of control against this species (Willadsen et al., 1989). However, more research proved the vaccine to be only partially effective against other species, and not all tick species that pose a risk to humans and other animals respond to these vaccines (Fragaso et al., 1998; de la Fuente et al.,
2007). Unfortunately, tick vaccines do not have the same knockdown effect as previously used acaricides (Almazan et al., 2018).
At present, researchers are beginning to take a genomics approach to vaccine development. Since the launching of the Human Genome Project in 1990, the genomes of thousands of organisms have already been sequenced (Almazan et al., 2018). Research has utilized reverse vaccinology for vaccine development (Almazan et. al. 2018). This technique scans the genome of a pathogen to identify antigens that could be used in vaccine design (Mak et al., 2014). Development of anti-tick vaccines has also begun to take this approach with some success (Almazan et al., 2018). However, the search for the next best control method is on-going, as researchers look for new and safe methods to contain tick and tick-borne diseases.
Newer research suggests biological control might be the better, more environmentally friendly option. This form of control would involve taking advantage of the microbiota of ticks and other vectors for disease to limit tick populations (Sicard et al., 2019). Although most tick research is geared towards the pathogens of the microbiome (pathobiome), certain
8 endosymbionts could possibly be utilized as methods of population control, such as the genus
Wolbachia, making this an important avenue of tick research.
Wolbachia
Wolbachia is a genus of α-Proteobacteria classified under the Rickettsiales order. The organisms within this genus are maternally transmitted endosymbionts that infect approximately two-thirds of insect species (Hilgenboecker et al., 2008). This genus includes 15 supergroups, with supergroups A and B most often found in insects (Lo et al., 2007). In the laboratory setting,
Wolbachia is often identified through detection of the filamenting temperature-sensitive mutant Z
(ftsZ) gene, chaperonin groEL [Escherichia coli] gene (groEl) and Wolbachia surface protein
(wsp) gene (Baldo et al. 2006; Paraskevopoulos et al., 2006).
Most research regarding Wolbachia concerns its effects on the host reproductive system.
Therefore, many Wolbachia samples that have been studied are taken from the reproductive organs of its hosts (Bi and Wang, 2019). However, Wolbachia can be found in many more somatic tissues and have other effects not pertaining to their influence on host reproduction (Bi and Wang, 2019). For example, one study found Wolbachia in the nervous tissue of certain fly species, such as Drosophila melanogaster and Drosophila simulans (Bi and Wang 2019). It is thought that their presence in these tissues may give them the ability to alter host behaviors (Bi and Wang, 2019). One of these behavioral alterations that could cause a significant decrease in the transmission of pathogens is a change in feeding behaviors. This is believed to be caused by a specific strain of Wolbachia, wMelPop-CLA, specifically in the mosquito species Aedes aegypti
(Bi and Wang, 2019). This particular Wolbachia strain resulted in a decrease in the number of meals the mosquitos took, as well as how much blood they ingested (Bi and Wang, 2019). A
Wolbachia infection was also shown to result in a bent proboscis when older mosquitos
9 attempted to feed. Ultimately, an alteration in feeding behaviors could decrease the transmission rate of harmful pathogens and viruses to hosts, such as humans (Bi and Wang, 2019).
Wolbachia has been used in recent years to control mosquito populations and the spread of mosquito-borne diseases (Sicard et al., 2019). Extensive research has shown Wolbachia to be capable of interfering with their host’s reproduction. By doing so, Wolbachia is spread throughout the host population (Sicard et al., 2019). One of the most common mechanisms this endosymbiont employs is cytoplasmic incompatibility. Cytoplasmic incompatibility occurs when infected males mate with uninfected females (Fig. 2). Wolbachia ultimately renders the male host sterile in these situations, for the offspring that are produced are non-viable and unable to hatch (Stouthamer et al., 1999). Wolbachia also has the ability to eliminate male offspring during embryogenesis, a phenomenon known as male killing (Riparbelli et al., 2012). It can also cause an incidence called parthenogenesis, which involves the reproduction of infected females without fertilization by a male. (Stouthamer et al., 1994).
Figure 2. Application of Wolbachia in reducing mosquito population and pathogen transmission. Wolbachia causes cytoplasmic incompatibility in mosquitos, which arises from the production of nonviable offspring when Wolbachia infected males mate with uninfected females. Production of pathogen resistant offspring also arises when Wolbachia infected females mate with uninfected males.
10 Besides Wolbachia’s ability to alter host reproduction, it has also been shown to influence the growth of other pathogens residing in the host. The mosquito species, Ae. aegypti, transmits the dengue virus (DENV), yellow fever virus, and Zika virus (WHO, 2020). The transmission of these viruses is the cause of deadly infections and other detrimental consequences to human health. Like ticks, most methods to control mosquitos and mosquito- borne pathogens involved the use of chemical pesticides. As an alternative, researchers are considering Wolbachia as a method of biocontrol. Wolbachia’s effect on host reproduction can be utilized to decrease mosquito populations. However, more research on Wolbachia and the effect they have on mosquitoes suggests the endosymbiont can also be used to prevent the establishment of other pathogens within the mosquito microbiome, such as DENV (Moreira et al., 2009). For example, studies done on Ae. aegypti, suggests Wolbachia infected mosquitos decreases replication of the virus within the host (Hedges et al., 2011; Glaser and Meola, 2010).
Wolbachia inhibits the ability of viruses to enter a host cell and replicate. Research has also reported that Wolbachia can increase the same species of mosquito’s resistance to DENV or block the virus. Though trials that have transinfected Ae. aegypti with Wolbachia as a method of biocontrol are already underway, there is still many unknowns that surround this method. One of these unknowns is how the rest of the mosquito microbiome responds to the blocking of DENV.
A study conducted by (Audsley et al., 2017) set out to address these questions. In this study, the microbiome of laboratory-reared Ae. aegypti mosquitos infected with the Wolbachia strain, wMel, was compared to the microbiome of uninfected laboratory reared Ae. aegypti mosquitos.
This study found that an infection with Wolbachia caused little changes to the mosquito microbiome (Audsley et al., 2017). The study also investigated whether changes to the microbiome of mosquitos infected with Wolbachia would alter the blocking of DENV. By
11 treating the mosquitos with an antibiotic, the study was able to create these changes and ultimately concluded that they had no effect on Wolbachia’s ability to block DENV (Audsley et al., 2017).
As it has been done for mosquitos, biocontrol might be the next big weapon when it comes to the control of ticks and tick-borne diseases. If Wolbachia was able to be utilized to decrease mosquito populations and block the transmission of pathogens, the same methods could also possibly be used in ticks. Currently, very little research has been done when it comes to
Wolbachia and its presence in ticks. Questions, such as what strains of the pathogen are present in the ticks and what species of tick are more susceptible to it, require answering. Other questions include how this pathogen would affect the tick and its microbiome. Identifying and characterizing Wolbachia ticks may encourage study of these organisms as a potential method of biocontrol.
12 Chaper 3: Methodology
Figure 3: Illustration of experimental design and methodology.
Tick Collection
All ticks used in this study were either field collected or colony reared under laboratory conditions. Hyalomma anatolicum anatolicum and Rhipicephalus microplus ticks were carefully removed while feeding on cattle from the Punjab province of Sialkot [32°29′33.7″N,
74°31′52.8″E], Gujrat [32°34′22″N, 74°04′44″ E], Gujranwala [32°9′24″N, 74°11′24″E], and
Sheikhupura [31°42 47″ N, 73°58′41″ E], Pakistan. Amblyomma maculatum, Amblyomma americanum, and Ixodes scapularis ticks were all maintained at room temperature with approximately 90 % relative humidity under a photoperiod of 14 h light/10 h dark to shorten the reproductive cycle.
13 Tick Identification
Morphological identification of the exotic tick species was carried out by an expert taxonomist (Dmitry A. Apanaskevich) at the United States National Tick Collection (USNTC) as previously described (Hoogstrall et al., 1966; Karim et al., 2017; Mukherjee et al., 2014).
Laboratory reared ticks were visually differentiated using phenotypes such as tick size and distinct landmarks on the cuticles. Ixodes scapularis are distinctly reddish and have a solid black dorsal shield with long, thin mouth parts. Amblyomma americanum are medium-sized, with a very round body, reddish-brown color, and long thin mouthparts. The most easily identifiable characteristic is an obvious white dot on the female's dorsal shield, which gives the tick species its name. Amblyomma maculatum have a reduced dark brown scutum, a plate on their back, with silvery white posterior ornamentation that includes one median and two lateral interrupted stripes. Rhipicephalus microplus have a short, straight capitulum. The legs are pale cream and there is a wide space between first pair of legs and the snout. The body is oval to rectangular and the shield is oval and wider at the front. The snout is short and straight. Hyalomma anatolicum anatolicum have large mouthparts and integuments striations. The scutum are colored brown.
The slender legs usually have pale rings. A total of 305 ticks were morphologically identified and used for subsequent isolation of genetic material. Based on the morphologies of the ticks, 80 ticks were identified as Amblyomma americanum, 40 as Amblyomma maculatum, 90 as
Hyalomma anatolicum anatolicum, 20 as Ixodes scapularis, and 75 as Rhipicephalus microplus.
All ticks were adult female ticks and either fully fed or pulled off prior to being fully fed.
Genomic DNA Extraction
Prior to DNA extraction, individual ticks were surface sterilized in a series of steps.
Briefly, a 10% solution of sodium hypochlorite was used to clean the individual surfaces of ticks
14 followed by rinsing in 70% ethanol. A final cleaning was done using sterile water. Genomic
DNA was extracted from each individual tick homogenate using a DNeasy blood and tissue kit
(Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The concentrations of the extracted genomic DNA samples were quantified using a Nanodrop ND-100 instrument and
DNA was stored in −20°C till further needed.
Molecular Identification of Tick Species
To further confirm morphological identification, two DNA representing the five tick species were subjected to molecular identification by amplifying the highly conserved 708 bp mitochondrial cytochrome oxidase I (COI) gene. Briefly, a reaction mix containing 1 L DNA template, 12.5 L of One-Taq polymerase Master Mix, 9.5 L of nuclease free H2O, 1 L each of the COI gene specific forward primer (5’−GGT CAA CAA ATC ATA AAG ATA TTG
G−3’)) and reverse primer (5’−TAA ACT TCA GGG TGA CCA AAA AAT CA−3’) was set up and subjected to the following PCR conditions; 94 C for 2 min, 94 C for 30 sec, 50 C for 45 sec, 72 C for 1 min (x 35), 72 C for 10 min, and 4C on hold using a Bio-Rad T100TM Thermal
Cycler (Dougherty and Li., 2017). The presence of the COI gene in the PCR product was confirmed with gel electrophoresis using a 3% agarose gel and 10 L of ethidium bromide.
Molecular Detection of Wolbachia
The presence of Wolbachia species was confirmed by amplifying the highly conserved
438 bp Wolbachia 16S ribosomal RNA gene and the 800 bp heat shock (GroEL) gene (Masui et al., 1997).
Detection of Wolbachia 16S rRNA
For identification of the Wolbachia 16S rRNA gene, a reaction mix of 1 L of DNA, 12.5
L of One-Taq polymerase Master Mix, 9.5 L of nuclease free H2O, 1 L each of the
15 Wolbachia 16S rRNA gene specific forward (5’−CAT ACC TAT TCG AAG GGA TAG−3’) primer and reverse (5’−AGC TTC GAG TGA AAC CAA TTC−3’) primer was set up. A negative control was made by replacing the cDNA sample with 1 L of nuclease free H2O
(Baldini et al., 2014). A positive control was made by replacing the tick cDNA sample with 1 L of DNA extracted from a mosquito infected with Wolbachia. Polymerase chain reaction (PCR) was performed using a Bio-Rad T100TM Thermal Cycler under the following conditions: 94 C for 2 min, 94 C for 30 sec, 55 C for 45 sec, 72 C for 1 min (x40), 72 C for 10 min, and 4 C on hold. The presence of the Wolbachia 16S rRNA gene in the PCR product was confirmed with gel electrophoresis using a 3% agarose gel and 10 L of SYBR Safe DNA Gel Stain.
Detection of Wolbachia GroEL
For identification of the Wolbachia GroEl gene, a reaction mix of 1 L of cDNA, 12.5
L of One-Taq polymerase Master Mix, 9.5 L of nuclease free H2O, 1 L of the Wolbachia
GroEl gene specific forward (5’−TGT ATT AGA TGA TAA CGT GC−3’) and reverse (5’−CCA
TTT GCA GAA ATT ATT GCA−3’) primer was set up. A negative control was made by replacing the cDNA sample with 1 L of nuclease free H2O (Masui et al., 1997). A positive control was made by replacing the tick cDNA sample with 1 L of cDNA extracted from a mosquito infected with Wolbachia. Polymerase chain reaction (PCR) was performed using a
Bio-Rad T100TM Thermal Cycler under the following conditions: 94 C for 2 min, 94 C for 60 sec, 56 C for 60 sec, 72 C for 2 min (x 35), 72 C for 10 min, and 4 C on hold. The presence of the Wolbachia GroEl gene in the PCR product was confirmed with gel electrophoresis using a
3% agarose gel and 10 L of SYBR Safe DNA Gel Stain.
16 PCR Sequencing and Phylogeny
The PCR products of the samples positive for the Wolbachia 16S rRNA gene and
Wolbachia GroEl gene, as well as the COI gene, were purified using the QIAquick PCR
Purification Kit in accordance with the provided protocol. The purified products were then sent for sequencing to Eurofins Genomics. The evolutionary history for Wolbachia 16S rRNA and
GroEL sequences was inferred using the Neighbor-Joining method (Saitou and Nei, 1987), while the tick’s COI sequences were inferred using the UPGMA method (Sneath and Sokal, 1973). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) and were in the units of the number of base differences per site. This analysis involved 11 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair
(pairwise deletion option). There were a total of 1,534 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The Klebsiella pneumonia 16S ribosomal RNA genes sequence was used as outliers when constructing the phylogenetic trees.
Microbial Profiling
All tick DNA positive for Wolbachia and representative of non-positive ticks were analyzed for their microbiome composition. Briefly, PCR sequencing of the V1-V3 variable region of the bacterial 16S rRNA gene were amplified using barcoded primers 27F/519R as outlined by the 16S Illumina’s MiSeq protocol (www.mrdnalab.com, Shallowater, TX, USA).
Briefly, PCR was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 min, followed by 30-35 cycles of 94 °C for 30 s, 53 °C for 40 s, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. After
17 amplification, PCR products were electrophoresed in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then, the pooled and purified PCR product was used to prepare Illumina DNA library. Sequencing was performed at MR DNA
(www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer’s guidelines.
Data Analyses
Sequence analysis was done using the Quantitative Insights into Microbial Ecology
(QIIME 2) (Bolyen et al., 2019), unless stated otherwise. Briefly, processing of raw FASTQ files were demultiplexed. The Atacama soil microbiome pipeline was incorporated for quality control of demultiplexed paired-end reads using the DADA2 plugin as previously described (Callahan et al., 2016). Sequence alignment and subsequent construction of phylogenetic trees from representative sequences were done using the MAFFT v7 and FasTree v2.1 plugin (Price et al.,
2010). Operational taxonomic assignment was done using the qiime2 feature-classifier plugin v7.0 (Bokulich et al., 2018) which was previously trained against the SILVA 132 database pre- clustered at 99% (Quast et al., 2013). Diversity analysis was done by rarifying individual sequences to a depth of 1,000 to get adequate coverage of all samples analyzed and samples with insufficient reads subsequently screened out. Faith phylogenetic distance (Faith_pd) and the
Shannon index was used in assessing alpha diversity. The Shannon index is a measure of diversity which takes into account both species richness and evenness in measuring alpha diversity. The higher the Shannon index, the more diverse a microbial community is presumed to be. Box plots were generated using GraphPad Prism (v8). Kruskal-Wallis (pairwise) test of one-
18 way ANOVA was used to test the significance of alpha-diversity between infected and uninfected mosquitos. Raw data from this analysis were part of a bigger microbiome study
(Adegoke et al., 2020 preprint) and has been deposited and assigned the GenBank BioProject number PRJNA600935.
19 Chapter 4: Results
Tick Species Studied
The identification of tick species used in this study was carried out using two independent approaches. The use of distinct, genus specific features such as cuticular coloration, unique markings and mouthpart conformation aided in morphological identification of the tick species to genus level (Fig. 4A), while the use of a molecular approach allowed for identification of the ticks to species taxonomic level. The 706 bp of the highly conserved arthropod’s COI gene was used for identification (Fig. 4B).
Figure 4: Morphological identification A), and molecular confirmation B), of tick species using the mitochondrial COI gene. Lane 1 represents the DNA ladder, 2 the negative control, 3 and 4 Ix. scapularis DNA, 5 and 6 A. maculatum DNA, 7 and 8 A. americanum DNA, 9 and 10 H. anatolicum anatolicum DNA, 11 and 12 R. microplus DNA.
20 The evolutionary relationship between the representative COI gene sequence of ticks from this study were further compared amongst each other (Fig. 5A) as well as from other ticks’ COI gene from the GenBank database (Fig. 5B to 5E)
21
Figure 5: Phylogenetic analysis of amplified COI sequences with tick species from this study 5A) and Ix. scapularis 5B), A. americanum 5C), A. maculatum 5D), and Hyalomma anatolicum, and Rhipicephalus microplus 5E) with other arthropod's COI genes from GenBank. Phylogenetic trees were evaluated using the UPGMA method and a bootstrap consensus of 1000 replicates.
Wolbachia Prevalence
The prevalence of Wolbachia species identified from the tick species was estimated to be very low. Overall, out of 305 ticks tested, three were found positive for Wolbachia species. All the North American ticks tested, Am. americanum, Am. maculatum, and Ix. Scapularis, were all negative for Wolbachia whereas three of the exotic ticks, H. anatolicum anatolicum and R. microplus, were positive for Wolbachia (Fig. 6A and B). Based on these results, it can be concluded that all laboratory-reared ticks were Wolbachia uninfected. The few ticks positive for
Wolbachia were wild ticks. Evolutionary relationship between the identified Wolbachia
22 sequences and sequences from public databases was compared and phylogenetic trees were made. (Fig 7A and B).
Figure 6: PCR analysis of Wolbachia species A) 438bp 16S ribosomal RNA and B), 800bp heat shock GroEL genes. Lanes 1-3 on both gels represent the DNA ladder, negative control and positive control, respectively. Lanes 4-6 of Figure A correspond to tick DNA positive for 16S rRNA gene, whereas lanes 6 and 7 of B show positive band for GroEL gene of Wolbachia.
Figure 7: Genetic relationship of identified Wolbachia A), 16S rRNA and B), GroEL genes compared with selected sequences from GenBank. The 438bp and 800bp sequences of the 16S rRNA and GroEL genes respectively, were analyzed using Neighbor Joining method
23 The tree shown in Fig. 7 suggests the heat shock GroEl gene identified in the sample of
H. anatolicum is closely related to the GroEl gene of a Wolbachia pipientis strain wAlbB-FL2016 and a Wolbachia endosymbiont of Aedes albopictus (Fig. 7B), whereas the 16S ribosomal RNA gene from R. microplus showed distinct similarity to a Wolbachia endosymbiont of Kleidocerys resedae and Chrysoma megacephala (Fig. 7A).
Microbiome Composition
Summary
Analysis of the demultiplexed paired-end-reads generated 2902862 reads, which ranged from
17,063 to 123,731 with an average of 65,609 reads. Sequences from samples of Wolbachia uninfected ticks generated the most reads of 1,594,076 while sequences from Wolbachia infected ticks generated 1,308,786 reads. Taxonomic classification using the SILVA reference base identified 30 and 85 OTUs generated from Wolbachia uninfected and infected ticks.
Bacteria Species Abundance
Ticks infected with Wolbachia had a very high abundance of the bacteria Candidatus
Midichloria mitochondrii (CMM, 24%), Francisella-like endosymbiont (FLE, 10%), and
Propionibacterium acnes (9%). The microbiome of Wolbachia uninfected ticks was dominated by Bacillus pumilus (19%), Staphylococcus sciuri (11%), and Empedobacter falsenii (12%).
Bacteria species found to be commonly shared amongst the two groups were Staphylococcus sciuri, Propionibacterium acnes, and Corynebacterium species (Fig. 8A).
24
Figure 8: Relative abundances of bacteria at the species taxonomic differences.
Diversity Analysis
Estimation of the number of operational taxonomic units (OUT), which is an estimation of bacteria richness, was done by plotting rarefied curves of Wolbachia infected and uninfected ticks (Fig 9A). This clearly shows that bacteria richness was much higher in the Wolbachia uninfected ticks compared to the infected ticks. Subsequently, species diversity was compared between the infected and uninfected tick groups. This also shows that Wolbachia infected ticks have a less diverse microbiome compare to the uninfected ticks (Fig 9B and C).
25
Figure 9: Rarefaction analysis of Wolbachia infected and uninfected ticks 9A. Each curve was rarefied to a depth of 650. Diversity analysis using A), Faith_phylogenetic (Kruskal-Wallis, p=0.007) distance and B), Shannon Index (Kruskal-Wallis, p=0.05) were both used as metrics to measure alpha diversity.
26 Chapter V: Discussion and Conclusion
The aim of this study was to identify and characterize the -Proteobacteria endosymbiont, Wolbachia, in six different species of ticks. In addition to characterization of
Wolbachia, this study set out to investigate differences between the microbiomes of ticks infected with Wolbachia and those that were negative for the endosymbiont. This study included three tick species prevalent in the United States of America and two tick species prevalent in
Pakistan. Based on microbiome analysis, this study supports my hypothesis that the native microbiome will be altered by the presence of Wolbachia.
PCR and gel analysis, as well as sequencing results, revealed that ticks do harbor the
Wolbachia endosymbiont, but at a very low prevalence. All Ambylomma and Ixodes ticks (0/140) were negative for Wolbachia whereas Hyalomma and Rhipicephalus ticks (3/165) tested positive for Wolbachia. All three of those samples of tick DNA were found to be positive for the 16S rRNA gene and heat shock GroEl gene, which are both characteristic genes found in all
Wolbachia strains. Analysis of genetic relationship revealed that the Wolbachia sequences isolated from the samples of tick DNA have a close relationship with the Wolbachia pipientis strain wAlbB-FL2016. The sequences were also found to be closely related to Wolbachia symbionts found in Ae. aegypti, Ae. albopictus, and Chrysoma (C.) megacephala. How the presence of the Wolbachia endosymbiont affects C. megacephala in terms of reproduction and microbiome is relatively unknown. Much of the current research that exists on Wolbachia involves how it affects mosquito species and their microbiome and feeding. As previously mentioned, Wolbachia has been proven to cause cytoplasmic incompatibility in mosquitos, which can ultimately lead to a decrease in mosquito populations (Stouthamer et al., 1999). It has also been shown to block the replication of other pathogens and viruses in the mosquito, such as
27 DENV (Audsley et al., 2016). Since the Wolbachia strains isolated in ticks by this study were closely related to the Wolbachia strains present in mosquitos, it raises the question as to if
Wolbachia would behave similarly in ticks as it does in mosquitos. More research is required to determine whether Wolbachia is capable of rendering male ticks sterile through cytoplasmic incompatibility or even affecting the replication and growth of other pathogens in ticks. If this is the case, Wolbachia could possibly become the next step when it comes to biocontrol of tick populations.
Out of all the ticks tested for the presence of Wolbachia, only three were found to be positive for the endosymbiont. All three of those positive samples were from Hyalomma and
Rhipicepihalus tick species. A study conducted by Gal and colleagues (2017) compared the microbiomes of lab-reared and field populations of Dermacentor andersoni ticks over the course of three generations. The study found significant differences between the microbiome of laboratory-reared ticks and microbiome of field ticks (Gall et al., 2017). They also found that the laboratory setting stabilized the tick microbiome. In comparison, the microbiome of the field tick populations was influenced by the environment from which they were collected (Gall et al.,
2017). In my studies, all the North American ticks were raised under laboratory conditions, whereas the Pakistani ticks were collected from the field. This could possible explain the absence of Wolbachia from the North American ticks. Based on the evidence presented by Gall and colleagues (2017), a possible theory for why Wolbachia was only isolated from field ticks could be related to the idea of the bacterial microbiomes of field ticks being influenced by their environment. Wolbachia could have been acquired from the environment the field ticks were collected from, which would explain why it was not present in the laboratory-reared ticks.
28 One possible explanation for all Amblyomma and Ixodes tick samples testing negative for
Wolbachia may be related to a relationship between Wolbachia and Rickettsial endosymbionts.
Both Amblyomma and Ixodes ticks are often colonized by members of the Rickettsia genus, which is closely related to the Wolbachia genus (Parola et al., 2013). The Rickettsia genus includes many pathogens known to cause harmful human diseases (Parola et al., 2013). In recent years, several studies have suggested members of the Rickettsia genus to be capable of interfering with the reproduction of arthropods, similarly as is done by Wolbachia (Perlman et al., 2006). This raises questions as to if the existence of two reproductive manipulators places a heavy burden on the host. Though Amblyomma and Ixodes ticks are known to be colonized by members of the Rickettsia genus, the presence of Wolbachia could be too much for the host’s system to handle. This could be a possible reason as to why all Amblyomma and Ixodes ticks were found to be negative for Wolbachia.
Other studies have identified Wolbachia in certain species of ticks and have provided some insight as to whether Wolbachia is a natural inhabitant of the tick microbiome or if it’s a consequence of another infection. Multiple studies have reported naturally Wolbachia infected populations in Ae. aegypti, however many efforts of biocontrol in Ae. aegypti populations have involved introducing Wolbachia isolated from Drosophila melanogaster to these populations
(Ross et al., 2020; Moreira et al., 2009). Wolbachia is found in the cytoplasm of cells and is passed from mother to offspring through vertical transmission (Jiggin et al., 2017). The idea that
Wolbachia exists in the cytoplasm of cells suggests why it cannot be transferred from male to offspring, for there is little cytoplasm found in sperm compared to oocyte (Stouthamer, 2009). It has also been documented that Wolbachia can be passed to an un-infected organism through the ingestion of an infected one. In this case, Wolbachia was capable of passing the intestinal lining
29 and infecting the reproductive organs (Le Clec’h et al., 2013). Incidences where Wolbachia was introduced into an organism though parasitism has been seen in multiple species, including ticks.
A study conducted by Plantard and colleagues (2012) set out to determine if there was a link between Wolbachia detection and the presence of the endoparasitoid Ixodiphagus hookeri in the tick species Ixodes ricinus. The study detected the presence of Wolbachia when Ix. hookeri was present in ticks but noticed no Wolbachia was present when the parasite was absent. This study suggests that the parasite harbors Wolbachia and introduced it to the tick through their association (Plantard et al., 2012). Moreover, the ticks used in that study were collected from the environment and were already parasitized. Therefore, no lab-reared ticks were used. The work by
Plantard et al. (2012) could support our hypothesis as to why North American ticks, or lab-reared ticks, were found to be negative for Wolbachia, while a few of our exotic ticks, or field ticks, were positive for the endosymbiont. Although the North American ticks were hatched and reared in a lab, the exotic ticks were collected from the environment. As the study by Plantard and colleagues (2012) suggested, Wolbachia could be introduced to ticks through other organisms the ticks comes in contact within their environment. Similarly, the presence of Wolbachia in mosquitos infected with Dirofilaria immitis, the nematode responsible for canine heartworm disease in dogs in the United States (Bowman and Atkins, 2009), has been associated with
Wolbachia infecting the nematode (Bandi et al., 2001).
Candidatus Midichloria mitochondrii (CMM), Francisella-like endosymbiont (FLE) and
Propionibacterium acnes were detected in relatively higher abundances in Wolbachia infected ticks. CMM and FLE are known obligate, vertically maintained in the phylum Proteobacteria endosymbiont of hard ticks with previous studies reporting them in ticks (Budachetri et al., 2018;
Williams-Newkirk et al., 2012; Ivanov et al., 2011). Both CMM and FLE have been reported to
30 support the ticks’ nutritional requirement as well as prevent pathogen colonization within the tick
(Budachetri et al., 2018; Ahantarig et al., 2013). The presence of these symbionts in Wolbachia infected ticks could support a possible synergism where different bacteria coexist together without interfering with the one another. Also, this could further be explained by the fact that all three of them are all in the same phylum Proteobacteria.
Uninfected ticks have a different bacterial composition within their microbiome. Bacillus pumilus (B.), Staphylococcus (S.) sciuri, and Empedobacter falsenii (F.) were all present in relatively high abundances in uninfected ticks. S. sciuri are part of normal flora of various livestock and wild animals. Previous report also detected these bacteria in the microbiome of
Rhipicephalus microplus collected from cattle (Andreotti et al., 2011). Numerous bacteria in the genus Bacillus have also been reported in the microbiome of ticks. Lee and colleagues (2019) reported the presence of B. pumilus and B. chungangensis in Amblyomma gemma ticks. Bacillus thuringiensis, which is known for its entomopathogenic Bt toxin, have also been tested as a possible biological control measure in Ixodes ricinus ticks (Szczepańska et al., 2018).
An overall difference in the species richness was also observed when the diversity was compared between the two groups. Wolbachia infected ticks have a least diverse and species rich microbiome composition in contrast to uninfected ticks. I observed a difference in the individual abundances of bacteria such as Corynebacterium, Bacillus carboniphilus, and Acinetobacter indicus, which were relatively higher in uninfected ticks when compared to infected ticks. My observation was also supported by a similar study in Aedes aegypti mosquitos (Audsley et al
2017). The study revealed that the presence of Wolbachia reduces the relative abundances of several bacterial taxa in adult mosquitos. This observation was associated to the pathogen blocking effect that has been previously reported for Wolbachia.
31 Though this study does answer questions as to how the microbiome of Wolbachia infected and un-infected ticks differ, more research is required to offer up further comparisons.
This study was unable to analyze the microbiome of the blood from which the ticks were fed, as well examine the microbiome of the tick in different tissues or life stages. Though these are common and popular approaches in microbiome research, technical challenges did not allow for this to be possible in this study. Limitations in this study have raised more questions and left the need for more research, as well. For example, a small sample size was used when identifying
Wolbachia in tick species, which suggests our results are not completely representative of larger tick populations. These limitations have left questions open for future research.
Though many questions remain, this study did show that Wolbachia is present in tick species and has an influence over the microbiome of the tick. The strains identified in these ticks were closely related to the strains seen in mosquito species, where Wolbachia is already utilized as a method of biocontrol. The hope is that the conversation remains open as to the host- endosymbiont relationship between Wolbachia and ticks, as well the possibility of Wolbachia’s use as a weapon for control of ticks and tick-borne diseases.
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INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
118 College Drive #5116 | Hattiesburg, MS 39406-0001 Phone: 601.266.5997 | Fax: 601.266.4377 | [email protected] | www.usm.edu/iacuc
NOTICE OF COMMITTEE ACTION
The proposal noted below was reviewed and approved by The University of Southern Mississippi Institutional Animal Care and Use Committee (IACUC) in accordance with regulations by the United States Department of Agriculture and the Public Health Service Office of Laboratory Animal Welfare. The project expiration date is noted below. If for some reason the project is not completed by the end of the approval period, your protocol must be reactivated (a new protocol must be submitted and approved) before further work involving the use of animals can be done.
Any significant changes should be brought to the attention of the committee at the earliest possible time. If you should have any questions, please contact me.
PROTOCOL NUMBER: 15101501.1 (Replaces 15101501) PROJECT TITLE: Tick Sialome PROPOSED PROJECT DATES: 10/2018 – 09/2020 PROJECT TYPE: Renewal PRINCIPAL INVESTIGATOR(S): Shahid Karim DEPARTMENT: School of Biological, Environmental, and Earth Sciences FUNDING AGENCY/SPONSOR: USDA NIFA/NIH/USAID IACUC COMMITTEE ACTION: Designated Review Approval PROTOCOL EXPIRATON DATE: September 30, 2020
Jake Schaefer, PhD Date: October 25, 2018 IACUC Chair
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