Effect of short photoperiod on the development of Haemaphysalis longicornis (Ixodida: Ixodidae).

The invasive tick, Haemaphysalis longicornis Neumann, is now present across most of the mid-Atlantic States in the eastern United States. This tick ends its seasonal activity in late October to early November, with larvae being the last life-stage observed questing. Previous research has revealed that the activity of H. longicornis is influenced by photoperiod: short daylight lengths trigger diapause in nymphs, marking it as the primary overwintering stage. However, whether engorged larvae can enter diapause or are affected by short daylight is unclear. In this study, we tested in the laboratory whether the photoperiod Affects the development of H. longicornis engorged larvae and engorged nymphs under constant temperature and humidity. The results showed that engorged larvae molted significantly faster (3 days faster) when the photoperiod was 9 h of light as opposed to 14 h. In contrast, changes in the photoperiod did not affect the molting of engorged nymphs. Our results demonstrate that engorged larvae respond to short daylight length, by molting faster. These results suggest that engorged larvae are unlikely to overwinter under field conditions and support the expectation that nymphs are the primary overwintering stage for H. longicornis in the United States.

Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

Baseline susceptibility of Haemaphysalis longicornis to organophosphate, carbamate, and pyrethroid acaricides.

BACKGROUND: The Asian longhorned tick, Haemaphysalis longicornis, continues to expand its range in North America, and synthetic acaricides are likely to play an increasing role in managing this species. Acaricide resistance is common in some tick species that infest livestock. However, baseline acaricide susceptibility has not previously been examined in this invasive tick. RESULTS: We used a standard larval packet test to evaluate the susceptibility of the Asian longhorned tick to acaricides currently or formerly used in tick control: propoxur, carbaryl, bifenthrin, permethrin, and coumaphos. Discriminating concentrations were estimated at 6.5, 27.9, 988, 2242, and 808 ppm, respectively. The half-maximal lethal concentration (LC50 ) values for propoxur, carbaryl, permethrin and coumaphos were compared with data available for other tick species and showed that H. longicornis was more susceptible to propoxur, carbaryl and coumaphos, and had a similar susceptibility to permethrin. CONCLUSIONS: The results indicate that resistance to these acaricides is not currently a concern for H. longicornis in the United States. However, responsible integrated management and early detection of resistance can help ensure the long-term efficacy of products used for controlling this tick species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Seasonal Dynamics of Tick Species in the Ecotone of Parks and Recreational Areas in Middlesex County (New Jersey, USA).

People often use parks and other forested areas for outdoor activities such as hiking and walking their dogs. Areas of primary use are paths or grassy meadows on the edges of the forests that constitute transitional areas between different plant communities (aka ecotones). In this study, we monitored the seasonal dynamics of questing ticks in forest/meadow and forest/path ecotones in five areas in Middlesex County, New Jersey (NJ). We found anthropophilic species such as Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis coexisting with Haemaphysalis longicornis, an invasive tick species first detected in NJ in 2017. Surveillance was conducted weekly from March to November 2020, and collected ticks were identified. The most abundant tick species was H. longicornis (83%), followed by A. americanum (9%), I. scapularis (7%), and D. variabilis (<1%). The seasonal dynamics of A. americanum and I. scapularis in the ecotone were similar to previous surveys in forest habitats. The presence of anthropophilic ticks, particularly I. scapularis, suggests the need for specific control approaches to target these habitats. In addition, the extraordinarily high numbers of H. longicornis collected in ecotones (1.70 ticks/m2) and frequent reports of this species on dogs highlight the importance of monitoring its expansion due to its potential as a vector of animal and human diseases.

Rickettsia and relapsing fever Borrelia in Alectorobius kelleyi (Ixodida: Argasidae) from peri domestic bats in the northeastern United States.

The soft ticks (Argasidae) are known vectors of human and animal pathogens around the globe and are relatively understudied. Our aim was to assess the presence of Rickettsia and Borrelia bacteria in Alectorobius kelleyi (Argasidae) parasitizing synanthropic bats in the highly urbanized northeastern United States. By collaborating with parasitologists, bat scientists and wildlife rehabilitators we were successful in obtaining A. kelleyi from five states. Since Argasid larvae will attach to their hosts for many days, most A. kelleyi examined (92%) were larvae collected from sick or injured big brown bats, Eptesicus fuscus, undergoing care at rehabilitation centers. In addition, we obtained adult A. kelleyi captured in residential living areas and trapped in attics. An in-depth analysis of a A. kelleyi found to be infected with a spotted fever group Rickettsia (SFGR) revealed a dual infection with a R. belli-like taxon (ancestral group) as well as an SFGR closely related to R. peacockii, likely the same previously found in A. kelleyi from Iowa and Kansas. We found that 36% of the A. kelleyi tested carried the SFGR. Furthermore, we detected a relapsing fever spirochete, likely Candidatus Borrelia johnsonii, in 25% of the A. kelleyi from Pennsylvania. While it is unclear if these bacteria constitute a health risk to either bats or humans, our study indicates that human exposure to ectoparasites infesting peridomestic wildlife should be considered in the epidemiology of tick-borne diseases.

Dietary intake and eating behavior in the Chilean population during the COVID-19 lockdown

Lockdown and social distancing due to COVID-19 affected the mental health and lifestyle of the population. However, there is insufficient evidence of alterations in eating behavior. Our study seeks to describe the relationship between eating behavior and eating habits among Chilean adults during the confinement period. A sample of 760 Chilean subjects was analyzed, who answered surveys using Google Forms, considering demographic characteristics, social distancing, dietary habits and EB. More than half of the participants consider that their dietary intake increased during confinement. Changes in dietary intake were analyzed according to food group, and a decrease in the consumption of fish, fruits and dairy products was observed, while legumes, processed foods and soft drinks showed an increase, which represents risk factors for the development of cardiovascular diseases. When analyzing eating behavior, a greater difficulty in stopping eating was observed when faced with external stimuli; increased intake associated with complex emotional situations, and when isolating the group that decreased their intake of unhealthy foods, a greater ability to limit their intake for weight control was reported. Our results are similar to other studies, and they reinforce that confinement is related to eating behavior, leading to changes in eating habits, which indicates that, at the public health level, post-pandemic nutritional strategies, should be focused on regulating eating behavior in order to guide habits towards healthy eating.

El confinamiento y distanciamiento social debido al COVID-19 afectó la salud mental y hábitos de vida de la población. Sin embargo, no existe suficiente evidencia de alteraciones en la conducta alimentaria (CA). Nuestro estudio busca establecer la relación entre la conducta alimentaria y los hábitos alimentarios en adultos chilenos durante el periodo de confinamiento. Se analizó una muestra de 760 sujetos chilenos, quienes contestaron encuestas mediante Google Forms, considerando características demográficas, distanciamiento social, hábitos dietéticos y (CA). Más de la mitad de los participantes considera que su ingesta alimentaria aumentó durante el confinamiento. Se analizó los cambios en ingesta según grupo de alimentos, y se observó una disminución en la ingesta de pescados, frutas y lácteos, mientras que legumbres, alimentos procesados y bebidas azucaradas presentaron un aumento, lo cual representa factores de riesgo para el desarrollo de enfermedades cardiovasculares. Al analizar la CA se observó una mayor dificultad para dejar de comer frente a estímulos externos, aumento de la ingesta asociado a situaciones emocionales complejas, y al aislar al grupo que disminuyó su ingesta de alimentos no saludables se reportó una mayor capacidad para limitar la ingesta para el control del peso. Nuestros resultados son similares con otros estudios, y refuerzan que el confinamiento se relaciona con la CA, conduciendo a cambios en los hábitos de alimentación, lo cual nos indica que, a nivel de salud pública, las estrategias nutricionales post-pandemia, deben estar enfocadas en regular la CA con el objetivo de guiar los hábitos a una alimentación saludable.

Field applications of granular and liquid pyrethroids, carbaryl, and IGRs to control the asian longhorned tick (Haemaphysalis longicornis) and impacts on nontarget invertebrates.

Few documented control strategies exist for the invasive tick, Haemaphysalis longicornis, despite its potential to reach extremely high numbers and vector human and animal pathogens. In 2020, we evaluated the effects of single applications of five granular and liquid acaricides on H. longicornis in a public park in northern New Jersey. Acaricides tested included pyrethroids (lambda-cyhalothrin, bifenthrin), a carbamate (carbaryl), and the insect growth regulators (IGRs) pyriproxyfen and novaluron. We also monitored the impact of each treatment on non-target soil and above-ground invertebrate species using pitfall and sticky traps, respectively. We recorded over 70,000 H. longicornis ticks in the study area from July to October 2020. An average of 99% control was achieved with lambda-cyhalothrin spray and 95% with granular bifenthrin. In contrast, granular carbaryl did not significantly reduce any life stages of H. longicornis. The IGR (pyriproxyfen/novaluron) resulted in a significant 45% reduction of the larval stage following treatments in July. No other stages were significantly impacted by pyriproxyfen alone or in combination with novaluron. Analysis of non-target species revealed that the community composition of soil-dwelling arthropods was strongly impacted by pyrethroid treatments and, to a lesser extent, by the carbamate treatment. The granular pyrethroid bifenthrin had more pronounced effects and impacted a broader range of non-target groups in the pitfall traps than the liquid pyrethroid lambda-cyhalothrin. Arthropod groups that were negatively impacted included Isopoda, Formicidae, Coleoptera, Araneae, Acari, and Grylloidea. Collembola numbers, however, were elevated in both pyrethroid treatments. The community composition of arthropods collected on the above-ground sticky traps was strongly impacted only in the liquid lambda-cyhalothrin treatment. The primary groups impacted in the sticky trap analysis were Collembola and Hemiptera. Community composition in traps remained distinct in the pyrethroid treatments through the entire survey period up to 62 days post-treatment. The results of this study indicate that pyrethroid acaricides were highly effective at controlling H. longicornis, while other compounds, including carbaryl and IGRs, did not achieve consistent levels of control. Further research is needed to find effective and environmentally sustainable alternatives. Integrated management programs can include the judicious use of pyrethroids to control H. longicornis.

Repellency of novel catnip (Nepeta cataria) cultivar extracts against Ixodes scapularis and Haemaphysalis longicornis (Acari: Ixodida: Ixodidae).

Tick bites are a major public health concern due to the vector role that many tick species have in transmitting human pathogens. Synthetic repellents such as N­diethyl-meta-toluamide (DEET) remain the standard for repellency. Still, there is a need for natural commercial alternatives with similar or better properties than DEET. We evaluated the repellency of two extracts, CR3 and CR9, derived for newly developed catnip cultivars on two tick species, Ixodes scapularis and Haemaphysalis longicornis. Dose-response in vitro assays showed that CR3 and CR9 extracts have similar repellency properties to DEET. At a 20% concentration, both CR3 and CR9 extracts exhibited a repellency of 100%. Catnip extracts maintained their repellency properties for at least 8 h. In a two-choice assay, I. scapularis, but not H. longicornis, was more sensitive to CR3 than DEET. In addition, CR3 reduces the life span of I. scapularis, suggesting that it has an acaricidal effect on ticks. In summary, CR3 and CR9 catnip extracts are promising tick repellents that should be further developed, alone or in combination with other tick repellents, and tested for their use as tick repellents for humans.

Borrelia burgdorferi Surface Exposed GroEL Is a Multifunctional Protein.

The spirochete, Borrelia burgdorferi, has a large number of membrane proteins involved in a complex life cycle, that includes a tick vector and a vertebrate host. Some of these proteins also serve different roles in infection and dissemination of the spirochete in the mammalian host. In this spirochete, a number of proteins have been associated with binding to plasminogen or components of the extracellular matrix, which is important for tissue colonization and dissemination. GroEL is a cytoplasmic chaperone protein that has previously been associated with the outer membrane of Borrelia. A His-tag purified B. burgdorferi GroEL was used to generate a polyclonal rabbit antibody showing that GroEL also localizes in the outer membrane and is surface exposed. GroEL binds plasminogen in a lysine dependent manner. GroEL may be part of the protein repertoire that Borrelia successfully uses to establish infection and disseminate in the host. Importantly, this chaperone is readily recognized by sera from experimentally infected mice and rabbits. In summary, GroEL is an immunogenic protein that in addition to its chaperon role it may contribute to pathogenesis of the spirochete by binding to plasminogen and components of the extra cellular matrix.

Applications of artificial membrane feeding for ixodid ticks.

Ticks are obligatory hematophagous ectoparasites that feed on a large variety of vertebrates. In the laboratory, animals (mainly mice and rabbits) are used to maintain tick colonies. However, the use of animals to rear ticks can be expensive and requires dedicated animal facilities. In addition, research institutions are committed to the principle of 3Rs (Replacement, Reduction and Refinement), which encourages the use of alternatives to animals when possible. The development of artificial membrane systems has provided an alternative to animals, at least for some tick species. Over the years, different modifications in artificial feeding systems have led to new applications, including acaricide testing, tick-pathogen interaction, and novel approaches to study tick physiology. Although artificial membrane feeding still has some limitations, the method can provide numerous advantages, including the standardization of acaricide treatments under controlled conditions, an alternative to animals for tick rearing, and reduction of cost associated with animals and animal housing facilities. In this review, we summarized the evolution of tick feeding membranes and their applications over time, explaining the modifications incorporated to study tick physiology, tick-pathogen interactions, and acaricide testing.

A life stage-targeted acaricide application approach for the control of Haemaphysalis longicornis.

Haemaphysalis longicornis Neumann is an invasive tick species that has recently been detected in the eastern United States. We designed field studies to monitor the population dynamics of H. longicornis over a year (2019) in New Jersey, and to assess the efficacy of a pyrethroid acaricide (lambda-cyhalothrin) in controlling this tick using a stage-treatment approach. Nymphs were the most abundant life stage found host-seeking from May through early July, followed by a brief period of high adult activity in mid-July, and a very high larval peak extending from late July through October. Overall, 542 adults, 1910 nymphs, and 69,238 larvae were recorded during the sampling. In the present study, the efficacy of acaricide treatments to suppress host-seeking ticks was assessed by applying lambda-cyhalothrin once during each of the three periods of activity for nymphs (June), adults (July), and larvae (August), or sequentially during all three months. Control plots were left untreated and used for phenology studies. Applications in June and July provided 100 % control of all life stages, including the dominant nymphal and adult stages for 42 and 35 d, respectively. Ticks re-established at normal or reduced levels following applications in June or July, respectively, compared to untreated controls. The application in August provided 100 % control for 49 d, and a high level of suppression (>99 % control) remained through the end of the tick season in October. This study therefore supports that single pyrethroid applications can provide 100 % control of H. longicornis for up to 7 wk, and a single late-summer application towards the end of host-seeking adult activity can provide near complete control of the larval population. An evaluation of single applications over the course of the season revealed that treatments in July or August did not lead to significant reductions in the nymphal population. However, multiple sequential treatments targeting all life stages provided 66 %, 97 %, and >99 % control of adults, nymphs, and larvae, respectively through the season of H. longicornis activity.

Emerging tick-borne pathogens of public health importance: a mini-review.

Ticks are the most important vectors of human pathogens, leading to increased public health burdens worldwide. Tick-borne pathogens include viruses (e.g. tick-borne encephalitis and Powassan); bacteria, such as the causative agents of Lyme disease, spotted fever rickettsiosis and human anaplasmosis; and malaria-like protozoan parasites causing babesiosis. Tick-borne diseases are emerging due to the geographical expansion of their tick vectors, especially in the northern hemisphere. Two examples of this phenomenon are Ixodes scapularis and Amblyomma americanum, which have expanded their ranges in the USA in recent decades and are responsible for the continuous emergence of Lyme disease and human ehrlichiosis, respectively. This phenomenon is also occurring worldwide and is reflected by the increasing number of tick-borne encephalitis and haemorrhagic fever cases in Europe and Asia. In this review, we provide a concise synopsis of the most medically important tick-borne pathogen worldwide, with a particular emphasis on emerging public health threats.

Rapid and Highly Sensitive DNA Flow Technology Platform to Detect Tick-Borne Bacterial Pathogens in Clinical Samples.

Zoonotic diseases represent a significant public health concern worldwide due to the emergence/re-emergence of vector-borne diseases in the last decade. Ticks are the most important vectors in the northern hemisphere and can transmit diseases such as Lyme disease, human granulocytic anaplasmosis, and spotted fever rickettsioses, among others. Therefore, there is a growing need to develop better and faster diagnostic tools that can detect zoonotic human pathogens in clinical samples. In this study, we present the results for a new kit tick-borne bacteria flow chip (TBFC), which allows the simultaneous screening of seven different bacterial pathogens in human samples using a DNA flow technology platform (hybriSpot system). The analytical sensitivity and specificity of the TBFC were calculated spiking bacterial DNA in human DNA samples, and the results were compared with an in-house single PCR-reverse line blot (RLB) routinely used for diagnosis at the National Center for Microbiology in Spain. The analytical sensitivity and specificity of the TBFC were almost identical to the PCR-RLBs used in diagnosis. In addition, samples from patients (n = 212) with a wide range of clinical signs/symptoms consistent with multisystem disorders suggestive of a tick-borne infection were tested using the TBFC, and the results were compared with those obtained by PCR-RLB. The concordance of both methods using patient samples was 97.2%. The TBFC kit is a rapid new and cost-efficient diagnostic molecular tool capable of detecting tick-borne pathogens in clinical samples.

Modeling of crop wild relative species identifies areas globally for in situ conservation.

The impact of climate change is causing challenges for the agricultural production and food systems. More nutritious and climate resilient crop varieties are required, but lack of available and accessible trait diversity is limiting crop improvement. Crop wild relatives (CWR) are the wild cousins of cultivated crops and a vast resource of genetic diversity for breeding new, higher yielding, climate change tolerant crop varieties, but they are under-conserved (particularly in situ), largely unavailable and therefore underutilized. Here we apply species distribution modelling, climate change projections and geographic analyses to 1261 CWR species from 167 major crop genepools to explore key geographical areas for CWR in situ conservation worldwide. We identify 150 sites where 65.7% of the CWR species identified can be conserved for future use.

HtrA of Borrelia burgdorferi Leads to Decreased Swarm Motility and Decreased Production of Pyruvate.

Borrelia burgdorferi HtrA (HtrABb) is a serine protease that targets damaged or improperly folded proteins. In our previous studies, HtrABb specifically degraded basic membrane protein BmpD, chemotaxis phosphatase CheX, and outer membrane protein P66. In addition, HtrABb degrades virulence factor BB0323 and components of the extracellular matrix fibronectin and aggrecan. A proteomics-based analysis (two-dimensional difference gel electrophoresis [2-D DIGE], liquid chromatography-mass spectrometry [LC-MS]) of an HtrABb-overexpressing strain of B. burgdorferi (A3HtrAOE) revealed that protein levels of P66 were reduced in comparison to wild-type B. burgdorferi, confirming its status as an HtrABb substrate. Hbb, a P66-DNA-binding transcription factor, was specifically degraded by HtrABb, providing supportive evidence for a role for both in the regulation of P66. A3HtrAOE exhibited reduced motility in swarm assays, a possible link between overabundance of HtrABb and its enzymatic specificity for P66. However, the ΔP66 strain did not have reduced motility in the swarm assays, negating a role for this protein. The proteomics analyses also identified three enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol-3-phosphate dehydrogenase (GPDH), and glycerol kinase (GK), and one enzyme involved in carbohydrate metabolism, diphosphate-fructose-6-phosphate 1-phosphotransferase, which were reduced in A3HtrAOE. Consistent with its reduced protein levels of these glycolytic enzymes, A3HtrAOE was also deficient in production of pyruvate. We propose a model for a role for HtrABb in contributing to a decrease in metabolic activity of B. burgdorferiIMPORTANCE Being a vector-borne bacterium, B. burgdorferi must remodel its protein content as it transfers from tick to mammal. Proteolysis is a mechanism whereby remodeling can be accomplished. HtrABb degrades a number of proteins whose disappearance may help in preparing this organism for a stage of low metabolic activity.

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins.

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

Analysis of Lipids and Lipid Rafts in Borrelia.

Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.

Ordered Membrane Domain-Forming Properties of the Lipids of Borrelia burgdorferi.

Co-existing disordered and ordered (raft) membrane domains exist in Borrelia burgdorferi, the causative agent of Lyme disease. However, although B. burgdorferi contains cholesterol lipids, it lacks sphingolipids-a crucial component of rafts in eukaryotes. To define the principles of ordered lipid domain formation in Borrelia, the domain forming properties of vesicles composed of its three major lipids, acylated cholesteryl galactoside (ACGal), monogalactosyl diacyglycerol (MGalD), and phosphatidylcholine (PC) and/or their mixtures were studied. Anisotropy and fluorescence resonance energy transfer measurements were used to assay membrane order and ordered-domain formation. ACGal had the highest potential to form ordered domains. Interestingly, mixtures of ACGal with B. burgdorferi PC formed ordered domains more readily than mixtures of ACGal with MGalD. This appears to reflect the relatively high level of saturation observed for B. burgdorferi PC, as vesicles containing ACGal and PC, but in which the unsaturated lipid dioleoyl PC was substituted for Borrelia PC, failed to form ordered domains. In addition, the properties of ACGal were compared to those of cholesterol. Depending on what other lipids were present, ordered-domain formation in the presence of ACGal was greater than or equal to that in the presence of cholesterol. Giant unilamellar vesicles formed from ACGal-containing mixtures showed rounded domain shapes similar to those in analogous vesicles containing cholesterol, indicative of liquid-ordered state rather than solid-like gel-state domain formation. Over all, principles of ordered-domain formation in B. burgdorferi appear to be very similar to those in eukaryotes, with saturated PC taking the place of sphingolipids, but with ACGal being the main lipid component inducing ordered-domain formation.

Borrelia burgdorferi†HtrA: evidence for twofold proteolysis of outer membrane protein p66.

In prokaryotes, members of the High Temperature Requirement A (HtrA) family of serine proteases function in the periplasm to degrade damaged or improperly folded membrane proteins. Borrelia burgdorferi, the agent of Lyme disease, codes for a single HtrA homolog. Two-dimensional electrophoresis analysis of B. burgdorferi B31A3 and a strain that overexpresses HtrA (A3HtrAOE) identified a downregulated protein in A3HtrAOE with a mass, pI and MALDI-TOF spectrum consistent with outer membrane protein p66. P66 and HtrA from cellular lysates partitioned into detergent-resistant membranes, which contain cholesterol-glycolipid-rich membrane regions known as lipid rafts, suggesting that HtrA and p66 may reside together in lipid rafts also. This agrees with previous work from our laboratory, which showed that HtrA and p66 are constituents of B. burgdorferi outer membrane vesicles. HtrA degraded p66 in vitro and A3HtrAOE expressed reduced levels of p66 in vivo. Fluorescence confocal microscopy revealed that HtrA and p66 colocalize in the membrane. The association of HtrA and p66 establishes that they could interact efficiently and their protease/substrate relationship provides functional relevance to this interaction. A3HtrAOE also showed reduced levels of p66 transcript in comparison with wild-type B31A3, indicating that HtrA-mediated regulation of p66 may occur at multiple levels.