Development of Genetic Barcodes for Hosts of the Blacklegged Tick (Ixodes scapularis) in Southern New York
In the northeastern United States, blacklegged ticks (Ixodes scapularis) transmit a number of tick-borne pathogens to humans. These ticks are generalist feeders and have a wide range of potential hosts, each of which differs in their ability to harbor and transmit pathogens. Studies that examine the role of different hosts are important for understanding tick-borne disease transmission and risk. A recent study by Alcaide et al. (2009) demonstrated that a segment of the vertebrate mitochondrial Cytochrome c Oxidase Subunit I (COI) gene could be used to detect bloodmeal sources from a variety of hematophagous arthropods. This technique was tested on a number of biting fly species and one tick species, but was not tested on blacklegged ticks.
The goal of this study was to develop DNA barcodes for the COI gene for vertebrate species found in southern New York, that may act as potential bloodmeals sources for blacklegged ticks. Results from this study are needed for the development of DNA barcoding projects aimed at using tick bloodmeals to examine the role of different host species in tick-borne disease dynamics in southern New York State.
Vertebrate tissue samples were collected in southern New York where blacklegged ticks are highly prevalent. DNA was extracted from the tissues of 60 individuals (31 species). Primer pools used to amplify a segment of the COI gene were used as described by Alcaide et al. (2009). The universal-forward primer (HAA YCA YAA RGA YAT YGG) was designed for a conserved nucleotide position at the 5í end of the COI gene and the reverse primer (GCY CAN ACY ATN CCY ATR TA) is specific for vertebrate COI sequences. I followed the optimized reaction protocol from Alcaide et al. (2009) with slight modification. All PCR products were purified and sequenced in forward and reverse directions to confirm accuracy of the sequences generated and compared to the Nucleotide Collection in NCIB BLAST.
Two rounds of PCR were required to amplify this gene segment from the vertebrate tissues (Figure 1). DNA isolates from 37 individuals (22 species) yielded PCR products during the second round of amplification (Figure 2). The DNA of 24 individuals (17 species) matched the expected species in GenBank. The DNA of 11 individuals (5 species) matched to the appropriate order but there were no matching sequences for lower taxonomic groups (family, genus, species) for these species. These species all were members of the orders Rodentia (rodents) or Lagomorpha (rabbits and hares). Two samples did not match expected species.
Of the 24 isolated DNA samples that matched expected species, six (five species) had unique single nucleotide polymorphisms (SNPs) when compared to species sequences in GenBank (Figure 3). Most of the nucleotide differences were purine to purine, or pyrimidine to pyrimidine differences, with the exception of one SNP for the raccoon DNA that was thymine to guanine (Figure 4). For all nucleotide differences between the sequenced vertebrate samples in this study and the species they aligned with in GenBank, none resulted in changes in the protein sequence.
This study resulted in the successful extraction and sequencing of vertebrate DNA from various tissues sources. Genetic barcodes were generated for 22 species (five of which were new species barcodes not previously published in GenBank) that are found in southern New York and are potential hosts of blacklegged ticks. Generation of the five new species barcodes is of particular importance for tick-borne disease research, since these five species are known to harbor large numbers of ticks and are also important in disease amplification in wildlife populations (LoGiudice et al. 2003).
Additionally, two individual DNA sample sequences matched incorrect species in GenBank, which is likely to be an error in GenBank. The unique SNPs found in five species demonstrates the possible sequence variation in these species or a possible polymerase error during replication. This illustrates the importance of sequencing DNA from multiple individuals within each species, a goal that was attempted in this study. It is important to note that none of the SNPs changed the protein sequence.
The information generated from this study will be useful in future studies that aim to use genetic barcodes for potential host species of blacklegged ticks, such as studies that wish to determine bloodmeal sources of these ticks within southern New York State.
Alcaide, M., C. Rico, S. Ruiz, R. Soriguer, J. Munoz, and J. Figuerola. 2009. Disentangling vector-borne transmission networks: A universal DNA barcoding method to identify vertebrate hosts from arthropod bloodmeals. PLos ONE 4:1-6.
LoGiudice, K., R. S. Ostfeld, K. A. Schmidt, and F. Keesing. 2003. The ecology of infectious disease: Effects of host diversity and community composition on Lyme disease risk. PNAS 100: 567-571.
Figure 1-PCR product (run on 1% agarose gel) from 10 samples after two rounds of PCR amplification. All samples with PCR product were sent for sequencing.
Figure 2-Number of each species with sequenced DNA and sequence matches in GenBank.
Figure 3-Sequenced DNA from species with single nucleotide polymorphisms (SNPs).
Figure 4-Location of each single nucleotide polymorphism (SNP) from each sequenced DNA compared to published GenBank sequences (SNPs are in red). Note that eight out of the nine SNPs are either purine to purine or pyrimidine to pyrimidine changes and that none of the base changes resulted in differences in the amino acid sequence.
In the northeastern United States, blacklegged ticks (Ixodes scapularis) are responsible for transmitting several disease-causing pathogens to humans. Studies that examine the role of host species in pathogen transmission cycles are important for understanding tick-borne disease dynamics, and subsequently disease risk to humans. Previous work has been conducted on developing molecular protocols to test ticks for bloodmeal sources. Use of these techniques would provide direct evidence as to the host species of each blacklegged tick and elucidate the role that different hosts have on tick-borne disease transmission and risk.
The purpose of this study was to develop genetic barcodes for hosts of the blacklegged tick in southern New York State (an area with high prevalence of blacklegged ticks). Barcodes were created by amplifying a segment of the Cytochrome c Oxidase Subunit I (COI) gene from various vertebrate tissue sources and comparing them to published sequences in GenBank. Successful extraction and sequencing of DNA from these tissues generated barcodes for 22 species, five of which were new species barcodes not currently present in GenBank. Furthermore, identification of single nucleotide polymorphisms in the sequences of five species demonstrates genetic variation that may indicate different populations of these species.
I would like to thank Dr. Berish Rubin for his guidance with this projectís conception and development and for training in molecular techniques. Thank you to Alex Bulanov and Xie Xie for their assistance with all laboratory and computer work as well as their advice throughout the project.