Diversity of Rickettsiales in the Microbiome of the Lone Star Tick,Amblyomma americanum

Ticks are important vectors for many emerging pathogens. However, they are also infected with many symbionts and commensals, often competing for the same niches. In this paper, we characterize the microbiome of Amblyomma americanum (Acari: Ixodidae), the lone star tick, in order to better understand the evolutionary relationships between pathogens and nonpathogens. Multitag pyrosequencing of prokaryotic 16S rRNA genes (16S rRNA) was performed on 20 lone star ticks (including males, females, and nymphs). Pyrosequencing of the rickettsial sca0 gene (also known as ompA or rompA) was performed on six ticks. Female ticks had less diverse microbiomes than males and nymphs, with greater population densities of Rickettsiales. The most common members of Rickettsiales were “Candidatus Rickettsia amblyommii” and “Candidatus Midichloria mitochondrii.” “Ca. Rickettsia amblyommii” was 2.6-fold more common in females than males, and there was no sequence diversity in the sca0 gene. These results are consistent with a predominantly vertical transmission pattern for “Ca. Rickettsia amblyommii.”     

The Microbiome of Ehrlichia-Infected and Uninfected Lone Star Ticks (Amblyomma americanum)

The Lone Star tick, Amblyomma americanum, transmits several bacterial pathogens including species of Anaplasma and Ehrlichia. Amblyomma americanum also hosts a number of non-pathogenic bacterial endosymbionts. Recent studies of other arthropod and insect vectors have documented that commensal microflora can influence transmission of vector-borne pathogens; however, little is known about tick microbiomes and their possible influence on tick-borne diseases. Our objective was to compare bacterial communities associated with A. americanum, comparing Anaplasma/Ehrlichia -infected and uninfected ticks. Field-collected questing specimens (n = 50) were used in the analyses, of which 17 were identified as Anaplasma/Ehrlichia infected based on PCR amplification and sequencing of groEL genes. Bacterial communities from each specimen were characterized using Illumina sequencing of 16S rRNA gene amplicon libraries. There was a broad range in diversity between samples, with inverse Simpson’s Diversity indices ranging from 1.28–89.5. There were no statistical differences in the overall microbial community structure between PCR diagnosed Anaplasma/Ehrlichia-positive and negative ticks, but there were differences based on collection method (P < 0.05), collection site (P < 0.05), and sex (P < 0.1) suggesting that environmental factors may structure A. americanum microbiomes. Interestingly, there was not always agreement between Illumina sequencing and PCR diagnostics: Ehrlichia was identified in 16S rRNA gene libraries from three PCR-negative specimens; conversely, Ehrlichia was not found in libraries of six PCR-positive ticks. Illumina sequencing also helped identify co-infections, for example, one specimen had both Ehrlichia and Anaplasma. Other taxa of interest in these specimens included Coxiella, Borrelia, and Rickettsia. Identification of bacterial community differences between specimens of a single tick species from a single geographical site indicates that intra-species differences in microbiomes were not due solely to pathogen presence/absence, but may be also driven by vector life history factors, including environment, life stage, population structure, and host choice.     

Microbial communities and interactions in the lone star tick,Amblyomma americanum

To quantify microbial composition and interactions, we identified prokaryotic communities in the lone star tick (Amblyomma americanum) based on 16S rRNA gene sequences and direct probing. The lone star tick is the vector of emerging diseases and host to additional symbionts of unknown activity, and is representative of other blood‐sucking arthropods. We evaluated the potential for vertical (transovarial) transmission by molecular analysis of microbial symbionts from egg and larval clutches. Direct probing of adults (N = 8 populations from the southeastern and midwestern USA, 900 ticks total) revealed three vertically transmitted symbionts: a Coxiella symbiont occurred at 100% frequency, Rickettsia species occurred in 45–61% of all ticks in every population and an Arsenophonus symbiont occurred in 0–90% of ticks per population. Arsenophonus and Rickettsia exhibited significant heterogeneity in frequency among populations. The human pathogens Ehrlichia chafeensis and Borrelia lonestari were rare in most populations. Additional microbes were detected sporadically. Most ticks (78%) were co‐infected by two or three microbes but statistical analysis indicated no significant deviation from random co‐occurrence. Our findings indicate that microbial communities within lone star ticks are diverse, and suggest that direct probing for a wider range of prokaryotes and application of quantitative polymerase chain reaction (PCR) may provide further insights into microbial interactions within disease vectors. Our results also emphasize the close phylogenetic relationship between tick symbionts and human pathogens, and consistent differences in their prevalence.     

A conceptual model of the Amblyomma americanum life cycle in northeast Missouri

The geographic distribution of Amblyomma americanum (the lone star tick) has increased as has its role as a pathogen vector. The objectives of this study were to determine seasonal activity patterns of each life stage of A. americanum in the northwestern part of the species range and the relationship of these activity patterns among life stages and degree days. Tick activity was monitored over four years since 2007 in a forest and old field habitat located in northeast Missouri. Every other week from February to December, ticks were collected using bait and drag methods. Autocorrelations demonstrated yearly seasonal patterns in each life stage and cross‐correlations between life stages depicted a relationship between activity at a life stage and the previous stage's activity. Cross‐correlations indicated that degree days were related to activity. These data indicated that A. americanum generally complete their life cycle in a minimum of two years in northeast Missouri, with overwintering occurring predominantly in the nymphal and adult stages. These data provide a baseline to compare the life cycle of A. americanum in northeast Missouri to populations in different parts of the species range or at different times in the region.     

Ultrastructure of Haller's organ in the tick Amblyomma americanum (L.)

Summary Haller's organ on the tarsus of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) was studied by scanning and transmission electron microscopy. It consists of a distal bristle group, (the anterior pit), and a proximal capsule which encloses several sensilla. The seven sensilla of the anterior pit (A1–A7) are all thick-walled and multi-innervated (2–9 neurons), but at least three different types can be differentiated. Sensilla A1 and A2 possess large, plugged pores (>1000 Å) and are the only sensilla with branching dendrites. A3 and A5 are characterized by a spoke-wheel arrangement of the cuticle wall and very fine pores (100–200 Å) penetrating the spokes centrally; A4, A6, and A7 do not exhibit any pore system but a single opening at the bristle tip is assumed.The capsule contains seven thin-walled, blunt-tipped sensilla, and several non-sensory cuticular projections (pleomorphs). All of these sensilla possess large plugged pores in the cuticle wall and numerous dendritic branches of several neurons (3–5) in the lumen. Glandular openings were found inside the capsule; their significance is discussed.The fine structure of Haller's organ supports the functions postulated by Lees (1948), namely olfaction for the capsule and humidity reception (among others) for the anterior pit.This research was supported in part by the Office of Naval Research, and by NIH Training grant ES 00069. Paper no. 3459 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Fine structural analysis of palpal receptors in the tick Amblyomma americanum (L.)

Summary Palps of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) were studied by scanning and transmission electron microscopy. The terminal palp segment (IV) bears the so-called palpal organ, a cluster of 10 short, blunt-tipped sensilla. All sensilla (except for the center sensillum) receive a dual innervation: 2 mechanoreceptive dendrites which terminate in the socket membrane plus several chemoreceptive dendrites (4–12) which enter the lumen. The thick-walled cuticular shaft possesses 2–3 small pore openings (100 Å) below the tip, thus establishing communication between dendrites and environment. Two structurally different types of palpal sensilla exist: The A-type has a characteristic doublelumen and always contains 4 dendrites, the B-type features a single lumen and a specially layered cuticular shaft with 6–12 dendrites. The fine structure of the tick palpal receptors corresponds closely to that of known contact chemoreceptors in insects.This research was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator), and by NIH Training grant ES 00069. Paper no. 3700 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Inferring the population structure and demographic history of the tick, Amblyomma americanum Linnaeus.

A hierarchial population genetic study was conducted on 703 individual Amblyomma americanum from nine populations in Georgia, U.S.A. Populations were sampled from the Coastal Plain, midland Piedmont region, and the upper Piedmont region. Twenty-nine distinct haplotypes were found. A minimum spanning tree was constructed that indicated these haplotypes comprised two lineages, the root of which was distinctly star-like. The majority of the variation found was among ticks within each population, indicating high amounts of gene flow and little genetic differentiation between the three regions. An overall F(ST) value of 0.006 supported the lack of genetic structuring between collection sites in Georgia. Mantel regression analysis revealed no isolation by distance. Signatures of population expansion were detected in the shapes of the mismatch distribution and tests of neutrality. The absence of genetic differentiation combined with the rejection of the null model of isolation by distance may indicate recent range expansion in Georgia or insufficient time to reach an equilibrium where genetic drift may have affected allele frequencies. Alternatively, the high degree of panmixia found within A. americanum in Georgia may be due to bird-mediated dispersal of ticks increasing the genetic similarity between geographically separated populations.     

Relative abundance and survival of the tick Amblyomma americanum collected from sunlit and shaded habitats

The population density of host-searching nymphal and adult lone star ticks, Amblyomma americanum (L.) (Acari: Ixodidae), was determined at the Robinson tract of the Kansas Ecological Reserves and a private farm 5 km north-west of the Robinson tract using standard drag cloth methods. Nymphs, males and females were counted and collected weekly from shaded habitats and adjacent sunlit habitats from mid-May through late July, 2003. Of the 1598 nymphs and 549 males collected by drag sampling, 74.0% and 72.1%, respectively, were collected from shaded sections of the habitats, whereas 77.3% of 472 females were found in sunlit sections. A. americanum collected during each sampling period were maintained unfed at >95% relative humidity and a 14 : 10 h photoperiod, and survival was recorded weekly until all ticks had died. Survival of nymphs, males and females did not differ between ticks collected in the shade vs. those collected in the sun. Nymphs survived significantly longer than adults, whereas male and female survival did not differ from each other. These results suggest that host-searching A. americanum populations may partition their environment to increase the chances of coming into contact with a potential vertebrate host.     

A dopamine sensitive adenylate cyclase in the salivary glands of Amblyomma americanum (L.)

1. Enzyme activity, basal or dopamine-stimulated (10 microM), was linear with time to 25 min and with protein concentration to 0.8 mg protein/ml of final assay volume. Activity was maximal between pH 7.0 and 7.5. 2. Mg2+ maximally stimulated basal or dopamine-sensitive adenylate cyclase activity at about 4 mM. 3. Adenylate cyclase had a Km of 0.042 mM for ATP and maximum velocities for basal and dopamine-stimulated activity of 107 and 179 pmol cyclic AMP formed/mg protein per min, respectively. 4. Half-maximal stimulation of the enzyme occurred at about 4.2 x 10(-7) M dopamine with the threshold being less than 10(-9) M. Dopamine increased the Vmax but had no effect on the Km of ATP. 5. Eighty-five to 90% of the adenylate cyclase activity was found in the particulate fraction. 6. Calcium ion produced a marked inhibition of adenylate cyclase activity above 0.04 mM and half-maximal inhibition occurred near 0.1-0.2 mM.     

Localization and visualization of a coxiella-type symbiont within the lone star tick, Amblyomma americanum

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.     

Localization and Visualization of a Coxiella-Type Symbiont within the Lone Star Tick, Amblyomma americanum

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.     

Host association and seasonal activity of Amblyomma americanum (Acari: Ixodidae) in Missouri

From June 1993 through June 1996, 2,260 adult, 4,426 nymphal, and 2,178 larval lone star ticks Amblyomma americanum (L.) were collected in Missouri from vertebrate hosts and by dragging a cloth over vegetation. Prevalence, mean intensity, and relative abundance of each stage varied among hosts. The relative abundance of adult lone star ticks was highest on white-tailed deer, but this stage was also collected from raccoons, opossum, red fox, coyotes, and wild turkey. Nymphs were collected from gray squirrels, eastern cottontail rabbits, opossums, red fox, Carolina wren, and bobwhite quail, but the highest relative abundance occurred on wild turkey, white-tailed deer, and raccoons. Eastern cottontail rabbits, white-tailed deer, raccoons, and squirrels had the highest relative abundance of larval lone star ticks, but they were also found on opossums and wild turkey. The activity of adult lone star ticks was greatest from May through July. The activity for nymphs was highest from May through August, and for larvae, July through September.     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Permeability of tarsal sensilla in the tick Amblyomma americanum L. (Acarina, Ixodidae)

Ticks were submerged in silver-protein solution, prior to fixation for electron microscopy, in order to trace the pathway of molecules in supposed tarsal chemoreceptors. Sensilla with radially arranged cuticular canals (100-200 A in diameter) leading to the centrally located dendrites show silver granules inside the canals and in the central lumen, thus directly making contact with the dendrites. Sensilla with large, plugged pores (1200 A) exhibit an accumulation of silver granules in the pore openings but no granules (about 50 A in diameter) were observed penetrating into the lumen. Apparently silver granules could diffuse in, but not through the material which suspends the pore plugs. It is suggested that this material corresponds to the 'pore tubules' in insect olfactory sensilla and that it may play an essential role in transmitting a chemical stimulus from the environment to the dendrites.     

Comparison of differentially expressed genes in the salivary glands of male ticks,Amblyomma americanum and Dermacentor andersoni

Genes expressed differentially in the salivary glands of unfed and fed male ticks, Amblyomma americanum (L.), were identified, cloned and sequenced, and some were compared with those expressed in the salivary glands of Dermacentor andersoni. Total protein and RNA increased sixfold in the salivary glands of fed male A. americanum, while in fed male D. andersoni salivary glands, RNA increased approximately 3.5 times. Feeding D. andersoni in the presence of females increased total RNA by 25% over those fed in the absence of females. Complementary DNAs were synthesized from RNA obtained from unfed and fed ticks and amplified using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) with three different primers in separate reactions. Differential display showed clear banding differences between the fed and the unfed ticks in A. americanum and D. andersoni. Sixty-one cDNA fragments that appeared to be from differentially expressed genes in A. americanum were isolated, cloned and sequenced. Hybridization reactions with labeled cDNA probes confirmed the differential expression of many of the genes in unfed and fed ticks' salivary glands; however, many of the bands contained more than one fragment and some of the fragments isolated from apparently differential bands were not specific. Sequences for 28 of the cDNA fragments (150-600 nucleotides in length) demonstrated similarity to genes in the databases, but nine of these were similar to sequences of unknown function. Some of the gene fragments identified may be important to tick feeding or tick salivary gland physiology, including a histamine-binding protein, an organic ion transporter, an apoptosis inhibitor, a cathepsin-B-like cysteine protease, proteins involved in gene regulation and several proteins involved in protein synthesis. Cross-hybridization of identified cDNAs from A. americanum with cDNA probes synthesized from D. andersoni total RNA did not show significant similarity between the two species.