Quentin Q. Fang

MOLECULAR PHYLOGENETIC ANALYSES INDICATE THAT THE IXODES RICINUS COMPLEX IS A PARAPHYLETIC GROUP

The Ixodes ricinus species complex is a group of ticks distributed in almost all geographic regions of the world. Lyme borreliosis spirochetes are primarily transmitted by tick species within this complex. It has been hypothesized that the Lyme vector ticks around the world are closely related and represent a monophyletic group. This implies that vector competence in ixodid ticks for Lyme agents might have evolved only once. To test this hypothesis, we used a molecular phylogenetic approach. Two fragments of mitochondrial 16S ribosomal deoxyribonucleic acid were sequenced from 11 species in the I. ricinus complex and from 16 other species of Ixodes. Phylogenetic analysis using Bayesian methodology indicated that the I. ricinus complex is not a monophyletic group unless 3 additional Ixodes species are included in it. The known major vectors of Lyme disease agents in different areas of the world are not sister taxa. This suggests that acquisition of the ability to transmit borreliosis agents in species of Ixodes may have multiple origins.

WOLBACHIA ENDOSYMBIONTS IN FLEAS (SIPHONAPTERA)

Intracellular endosymbionts, Wolbachia spp., have been reported in many different orders of insects and in nematodes but not previously in fleas. This is the first conclusive report of Wolbachia spp. within members of the Siphonaptera. Using nested polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in fleas collected from 3 counties in Georgia and 1 in New York. The prevalence of Wolbachia spp. detected varied among the 6 different species screened: 21% in the cat flea Ctenocephalides felis (n = 604), 7% in the dog flea C. canis (n = 28), 25% in Polygenus gwyni (n = 8), 80% in Orchopeas howardi (n = 15), 94% in Pulex simulans (n = 255), and 24% in the sticktight flea Echidnophaga gallinacea (n = 101). Wolbachia spp. infection in fleas was confirmed by sequencing positive PCR products, comparing sequenced 16S ribosomal DNA (rDNA) with Wolbachia spp. sequences in GenBank using BLAST search, and subjecting sequence data to phylogenetic analysis. For further confirmation, 16S rDNA–positive samples were reamplified using the wsp gene.

Prevalence of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from the Georgia Coast and Barrier Islands

Abstract Human monocytic ehrlichiosis is an emerging zoonosis caused by infection with Ehrlichia chaffeensis Anderson, Dawson, Jones & Wilson, which is transmitted to mammals by ixodid ticks. Prevalence of infected ticks and distribution of infection foci indicate relative risk of human exposure to ehrlichiosis and may be influenced by factors such as geographic isolation, human disturbance, and the availability of suitable mammalian reservoir hosts. To test this, individual and pooled lone star ticks, Amblyomma americanum (L.), were collected from three populations from the Georgia coast (1 mainland site and two barrier islands) and screened for E. chaffeensis. A species-specific, nested polymerase chain reaction (PCR) assay was used to amplify a 572 bp fragment of the E. chaffeensis 16S rRNA gene from infected ticks. PCR product specificity was confirmed by nucleotide sequencing. Our results showed the prevalence of infected ticks to be 0.0% (n = 151), 0.9% (n = 111), and 9.3% (n = 129) for Sapelo Island, St. Catherine’s Island, and Fort McAllister, respectively.

HARD TICK CALRETICULIN (CRT) GENE CODING REGIONS HAVE ONLY ONE INTRON WITH CONSERVED POSITIONS AND VARIABLE SIZES

Calreticulin (CRT ) is a unique eukaryotic gene. The CRT gene product, calreticulin, was first identified as a calcium binding protein in 1974, but further investigations have indicated that CRT protein performs many functions in cells, including involvement in evading the hosts immune system by parasites. Many studies of CRT have been published since the molecule was first discovered; however, the CRT gene exon-intron structure is only known for a limited number of ectoparasite species. In this study, we compared tick CRT genomic sequences to the corresponding cDNA from 28 species and found that 2 exons and 1 intron are present in the tick CRT gene. The intron position is conserved in 28 hard ticks, but intron size and nucleotide sequences vary. Three tick introns possess duplicated fragments and are twice as long as other introns. All tick CRT introns obey the GT-AG rule in the splice-site junctions and are phase 1 introns. By comparing tick CRT introns to those of fruit fly, mouse, and human, we conclude that tick CRT introns belong to the intron-late type. The number and size of CRT introns have increased through the evolution of eukaryotes.

Prevalence of the Agent of Human Granulocytic Ehrlichiosis in Ixodes scapularis (Acari: Ixodidae) in the Coastal Southeastern United States

Abstract Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne disease recently recognized in the United States. The blacklegged tick, Ixodes scapularis Say, is the principle vector in the eastern United States. The disease has been commonly reported in the northeastern and upper midwestern states; however, suitable vectors and reservoir hosts exist in the southeast. To assay the prevalence of the HGE agent in vector ticks, we screened 818 individual I. scapularis from 15 locations in South Carolina, Georgia, and Florida using nested polymerase chain reaction, which targets the HGE agent 16S rRNA gene. Prevalence among locations ranged from 0 to 20%. The overall average prevalence of 15 sites was 1.6% (n = 818). Verification by sequencing the 16S rDNA from the positive samples showed 99.8–100% nucleotide identities with the sequences of the HGE agent in GenBank. These results were supported by the phylogenetic analysis using 16S rDNA sequences.

CLONING AND SEQUENCING OF PUTATIVE CALRETICULIN COMPLEMENTARY DNAs FROM FOUR HARD TICK SPECIES

Calreticulin (CRT) is a calcium-binding protein and has many functions in eukaryotic cells. CRT is possibly involved in parasite host immune system evasion. To better understand the molecular basis of CRT in ticks, we cloned and sequenced 4 full-length complementary DNAs (cDNAs) from the hard tick species, Dermacentor variabilis, Haemaphysalis longicornis, Ixodes scapularis, and Rhipicephalus sanguineus, using the technique of rapid amplification of cDNA ends. The deduced amino acid sequences share high identities (between 77 and 98%) with 3 known tick CRT sequences. The major characteristics of known CRTs are observed in all 4 of our deduced tick CRTs. These include 3 major domains, a signal peptide sequence at the beginning of the coding region, 2 triplets of conserved regions, cysteine sites providing disulfide bridges for N-terminal folding, and a nuclear localization signal. Remarkably, the replacement of the endoplasmic reticulum retention signal KDEL by HEEL, which is believed to be associated with secretion of CRT into the host during feeding and was previously recorded only in 2 ticks and a hookworm, is also present in all 4 of our tick putative CRTs. In addition, the CRT gene is potentially useful for tick phylogenetic reconstruction.

Ferritin Gene Coding Sequences Are Conserved Among Eight Hard Tick Species (Ixodida: Ixodidae)

Abstract Eight complete cDNAs encoding for ferritins from the hard tick species Amblyomma americanum (L.), Amblyomma maculatum Koch, Boophilus microplus (Canestrini), Dermacentor albipictus (Packard), Dermacentor variabilis (Say), Hemaphysalis longicornis Neumann, Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille) were obtained using the techniques of rapid amplification of cDNA ends. The deduced amino acid sequences from these tick ferritin genes share high identities (84–98%) with those of two published tick ferritins. The sequences and positions of the iron-responsive element in the 5′-untranslated region of the cDNAs are highly conserved in ticks. Similar to other known animal ferritins, a ferroxidase center, consisting of seven conserved amino acid residues, was identified in the deduced tick ferritins. Sequence comparison indicates that these tick ferritin genes belong to the type of invertebrate cytosolic heavy chain homologue. Two separate phylogenetic analyses of ferritin amino acid sequences, one with 52 arthropod ferritin sequences and another one using 10 tick ferritins, suggested that ferritin is a suitable nuclear protein-encoding gene for tick phylogenetic reconstruction.

Cloning and Sequencing of Putative Acetylcholinesterase cDNAs from the American Dog Tick, Dermacentor variabilis, and the Brown Dog Tick, Rhipicephalus sanguineus (Acari: Ixodidae)

Abstract Two putative cDNAs of acetylcholinesterase (AChE), one from Dermacentor variabilis, and the other from Rhipicephalus sanguineus, were amplified and sequenced. The deduced amino acid sequences have high amino acid identities (between 70 and 94%) to known tick AChE sequences deposited in GenBank. Furthermore, these two AChEs also possess common features in their primary AChE structure such as catalytic active sites. A 2,220-bp contiguous sequence, containing a 1,791-bp open reading frame encoding an AChE precursor with 596 amino acid residues, was obtained from D. variabilis. The deduced proteins of R. sanguineus are different in size by 6 amino acids because of alternative splicing at the 5′ end. A gene tree deduced from phylogenetic analysis indicates that there are at least three lineages of AChE in arthropods.

The Use of the Nuclear Protein-Encoding Gene, RNA Polymerase II, for Tick Molecular Systematics

Phylogenetic studies of ticks have been increasing in recent years, particularly in the use of molecular data. However, all of the studies to date are either limited to the mitochondrial genome or to a few nuclear ribosomal genes. There is a need to explore the use of nuclear protein-encoding genes because these genes direct most aspects of the phenotypic traits in the development of an organism. We report here the test of a nuclear protein-encoding gene, RNA polymerase II, for the phylogenetic study of ticks. Thirty-eight ticks representing 26 species of hard and soft ticks were chosen for the study. The pairwise divergences among sampled species are ranged from 0.3 to 15.2% and most of the substitutions are transitions. In addition, the nucleotide composition is not obviously biased in POL II gene. The trees inferred from the POL II sequences using maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) by PAUP* and MrBayes are largely concordant with the existing phylogenies. Our study demonstrated that POL II gene sequences contain strong phylogenetic signals in ticks at the generic and higher levels. POL II has proven to be a useful gene for resolving tick phylogeny.

Anti-tick Vaccine Development: Status and Perspectives

Ticks (Acarina: Arachnida: Arthropoda) are obligate, nonpermanent ectoparasites of terrestrial vertebrates. In arthropods, ticks are second only to mosquitoes in the number of pathogens they transmit to people, domestic pets, livestock, and wild animals. Ticks are vectors to a variety of human diseases, including Rocky Mountain spotted fever, Lyme disease, Colorado tick fever, tickborne encephalitis, babesiosis, tularemia, and tick-borne relapsing fever. In addition, other maladies due to tick bites, such as tick paralysis, tick toxicosis, and anaphylaxis, are common. Each year, worldwide public health and agricultural industry costs due to tick-transmitted diseases are estimated to be in the range of several billion U.S. dollars. To date, the control of ticks has relied heavily on chemical pesticides (acaricides). The excessive use of pesticides in tick control has resulted in environmental contamination, food safety concerns, and pesticide resistance. Consequently, use molecular technology to develope anti-tick vaccines is an essential alternative strategy in tick and tick-borne disease control. Recent studies and the status of anti-tick vaccine development are reviewed in this chapter.