Distribution and molecular characterization of Wolbachia endosymbionts and filarial nematodes in Maryland populations of the lone star tick (Amblyomma americanum)

Abstract

The lone star tick Amblyomma americanum is host to a wide diversity of endosymbiotic bacteria. We identified a novel Wolbachia symbiont infecting A. americanum. Multilocus sequence typing phylogenetically placed the endosymbiont in the increasingly diverse F supergroup. We assayed a total of 1031 ticks (119 females, 78 males and 834 nymphs in 89 pools) from 16 Maryland populations for infection. Infection frequencies in the natural populations were approximately 5% in females and <2% (minimum infection rate) in nymphs; infection was not detected in males. Infected populations were only observed in southern Maryland, suggesting the possibility that Wolbachia is currently invading Maryland A. americanum populations. Because F supergroup Wolbachia have been detected previously in filarial nematodes, tick samples were assayed for nematodes by PCR. Filarial nematodes were detected in 70% and 9% of Wolbachia-positive and Wolbachia-negative tick samples, respectively. While nematodes were more common in Wolbachia-positive tick samples, the lack of a strict infection concordance (Wolbachia-positive, nematode-negative and Wolbachia-negative, nematode-positive ticks) suggests that Wolbachia prevalence in ticks is not due to nematode infection. Supporting this hypothesis, phylogenetic analysis indicated that the nematodes were likely a novel species within the genus Acanthocheilonema, which has been previously shown to be Wolbachia-free.

Introduction

Amblyomma americanum (the lone star tick) is broadly distributed, with a host range spanning the Midwest to the eastern United States (Childs & Paddock, 2003). Amblyomma americanum is a vector of pathogens that cause diseases of humans and domestic animals such as granulocytic ehrlichiosis, tularemia and borreliosis, and is associated with Southern tick-associated rash illness (Goddard & Varela-Stokes, 2009). Several endosymbiotic bacterial species (including Coxiella, Rickettsia and Arsenophonus) have been detected previously in A. americanum (Clay et al., 2008). Wolbachia is a common endosymbiotic associate of arthropods and filarial nematodes (Rowley et al., 2004; Baldo et al., 2007; Hilgenboecker et al., 2008; Werren et al., 2008) and has been described previously in the ticks Ixodes ricinus (Hartelt et al., 2004) and Ixodes scapularis (Benson et al., 2004). In this report, we identified a novel Wolbachia symbiont in A. americanum. The infection was found only in southern Maryland at a low frequency in female ticks and pooled nymphs, but was never observed in males. Multilocus sequence typing (MLST) placed the infection into the F supergroup. Filarial nematode infection was observed in some tick samples, but concordance between Wolbachia infection and nematode infection was not observed. Nematodes were phylogenetically placed into the genus Acanthocheilonema, members of which were previously shown to be Wolbachia-free.

Materials and methods

Tick collection information and sample sizes are listed in Table 1 and Fig. 1. All ticks were collected in 2008 using tick drags. Individual adult ticks and pooled nymphs (10 per pool) were homogenized using a TissueLyser II bead mill (Qiagen, Valencia, CA) with 5-mm stainless-steel bead. Genomic DNA was isolated using the MasterPure DNA purification kit (Epicentre Biotechnologies, Madison, WI) and eluted in 30 µL water. Samples were screened for Wolbachia infection using Wolbachia-specific 16S primers WSpecF and WSpecR (Sakamoto et al., 2006; Werren & Windsor, 2000) using a PTC thermocycler (Bio-Rad, Hercules, CA) with a program of 95 °C for 5 min; 40 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min; and a final extension of 72 °C for 5 min. Each 40 µL reaction consisted of 2.0 µL template DNA, 1 µM concentrations of all forward and reverse primers, 0.4mM dNTPs and 2.0U Taq polymerase. Infections were confirmed using wsp gene amplification using primers wspF1, wspR1 (Wolbachia MLST website: http://pubmlst.org/wolbachia/). The PCR protocol used was 94 °C for 2 min, followed by 37 cycles of 94 °C for 30 s, 59 °C for 45 s and 72 °C for 1.5 min and a final extension of 72 °C for 10 min (http://pubmlst.org/wolbachia/). Template DNA from a Wolbachia-infected mosquito cell line was used as a positive control; a sample containing deionized water instead of template DNA was used as a negative control. For Wolbachia MLST, the four MLST genes (coxA, gatB, hcpA and fbpA) were amplified using nested PCR. Primary PCR amplification was performed using F3/R3 ~64-fold degenerate primer sets (http://pubmlst.org/wolbachia/). Each 40 µL reaction consisted of 2 µL template DNA, 1 µM concentrations of all forward and reverse primers, 0.4mM dNTPs and 1.0U HotStar Taq polymerase (Qiagen). Primary PCR cycling conditions followed the ‘alternative protocols’ listed on the MLST website. Secondary PCR amplification was performed using the MLST website ‘standard protocols’ and F1/R1 primer sets for these four MLST genes (Baldo et al., 2006). The ftsZuniF/ftsZuniR (Lo et al., 2002) primer pair was used for the ftsZ gene PCR. PCR products were directly sequenced in both directions using an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). Sequences were deposited in GenBank under accession numbers HM061157–HM061163.

Table 1.

Collection site, tick stage and infection rate

Location	County	Stage	N	# Positive	% Infected
Idylwild WMA	Caroline	Female	19	2	10.5%
		Male	10	0	0
		Nymph	5 pools (44)	0	0
Linkwood WMA	Dorchester	Female	29	1	3.5%
		Male	19	0	0
		Nymph	3 pools (27)	0	0
Assateague SP	Worchester	Female	9	0	0
		Male	7	0	0
		Nymph	7 pools (68)	0	0
Pocomoke SP	Worchester	Nymph	7 pools (63)	3	4.76%*
Cedarville SF	PG and Charles	Female	2	0	0
		Male	1	0	0
		Nymph	6 pools (51)	3	5.88%*
Chapel Point SP	Charles	Female	3	0	0
		Nymph	6 pools (56)	4	7.14%*
Chapman SP	Charles	Female	17	2	11.8%
		Male	9	0	0
		Nymph	11 pools (103)	1	0.97%*
Purse SP	Charles	Nymph	5 pools (46)	1	2.17%*
Serpentine	Montgomery	Female	15	0	0
		Male	17	0	0
		Nymph	4 pools (40)	0	0
St Mary’s SP	St Mary’s	Female	4	1	25%
		Male	1	0	0
		Nymph	3 pools (29)	0	0
Greenwell SP	St Mary’s	Female	3	0	0
		Male	1	0	0
Sandy Point SP	Anne Arundel	Female	8	0	0
		Male	9	0	0
		Nymph	4 pools (38)	0	0
Millington WMA	Kent	Female	2	0	0
		Male	3	0	0
		Nymph	1 pool (11)	0	0
Calvert Cliffs SP	Calvert	Female	7	0	0
		Nymph	19 pools (183)	0	0
Elk Neck SP	Cecil	Female	1	0	0
		Nymph	3 pools (28)	0	0
Tuckahoe SP	Queen Anne’s	Male	1	0	0
		Nymph	5 pools (47)	0	0
	Total	Female	119	6	5.04%
		Male	78	0	0
		Nymph	89 pools (834)	12	1.44%*

^*Infection rate of nymphs is reported as the MIR; the number of infected pools divided by the total number of ticks tested.

Fig. 1.

Map of Maryland Amblyomma americanum collection sites. Closed circles indicate populations with infected ticks; open circles indicate populations where no infected ticks were collected.

Thirty-nine concatenated MLST sequences were retrieved from the Wolbachia MLST database. The Wolbachia strains are listed by host species name and concatenated MLST sequence ID number in Fig. 2. Sequence alignment and neighbor-joining phylogenetic analyses were conducted using MEGA version 4 (Tamura et al., 2007). Tree support was evaluated by bootstrapping with 1000 replications. Phylogenies were produced for concatenated and gene-specific data sets.

Fig. 2.

Neighbor-joining phylogenetic analysis of a 2079 bp alignment of five Wolbachia MLST concatenated gene sequences (coxA, gatB, hcpA, ftsZ and fbpA). Numbers at nodes indicate bootstrap support values (1000 replicates). Taxon names are host species. Values in parentheses represent MLST sequence ID numbers (searchable at http://pubmlst.org/wolbachia/). Letters represent Wolbachia supergroup designations. The tree is rooted by the supergroup D strain from Brugia malayi.

Tick samples were tested for the presence of filarial nematodes using specific 12S primers as described (Casiraghi et al., 2004). Template DNA from a Wuchereria bancrofti-infected Culex pipiens mosquito was used as a positive control; a sample containing deionized water instead of template DNA was used as a negative control. The positive amplicons obtained were directly sequenced. Phylogenetic placement of nematode sequences was conduced as described above.

Results and discussion

Out of 16 sampled Maryland populations, Wolbachia-infected ticks were found in eight populations. Infection rates within populations were low, ranging from 3.5% to 25% in females, and a minimum infection rate (MIR) from 1% to 7% in pooled nymphs. Wolbachia was not detected in males. The overall statewide infection rate was approximately 5% in females and 1.4% (MIR) in pooled nymphs (Table 1). Wolbachia infection was spatially structured; all infected populations were located in southern Maryland, while infection was not detected in central and northern populations (Fig. 1). This distribution suggests the possibility that Wolbachia is currently invading A. americanum populations in Maryland. Future sampling efforts will be required to confirm or refute this hypothesis.

The coxA, gatB, hcpA, ftsZ and fbpA gene amplicons were produced by PCR from sample #366 from Dorchester County. Additionally, the fbpA gene was amplified and sequenced from all positive samples, while the coxA, gatB, hcpA and ftsZ genes were amplified and sequenced from selected samples (Table 2). All sequences were identical to those obtained from sample #366. According to the phylogenetic analysis of these concatenated sequences, the Wolbachia strain found in A. americanum was placed into supergroup F and was 99% identical to Wolbachia from the ant Ocymyrmex picardi (Russell et al., 2009). Gene-specific phylogenetics analyses showed the same result (Supporting Information). This is the first report of supergroup F Wolbachia in ticks. Up to this point, F supergroupWolbachia infections have been detected in nematodes, scorpions and several insect hosts (termites, weevils, bush crickets, bedbugs, lice, louse flies, cockroaches and antlions) (Lo et al., 2002; Casiraghi et al., 2005; Dunn & Stabb, 2005; Sakamoto et al., 2006; Baldo et al., 2007; Covacin & Barker, 2007; Panaram & Marshall, 2007; Vaishampayan et al., 2007).

Table 2.

Sequences obtained from assayed ticks

Specimen	Stage assayed	Wolbachia	FbpA	CoxA	GatB	HcpA	ftsZ	Nematode
366	Adult female	+	×	×	×	×	×	Not tested
294	Adult female	+	×		×	×		−
295	Adult female	+	×			×		+
995	Adult female	+	×		×			+
998	Adult female	+	×					+
1178	Adult female	+	×					+
445–454	Pooled nymphs	+	×	×				−
465–474	Pooled nymphs	+	×					+
485–494	Pooled nymphs	+	×					+
790–799	Pooled nymphs	+	×					−
817–826	Pooled nymphs	+	×					−
827–836	Pooled nymphs	+	×					+
868–877	Pooled nymphs	+	×					+
927–932	Pooled nymphs	+	×					+
1016–1025	Pooled nymphs	+	×					+
1032–1041	Pooled nymphs	+	×					+
1042–1051	Pooled nymphs	+	×					+
1062–1071	Pooled nymphs	+	×					−
1337	Adult female	−						−
1338	Adult female	−						−
255–258	Pooled nymphs	−						−
475–484	Pooled nymphs	−						+
837–840	Pooled nymphs	−						−
907–916	Pooled nymphs	−						−
917–926	Pooled nymphs	−						−
997	Adult female	−						−
999	Adult female	−						−
1000	Adult female	−						−
1001	Adult female	−						−

F supergroup Wolbachia have been identified in filarial nematodes (genus Mansonella) in addition to arthropods. Although ticks collected for this study were collected by dragging, and were unlikely to be blood fed, we investigated the hypothesis that Wolbachia infection in Amblyomma was due to filarial nematode infection. All Wolbachia-positive tick samples and a random selection of Wolbachia-negative tick samples were assayed for filarial nematode infection by specific 12S PCR. Filarial nematodes were detected in both Wolbachia-positive and Wolbachia-negative adult female and nymphal ticks. Nematode infection was more common in Wolbachia-infected ticks (70% vs. 9%), but strict concordance of infection types was not observed (both Wolbachia-positive, nematode-negative ticks and Wolbachia-negative, nematode-positive ticks were identified) (Table 2). Phylogenetic analysis indicated that the filarial nematode infecting A. americanum was most closely related to nematodes in the genus Acanthocheilonema (formerly Dipetalonema) (Fig. 3). Acanthocheilonema nematodes have not been described previously from Amblyomma, but their transmission by both soft ticks (Ornithodoros) and hard ticks (Rhipicephalus) has been demonstrated (Londoño, 1976; Olmeda-García et al., 1993). Acanthocheilonema nematodes have also been shown previously to lack Wolbachia infection (Casiraghi et al., 2004). In total, these results indicate that Wolbachia prevalence in A. americanum is not explained by nematode infection.

Fig. 3.

Unrooted neighbor-joining phylogenetic analysis of of nematode 12S gene sequences. Amblyomma americanum nematode sequence has been deposited in GenBank under accession number JF732757. Alphanumeric codes represent GenBank ID numbers. Numbers at nodes indicate bootstrap support values (1000 replicates). Bold indicates nematode sequence identified in this study.

The frequency of Wolbachia infection is low in A. americanum. Some Wolbachia reproductive phenotypes, such as cytoplasmic incompatibility, tend to equilibrate at high frequencies in populations (Turelli & Hoffmann, 1999). In contrast, phenotypes such as male killing tend to equilibrate at low frequencies (Hurst & Jiggins, 2000).We did not detect any infected males in our study – this could reflect sex-specific infection differences (possibly related to male killing) or could simply be due to sampling. The reproductive phenotype of Wolbachia in A. americanum is unknown and needs further study.