Human Babesia microti Incidence and Ixodes scapularis Distribution, Rhode Island, 1998–2004

Distribution of nymphal Ixodes scapularis in Rhode Island was used as a logistical regressor for predicting presence of human babesiosis. Although the incidence of babesiosis is increasing in southern Rhode Island, large areas of the state are free of babesiosis risk

Functional Genomics Tool: Gene Silencing in \u3ci\u3eIxodes scapularis\u3c/i\u3e Eggs and Nymphs by Electroporated dsRNA

Background Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. Results The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. Conclusions Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

Background: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. Results: The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. Conclusions: Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks

Purification and characterization of a novel salivary antimicrobial peptide from the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP (I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3 kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3 kDa secreted protein (AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family

Duration of Exposure to Suboptimal Atmospheric Moisture Affects Nymphal Blacklegged Tick Survival

The biological processes affecting Ixodes scapularis Say survival are complex. Understanding these processes will be beneficial for predicting tick distribution and population dynamics. This research shows that the duration for which nymphal ticks are exposed to drying air is an important factor for their survival. Experimental analysis of variance results show that duration of exposure to dry air (duration) is as important as vapor pressure deficit (relative humidity) (duration, relative humidity, P \u3c 0.0001). Ticks do not survive when exposed to dry air for long periods; however, the return of humid air within 4–8 h has as large a positive impact on tick survival, as does constant humid air. This experiment exposes nymphal ticks to conditions of suboptimal humidity for different durations and then returns them to saturated conditions that are more typical of daily relative humidity fluctuations experienced during summer in southern New England forests

Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis

We report for the first time that saliva of the hard tick and Lyme disease vector,Ixodes scapularis,is a potent inhibitor of angiogenesis. Saliva (≤ 1:500 dilutions) or salivary gland (0.1–0.5 pairs/assay) dose-dependently inhibits microvascular endothelial cell (MVEC) proliferation. Inhibition was also detected with the saliva of the cattle tick Boophilus microplus but not with the salivary gland of Anopheles gambiae, An. stephensi, Lutzomyia longipalpis, Phlebotomus papatasi, Aedes aegypti, Culex quinquefasciatus, and Cimex lectularius. Inhibition of MVEC proliferation by I. scapularis saliva was accompanied by a change in cell shape (shrinkage of the cytoplasm with loss of cell-cell interactions) and apoptosis which was estimated by expression of phosphatidylserine using the Apopercentage dye, and by a typical pattern of chromatin margination, condensation, and fragmentation as revealed by nuclear staining with Hoechst 33258.The effect of saliva appears to be mediated by endothelial cell α5β1 integrin, because monoclonal antibodies against this but not αvβ3, αvβ5, α9β1, or α2β1 integrins remarkably block its effect. In addition, SDS/PAGE shows that saliva specifically degrades purified α5β1 but not αvβ5 or αvβ3 integrins. Incubation of saliva with EDTA and 1,10-phenanthroline, but not phenylmethylsulfonyl fluoride (PMSF), inhibits saliva-dependent degradation of purified α5β1 integrin, suggesting that a metalloprotease is responsible for the activity. Finally, saliva at ≤ 1:1,000 dilutions blocks sprouting formation from chick embryo aorta implanted in Matrigel, an in vitro model of angiogenesis. These findings introduce the concept that tick saliva is a negative modulator of angiogenesis-dependent wound healing and tissue repair, therefore allowing ticks to feed for days. Inhibition of angiogenesis was hitherto an unidentified biologic property of the saliva of any blood-sucking arthropod studied so far. Its presence in tick saliva may be regarded as an additional source of angiogenesis inhibitors with potential applications for the study of both vector and vascular biology

Is Selenoprotein K Required for \u3ci\u3eBorrelia burgdorferi\u3c/i\u3e Infection Within the Tick Vector \u3ci\u3eIxodes scapularis\u3c/i\u3e?

Background: Tick selenoproteins are involved in regulating oxidative and endoplasmic reticulum stress during prolonged tick feeding on mammalian hosts. How selenoproteins are activated upon tick-borne pathogen infection is yet to be defined. Methods: To examine the functional role of selenoprotein K in Borrelia burgdorferi infection within the tick host Ixodes scapularis, RNA interference (RNAi)-based gene silencing was performed. Results: Selenoprotein K is an endoplasmic reticulum (ER)-resident protein and a component of the ERAD complex involved in ER homeostasis. A qRT-PCR assay revealed the significant upregulation of selenogene K (selenoK) expression in B. burgdorferi-infected tick tissues. Silencing of the selenoK transcript significantly depleted B. burgdorferi copies within the infected tick tissues. Upon selenoK knockdown, another component of the ERAD complex, selenoprotein S (selenoS), was significantly upregulated, suggesting a compensatory mechanism to maintain ER homeostasis within the tick tissues. Knockdown of selenoK also upregulated ER stress-related unfolded protein response (UPR) pathway components, ATF6 and EIF2. Conclusions: The exact mechanisms that contribute to depletion of B. burgdorferi upon selenoK knockdown is yet to be determined, but this study suggests that selenoK may play a vital role in the survival of B. burgdorferi within the tick host

Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis

The full-length sequence of tick salivary gland cDNA coding for a protein similar to metalloproteases (MP) of the reprolysin family is reported. The Ixodes scapularis MP is a 488 amino acid (aa) protein containing pre- and pro-enzyme domains, the zinc-binding motif HExxHxxGxxH common to metalloproteases, and a cysteine-rich region. In addition, the predicted amino-terminal sequences of I. scapularis MPs were found by Edman degradation of PVDF-transferred SDS/PAGE-separated tick saliva proteins, indicating that these putative enzymes are secreted. Furthermore, saliva has a metal-dependent proteolytic activity towards gelatin, fibrin(ogen), and fibronectin, but not collagen or laminin. Accordingly, I. scapularis saliva has a rather specific metalloprotease similar to the hemorrhagic proteases of snake venoms. This is the first description of such activity in tick saliva and its role in tick feeding and Borrelia transmission is discussed

RNAi-mediated gene silencing in tick synganglia: A proof of concept study

<p>Abstract</p> <p>Background</p> <p>Progress in generating comprehensive EST libraries and genome sequencing is setting the stage for reverse genetic approaches to gene function studies in the blacklegged tick (<it>Ixodes scapularis</it>). However, proving that RNAi can work in nervous tissue has been problematic. Developing an ability to manipulate gene expression in the tick synganglia likely would accelerate understanding of tick neurobiology. Here, we assess gene silencing by RNA interference in the adult female black-legged tick synganglia.</p> <p>Results</p> <p>Tick β-Actin and Na⁺-K⁺-ATPase were chosen as targets because both genes express in all tick tissues including synganglia. This allowed us to deliver dsRNA in the unfed adult female ticks and follow a) uptake of dsRNA and b) gene disruption in synganglia. <it>In vitro </it>assays demonstrated total disruption of both tick β-Actin and Na⁺-K⁺-ATPase in the synganglia, salivary glands and midguts. When dsRNA was microinjected in unfed adult female ticks, nearly all exhibited target gene disruption in the synganglia once ticks were partially blood fed.</p> <p>Conclusion</p> <p>Abdominal injection of dsRNA into unfed adult female ticks appears to silence target gene expression even in the tick synganglia. The ability of dsRNA to cross the blood-brain barrier in ticks suggests that RNAi should prove to be a useful method for dissecting function of synganglia genes expressing specific neuropeptides in order to better assess their role in tick biology.</p

Seasonal Variation in Nymphal Blacklegged Tick Abundance in Southern New England Forests

In the northeastern United States, risk of human exposure to tick transmitted disease is primarily a function of the abundance of the blacklegged tick, Ixodes scapularis Say. We assessed seasonal variability in the abundance of nymphal stage I. scapularis over 13 yr, collected from several forested areas throughout Rhode Island. Specifically, we examined intraseasonal differences by using two temporally distinct tick collections made during the peak nymphal tick season. Intraseasonal factors significantly impacted tick abundance, with the June tick rate (mean = 40.42, SD = 14.79) significantly more abundant than the July tick rate (mean = 27.64, SD = 15.47). The greater variability in July (coefficient of variation: June, 36.61%; July, 55.95%) lead us to conclude June tick rates are relatively stable from year to year, whereas July tick rates contribute more to intraseasonal and yearly variation