Anaplasma marginale Actively Modulates Vacuolar Maturation during Intracellular Infection of Its Tick Vector, Dermacentor andersoni.

UNLABELLED: Tick-borne transmission of bacterial pathogens in the order Rickettsiales is responsible for diverse infectious diseases, many of them severe, in humans and animals. Transmission dynamics differ among these pathogens and are reflected in the pathogen-vector interaction. Anaplasma marginale has been shown to establish and maintain infectivity within Dermacentor spp. for weeks to months while escaping the complex network of vacuolar peptidases that are responsible for digestion of the tick blood meal. How this prolonged maintenance of infectivity in a potentially hostile environment is achieved has been unknown. Using the natural vector Dermacentor andersoni, we demonstrated that A. marginale-infected tick vacuoles (AmVs) concurrently recruit markers of the early endosome (Rab5), recycling endosome (Rab4 and Rab11), and late endosome (Rab7), are maintained near neutral pH, do not fuse with lysosomes, exclude the protease cathepsin L, and engage the endoplasmic reticulum and Golgi apparatus for up to 21 days postinfection. Maintenance of this safe vacuolar niche requires active A. marginale protein synthesis; in its absence, the AmVs mature into acidic, protease-active phagolysosomes. Identification of this bacterially directed modeling of the tick midgut endosome provides a mechanistic basis for examination of the differences in transmission efficiency observed among A. marginale strains and among vector populations. IMPORTANCE: Ticks transmit a variety of intracellular bacterial pathogens that cause significant diseases in humans and animals. For successful transmission, these bacterial pathogens must first gain entry into the tick midgut digestive cells, avoid digestion, and establish a replicative niche without harming the tick vector. Little is known about how this replicative niche is established and maintained. Using the ruminant pathogen A. marginale and its natural tick vector, D. andersoni, this study characterized the features of the A. marginale niche in the tick midgut and demonstrates that A. marginale protein synthesis is required for the maintenance of this niche. This work opens a new line of inquiry about the pathogen effectors and their targets within the tick that mediate tick-pathogen interactions and ultimately serve as the determinants of pathogen success.

Expression pattern of subA in different tissues and blood-feeding status in Haemaphysalis flava.

Tick-borne-diseases (TBD) pose a huge threat to the health of both humans and animals worldwide. Tick vaccines constitute an attractive alternative for tick control, due to their cost-efficiency and environmental-friendliness. Subolesin, a protective antigen against ticks, is reported to be a promising candidate for the development of broad-spectrum vaccines. However, the entire length of its gene, subA, and its gene expression pattern in different tissues and blood-feeding status (or different levels of engorgement) have not been studied extensively. In our study, the full-length of subA in Haemaphysalis flava, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Dermacentor sinicus was cloned by RACE-PCR. The subA expression pattern was analyzed further in H. flava in different tissues and blood-feeding status by RT-PCR. We found that the full-length of subA in H. flava, R. haemaphysaloides, R. microplus, and D. sinicus was 1318, 1498, 1316, and 1769 bp, respectively, with encoded proteins at 180, 162, 162, and 166 aa in length, respectively. The primary structure of subolesin in H. flava included three conserved regions and two hypervariable regions, with no signal peptide. SubA expression in female H. flava of different blood-feeding status was in the order of the fasted < the 1/4-engorged < the half-engorged < the fully-engorged (p < 0.01). Tissue expression of subA was in the order of salivary gland > midgut > integument (p < 0.01), but its expression in salivary glands was not statistically different from that in ovaries. We concluded that subolesin was a conserved antigen and that subA was expressed differentially in H. flava in different tissues and blood-feeding status. Those features made subolesin feasible as a potential target antigen for development of a universal vaccine for the control of tick infestations and a reduction in TBD.

Identification of host proteins involved in rickettsial invasion of tick cells.

Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

Defending the fort: a role for defensin-2 in limiting Rickettsia montanensis infection of Dermacentor variabilis.

The importance of tick defensins is evidenced by their expression in a wide variety of tick tissues and prevalence across many tick genera. To date, the functional and biological significance of defensin-2 as a rickettsiastatic or rickettsiacidal antimicrobial peptide has not been addressed. In a previous study, defensin-2 transcription was shown to increase in Dermacentor variabilis ticks challenged with Rickettsia montanensis. In the present study, the hypothesis that defensin-2 is functional as a rickettsiastatic and/or rickettsiacidal antimicrobial peptide is tested. We show that defensin-2 plays a role in reducing burden after acquisition of Rickettsia montanensis through capillary feeding. Moreover, defensin-2 is shown to associate with R. montanensis in vitro and in vivo, causing cytoplasmic leakiness.

Molecular characterization and tissue-specific gene expression of Dermacentor variabilis α-catenin in response to rickettsial infection.

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analysed. Comparative sequence analysis demonstrates a 3069-bp cDNA with a 2718-bp open reading frame with a sequence similar to Ixodes scapularisα-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin-binding region and ß-catenin-binding and -dimerization regions. Quantitative reverse-transcription PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated 12 h after exposure to R. montanensis, but not in Rickettsia amblyommii-exposed ovaries, compared with Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases.

Dermacentor variabilis: characterization and modeling of macrophage migration inhibitory factor with phylogenetic comparisons to other ticks, insects and parasitic nematodes.

We have identified and characterized the full length cDNA sequence of macrophage migration inhibitory factor (MIF) from the American dog tick, Dermacentor variabilis. The nucleotide and putative amino acid sequences from this study shared a high level of sequence conservation with other tick MIFs. The bioinformatics analysis showed across species conservation of the MIF amino acid sequence in ticks, insects and nematodes. The multiple sequence alignment identified Pro 1, 3, 55; Thr 7, 112; Asn 8, 72; Ile 64, 96; Gly 65, 110, Ser 63 and Leu 87 amino acids to be highly conserved among the sequences selected for this study. Tick MIF does not have the oxidoreductase domain as found in MIFs from other animals suggesting that tick MIF is not capable of performing as an oxidoreductase. The phylogenetic analysis revealed that tick MIFs share a closer evolutionary proximity to parasitic nematode MIFs than to insect MIFs.

Anaplasma marginale Infection of Dermacentor andersoni Primary Midgut Cell Culture Is Dependent on Fucosylated Glycans.

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Synganglion transcriptome and developmental global gene expression in adult females of the American dog tick, Dermacentor variabilis (Acari: Ixodidae).

454 Pyrosequencing was used to characterize the expressed genes from the synganglion and associated neurosecretory organs of unfed and partially fed virgin and mated replete females of the American dog tick, Dermacentor variabilis. A total of 14,881 contiguous sequences (contigs) was assembled, with an average size of 229 bp. Gene ontology terms for Level 2 biological processes were assigned to 4366 contigs. Seven acetylcholinesterases, a muscarinic acetylcholine (ACh) receptor, two nicotinic ACh receptor ß-subunits, two ACh unc-18 regulators, two dopamine receptors, two gamma aminobutyric acid (GABA) receptors, two GABA transporters, two norepinephrine transporters and an octopamine receptor are described. Microarrays were conducted to examine global gene expression and quantitative real-time polymerase chain reaction was used to verify expression of selected neuropeptides. Hierarchical clustering of all differentially expressed transcripts grouped part-fed and replete ticks as being more similar in terms of differentially expressed genes with unfed ticks as the outgroup. Nine putative neuropeptides (allatostatin, bursicon-ß, preprocorazonin, glycoprotein hormone α, insulin-like peptide, three orcokinins, preprosulphakinin) and a gonadotropin releasing hormone receptor were differentially expressed, and their developmental expression and role in reproduction was investigated. The presence of eclosion hormone, corazonin and bursicon in the synganglion, which in insects regulate behaviour and cuticle development associated with moulting, suggest that this system may be used in ticks to regulate blood feeding, cuticle expansion and development related to female reproduction; adult ticks do not moult.

Predicted iron metabolism genes in hard ticks and their response to iron reduction in Dermacentor andersoni cells.

For most organisms, iron is an essential nutrient due to its role in fundamental cellular processes. Insufficient iron causes sub-optimal metabolism with potential effects on viability, while high levels of iron are toxic due to the formation of oxidative radicals, which damage cellular components. Many molecules and processes employed in iron uptake, storage, transport and metabolism are conserved, however significant knowledge gaps remain regarding these processes in ticks due to their unique physiology. In this study, we first identified and sequenced 13 genes likely to be involved in iron metabolism in Dermacentor andersoni cells. We then developed a method to reduce iron levels in D. andersoni cells using the iron chelator 2,2'-bipyridyl and measured the transcriptional response of these genes to iron reduction. The genes include a putative transferrin receptor, divalent metal transporter 1, duodenal cytochrome b, zinc/iron transporters zip7, zip13, zip14, mitoferrin, ferrochelatase, iron regulatory protein 1, ferritin1, ferritin2, transferrin and poly r(C)-binding protein. Overall, the transcriptional response of the target genes to iron reduction was modest. The most marked changes were a decrease in ferritin2, which transports iron through the tick hemolymph, the mitochondrial iron transporter mitoferrin, and the mitochondrial enzyme ferrochelatase. Iron regulatory protein1 was the only gene with an overall increase in transcript in response to reduced iron levels. This work lays the foundation for an improved understanding of iron metabolism in ticks which may provide molecular targets for the development of novel tick control methods and aid in the understanding of tick-pathogen interactions.

Identification and evaluation of midgut protein RL12 of Dermacentor silvarum interacting with Anaplasma ovis VirD4.

Anaplasma ovis, a tick-borne intra-erythrocytic Gram-negative bacterium, is a causative agent of ovine anaplasmosis. It is known that Dermacentor ticks act as biological vectors for A. ovis. VirD4 is the machine component of Type IV Secretion System of A. ovis. To better understand the pathogen-vector interaction, VirD4 was used as a bait protein for screening midgut proteins of Dermacentor silvarum via yeast two-hybrid mating assay. As a result, a ribosomal protein RL12 was identified from the midgut cDNA library of D. silvarum. For further validation, using in vitro Glutathione S-transferase (GST) pull-down assay, interaction between the proteins, GST-RL12 and HIS-VirD4, was observed in Western blot analysis. The study is first of its kind reporting a D. silvarum midgut protein interaction with VirD4 from A. ovis. Functional annotations showed some important cellular processes are attributed to the protein, particularly in the stringent response and biogenesis. The results of the study suggest the involvement of the VirD4-RL12 interaction in the regulation of signaling pathways, which is a tool for understanding the pathogen-vector interaction.

Functional implication of heat shock protein 70/90 and tubulin in cold stress of Dermacentor silvarum.

BACKGROUND: The tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated. METHODS: The genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to - 20 °C lethal temperature (LT50), and then the mortality was calculated. RESULTS: Results indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709). CONCLUSIONS: The above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

De novo assembly and analysis of the transcriptome of the Dermacentor marginatus genes differentially expressed after blood-feeding and long-term starvation.

BACKGROUND: The ixodid tick Dermacentor marginatus is a vector of many pathogens wide spread in Eurasia. Studies of gene sequence on many tick species have greatly increased the information on tick protective antigen which might have the potential to function as effective vaccine candidates or drug targets for eco-friendly acaricide development. In the current study, RNA-seq was applied to identify D. marginatus sequences and analyze differentially expressed unigenes. METHODS: To obtain a broader picture of gene sequences and changes in expression level, RNA-seq was performed to obtain the whole-body transcriptome data of D. marginatus adult female ticks after engorgement and long-term starvation. Subsequently, the real-time quantitative PCR (RT-qPCR) was applied to validate the RNA-seq data. RESULTS: RNA-seq produced 30,251 unigenes, of which 32% were annotated. Gene expression was compared among groups that differed by status as newly molted, starved and engorged female adult ticks. Nearly one third of the unigenes in each group were differentially expressed compared to the other two groups, and the most numerous were genes encoding proteins involved in catalytic and binding activities and apoptosis. Selected up-regulated differentially expressed genes in each group were associated to protein, lipids, carbohydrate and chitin metabolism. Blood-feeding and long-term starvation also caused genes differentially expressed in the defense response and antioxidant response. RT-qPCR results indicated 6 differentially expressed transcripts showed similar trends in expression changes with RNA-seq results confirming that the gene expression profiles in transcriptome data is in consistent with RT-qPCR validation. CONCLUSIONS: Obtaining the sequence information of D. marginatus and characterizing the expression pattern of the genes involved in blood-feeding and during starvation would be helpful in understanding molecular physiology of D. marginatus and provides data for anti-tick vaccine and drug development for controlling the tick.

Identification and evaluation of UL36 protein from Dermacentor silvarum salivary gland and its interaction with Anaplasma ovis VirB10.

BACKGROUND: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. METHODS: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. RESULTS: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. CONCLUSIONS: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.

Using RNA interference to determine the role of varisin in the innate immune system of the hard tick Dermacentor variabilis (Acari: Ixodidae).

Defensins are an important component of the innate immune system of ticks. These small peptides are produced by various genera of ticks, and expressed in various tissues. In this study we used RNA interference to silence the expression of the defensin varisin produced by the hemocytes of the American dog tick, Dermacentor variabilis. Ticks were injected with double stranded varisin RNA prior to being placed on a rabbit. After feeding, the ticks were removed, bled, and the hemolymph plasma and hemocytes separated. Hemocytes were screened for the presence (or absence) of both varisin transcript and peptide. Varisin peptide was below detectable levels and the transcript showed a greater than 99% knockdown. The antimicrobial activity of the hemolymph plasma was reduced 2-4 fold compared to that of control injected ticks indicating varisin accounts for a large portion of the antimicrobial activity of the hemolymph.

Rickettsia peacockii, an endosymbiont of Dermacentor andersoni, does not elicit or inhibit humoral immune responses from immunocompetent D. andersoni or Ixodes scapularis cell lines.

Ixodes scapularis and Dermacentor andersoni cell lines were stimulated with heat-killed Escherichia coli and Micrococcus luteus to investigate whether infection by Rickettsia peacockii, an endosymbiont of D. andersoni, modifies humoral immune responses. Radial diffusion assays, western blotting, flow cytometry, and quantitative reverse-transcription PCR were used to determine if expression of bacteriolytic peptides, including lysozyme and defensin, was upregulated by bacterial stimulation or infection with R. peacockii. The I. scapularis line IDE12 upregulated expression of lysozyme and defensin following stimulation. The D. andersoni cell line DAE15 also expressed defensin and lysozyme, but only lysozyme was upregulated by bacterial stimulation. R. peacockii infection alone, or in cells stimulated with bacteria, did not modify defensin or lysozyme expression in either cell line. These results suggest tick endosymbionts may avoid recognition by the tick immune system, and infection may not affect humoral immune responses to bacteria not normally associated with ticks.

Transcriptome analysis of the salivary glands of Dermacentor andersoni Stiles (Acari: Ixodidae).

Amongst blood-feeding arthropods, ticks of the family Ixodidae (hard ticks) are vectors and reservoirs of a greater variety of infectious agents than any other ectoparasite. Salivary glands of ixodid ticks secrete a large number of pharmacologically active molecules that not only facilitate feeding but also promote establishment of infectious agents. Genomic, proteomic and immunologic characterization of bioactive salivary gland molecules are, therefore, important as they offer new insights into molecular events occurring at the tick-host interface and they have implications for development of novel control strategies. The present work uses complementary DNA (cDNA) sequence analysis to identify salivary gland transcripts expressed by the Rocky Mountain wood tick, Dermacentor andersoni, a vector of the human pathogens causing Rocky Mountain spotted fever, Colorado tick fever, tularemia, and Powassan encephalitis as well as the veterinary pathogen Anaplasma marginale. Dermacentor andersoni is also capable of inducing tick paralysis. Automated single-pass DNA sequencing was conducted on 1440 randomly selected cDNA clones from the salivary glands of adult female D. andersoni collected during the early stages of feeding (18-24h). Analysis of the expressed sequence tags (ESTs) resulted in 544 singletons and 218 clusters with more than one quality read and attempts were made to assign putative functions to tick genes based on amino acid identity to published protein databases. Approximately 25.6% (195) of the sequences showed limited or no homology to previously identified gene products. A number of novel sequences were identified which presented significant sequence similarity to mammalian genes normally associated with extracellular matrix (ECM), regulation of immune responses, tumor suppression, and wound healing. Several coding sequences possessed various degrees of homology to previously described proteins from other tick species. Preliminary nucleotide variation analysis of these and other tick sequences suggests extensive nucleotide diversity, which has implications for evolution of tick feeding. Intra-species diversity studies can be a promising tool for identifying sequence variations potentially associated with phenotypic traits affecting vector-host-pathogen interactions.

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration.

The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.

Molecular characterization, tissue-specific expression and RNAi knockdown of the first vitellogenin receptor from a tick.

This is the first full-length message for a vitellogenin receptor (VgR) sequenced from ticks. VgRs, members of the low-density lipoprotein receptor (LDLR) superfamily, mediate the uptake of the yolk protein, vitellogenin (Vg), from the hemolymph. The VgR message from the American dog tick, Dermacentor variabilis (GenBank accession No. DQ103506.4) comprised 5673 bp which coded for a 1798 aa deduced protein with a predicted 196.6 kDa molecular mass. After removing the 20 aa signal peptide, the 1778 aa deduced mature protein had a predicted 196.6 kDa molecular mass. BLAST comparisons showed the highest similarity to the VgR of the cockroach, Periplaneta americana. VgR message was expressed in mated female ovary but absent in female midgut and salivary glands or whole body mRNA from blood fed males, indicating that it is both sex and tissue specific. VgR transcript was absent in virgin (previtellogenic) females but present in ovaries of mated females following drop off. RNAi showed that unfed adult ticks injected with a VgR-dsRNA probe failed to lay eggs, develop brown eggs or fully express VgR transcript (Northern blots). In contrast, controls oviposited numerous normal brown eggs and showed strong expression of VgR transcripts. These results show that the expression of the VgR message is essential for Vg uptake and egg development in the American dog tick.