Phylogeography of the blacklegged tick (Ixodes scapularis) throughout the USA identifies candidate loci for differences in vectorial capacity.

The blacklegged tick (Ixodes scapularis (Journal of the Academy of Natural Sciences of Philadelphia, 1821, 2, 59)) is a vector of Borrelia burgdorferi sensu stricto (s.s.) (International Journal of Systematic Bacteriology, 1984, 34, 496), the causative bacterial agent of Lyme disease, part of a slow-moving epidemic of Lyme borreliosis spreading across the northern hemisphere. Well-known geographical differences in the vectorial capacity of these ticks are associated with genetic variation. Despite the need for detailed genetic information in this disease system, previous phylogeographical studies of these ticks have been restricted to relatively few populations or few genetic loci. Here we present the most comprehensive phylogeographical study of genome-wide markers in I. scapularis, conducted by using 3RAD (triple-enzyme restriction-site associated sequencing) and surveying 353 ticks from 33 counties throughout the species' range. We found limited genetic variation among populations from the Northeast and Upper Midwest, where Lyme disease is most common, and higher genetic variation among populations from the South. We identify five spatially associated genetic clusters of I. scapularis. In regions where Lyme disease is increasing in frequency, the I. scapularis populations genetically group with ticks from historically highly Lyme-endemic regions. Finally, we identify 10 variable DNA sites that contribute the most to population differentiation. These variable sites cluster on one of the chromosome-scale scaffolds for I. scapularis and are within identified genes. Our findings illuminate the need for additional research to identify loci causing variation in the vectorial capacity of I. scapularis and where additional tick sampling would be most valuable to further understand disease trends caused by pathogens transmitted by I. scapularis.

Isolation of a novel rhabdovirus and detection of multiple novel viral sequences in Culex species mosquitoes in the United States.

To increase our understanding of the diversity of the mosquito virome, 6956 mosquitoes of five species (Culex erraticus, Culex pipiens, Culex restuans, Culex tarsalis, and Culex territans) collected in Iowa in the United States in 2017 and 2020 were assayed for novel viruses by performing polyethylene glycol precipitation, virus isolation in cell culture, and unbiased high-throughput sequencing. A novel virus, provisionally named "Walnut Creek virus", was isolated from Cx. tarsalis, and its genomic sequence and organization are characteristic of viruses in the genus Hapavirus (family Rhabdoviridae). Replication of Walnut Creek virus occurred in avian, mammalian, and mosquito, but not tick, cell lines. A novel virus was also isolated from Cx. restuans, and partial genome sequencing revealed that it is distantly related to an unclassified virus of the genus Phytoreovirus (family Sedoreoviridae). Two recognized viruses were also isolated: Culex Y virus (family Birnaviridae) and Houston virus (family Mesoniviridae). We also identified sequences of eight novel viruses from six families (Amalgaviridae, Birnaviridae, Partitiviridae, Sedoreoviridae, Tombusviridae, and Totiviridae), two viruses that do not belong to any established families, and many previously recognized viruses. In summary, we provide evidence of multiple novel and recognized viruses in Culex spp. mosquitoes in the United States.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Target identification reveals lanosterol synthase as a vulnerability in glioma.

Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.

Twenty years of West Nile virus spread and evolution in the Americas visualized by Nextstrain.

It has been 20 years since West Nile virus first emerged in the Americas, and since then, little progress has been made to control outbreaks caused by this virus. After its first detection in New York in 1999, West Nile virus quickly spread across the continent, causing an epidemic of human disease and massive bird die-offs. Now the virus has become endemic to the United States, where an estimated 7 million human infections have occurred, making it the leading mosquito-borne virus infection and the most common cause of viral encephalitis in the country. To bring new attention to one of the most important mosquito-borne viruses in the Americas, we provide an interactive review using Nextstrain: a visualization tool for real-time tracking of pathogen evolution (nextstrain.org/WNV/NA). Nextstrain utilizes a growing database of more than 2,000 West Nile virus genomes and harnesses the power of phylogenetics for students, educators, public health workers, and researchers to visualize key aspects of virus spread and evolution. Using Nextstrain, we use virus genomics to investigate the emergence of West Nile virus in the U S, followed by its rapid spread, evolution in a new environment, establishment of endemic transmission, and subsequent international spread. For each figure, we include a link to Nextstrain to allow the readers to directly interact with and explore the underlying data in new ways. We also provide a brief online narrative that parallels this review to further explain the data and highlight key epidemiological and evolutionary features (nextstrain.org/narratives/twenty-years-of-WNV). Mirroring the dynamic nature of outbreaks, the Nextstrain links provided within this paper are constantly updated as new West Nile virus genomes are shared publicly, helping to stay current with the research. Overall, our review showcases how genomics can track West Nile virus spread and evolution, as well as potentially uncover novel targeted control measures to help alleviate its public health burden.

The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis.

BACKGROUND: The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. METHODS: In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. RESULTS: The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. CONCLUSIONS: This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

Skunk River virus, a novel orbivirus isolated from Aedes trivittatus in the United States.

The genomic organization and in vitro host range of a novel mosquito-associated orbivirus, designated Skunk River virus, is described. The virus was isolated from Aedes trivittatus collected in Iowa in the United States. Three recognized viruses were also recovered: Culex flavivirus (family Flaviviridae), Houston virus (family Mesoniviridae) and Umatilla virus (family Reoviridae). The genome of Skunk River virus contains 10 segments and its organization is characteristic of viruses in the genus Orbivirus (family Reoviridae). The coding region of each segment was fully sequenced, revealing that the greatest nucleotide identity was to the corresponding regions of Big Cypress orbivirus and Sathuvachari virus, two recently described mosquito-associated orbiviruses. The phylogenetic inference is in agreement with these findings. In vitro host range experiments revealed that Aedes, Anopheles and Culex cell lines, and select lepidopteran and rodent cell lines, are permissive to Skunk River virus replication. In conclusion, we provide evidence of a novel mosquito-associated orbivirus in Iowa.

Selective Recovery of Rare Earth Elements from Coal Fly Ash Leachates Using Liquid Membrane Processes.

Coal combustion residues and other geological waste materials have been proposed as a resource for rare earth elements (REEs, herein defined as the 14 stable lanthanides, yttrium, and scandium). The extraction of REEs from residues often generate acidified leachates that require highly selective separation methods to recover the REEs from other major soluble ions in the leachates. Here, we studied two liquid membrane processes (liquid emulsion membranes, LEM, and supported liquid membranes, SLM) and compared them to standard solvent extraction techniques for selective recovery and concentration of REEs from a leachate of coal fly ash. All separation methods involved an organic solution of di(2-ethylhexyl)phosphoric acid dissolved in kerosene or mineral oil and an acid strippant solution of 5 M nitric acid for the liquid-based separations. The LEM configuration, which separated REEs by immersing an acid-in-oil emulsion in the ash leachate, resulted in similar recovery percentages of individual REEs as the conventional solvent extraction approach. The recovery of REEs in the SLM configuration, which involved the impregnation of the solvent in a hydrophobic membrane, was slower than the LEM process. However, the SLM process was notably more selective for the heavy (and higher value) REEs, while the conventional extraction and LEM processes were more selective for the light REEs. A flux-based model of the extraction processes suggested that recovery rates were limited by REE affinity for the solvent chelator in the SLM, while the rates of REEs separation via LEM were limited by diffusive mass transfer across the liquid membrane. Altogether, these results help to identify specific steps in the recovery process that future work should target in the development of scalable liquid membrane separations for REE recovery.

Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron.

Insect kinins (leucokinins) are multifunctional peptides acting as neurohormones and neurotransmitters. In females of the mosquito vector Aedes aegypti (L.), aedeskinins are known to stimulate fluid secretion from the renal organs (Malpighian tubules) and hindgut contractions by activating a G protein-coupled kinin receptor designated "Aedae-KR." We used protease-resistant kinin analogs 1728, 1729, and 1460 to evaluate their effects on sucrose perception and feeding behavior. In no-choice feeding bioassays (capillary feeder and plate assays), the analog 1728, which contains α-amino isobutyric acid, inhibited females from feeding on sucrose. It further induced quick fly-away or walk-away behavior following contact with the tarsi and the mouthparts. Electrophysiological recordings from single long labellar sensilla of the proboscis demonstrated that mixing the analog 1728 at 1 mM with sucrose almost completely inhibited the detection of sucrose. Aedae-KR was immunolocalized in contact chemosensory neurons in prothoracic tarsi and in sensory neurons and accessory cells of long labellar sensilla in the distal labellum. Silencing Aedae-KR by RNAi significantly reduced gene expression and eliminated the feeding-aversion behavior resulting from contact with the analog 1728, thus directly implicating the Aedae-KR in the aversion response. To our knowledge, this is the first report that kinin analogs modulate sucrose perception in any insect. The aversion to feeding elicited by analog 1728 suggests that synthetic molecules targeting the mosquito Aedae-KR in the labellum and tarsi should be investigated for the potential to discover novel feeding deterrents of mosquito vectors.

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity.

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.

Hemocyte differentiation mediates the mosquito late-phase immune response against Plasmodium in Anopheles gambiae.

Plasmodium parasites must complete development in the mosquito vector for transmission to occur. The mosquito innate immune response is remarkably efficient in limiting parasite numbers. Previous work has identified a LPS-induced TNFα transcription factor (LITAF)-like transcription factor, LITAF-like 3 (LL3), which significantly influences parasite numbers. Here, we demonstrate that LL3 does not influence invasion of the mosquito midgut epithelium or ookinete-to-oocyst differentiation but mediates a late-phase immune response that decreases oocyst survival. LL3 expression in the midgut and hemocytes is activated by ookinete midgut invasion and is independent of the mosquito microbiota, suggesting that LL3 may be a component of a wound-healing response. LL3 silencing abrogates the ability of mosquito hemocytes to differentiate and respond to parasite infection, implicating hemocytes as critical modulators of the late-phase immune response.

Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

BACKGROUND: Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses. RESULTS: Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion. CONCLUSIONS: This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

Integrating tunable anion exchange with reverse osmosis for enhanced recovery during inland brackish water desalination.

For inland brackish water desalination by reverse osmosis or RO, concentrate or reject disposal poses a major challenge. However, enhanced recovery and consequent reduction in the reject volume using RO processes is limited by the solubility of ions present in the feedwater. One of the most common and stubborn precipitate formed during desalination is calcium sulfate. Reducing or eliminating the presence of sulfate would allow the process to operate at higher recoveries without threat to membrane scaling. In this research, this goal is accomplished by using an appropriate mixture of self-regenerating anion exchange resins that selectively remove and replace sulfate by chloride prior to the RO unit. Most importantly, the mixed bed of anion exchange resins is self-regenerated with the reject brine from the RO process, thus requiring no addition of external chemicals. The current work demonstrates the reversibility of the hybrid ion exchange and RO (HIX-RO) process with 80% recovery for a brackish water composition representative of groundwater in San Joaquin Valley in California containing approximately 5200 mg/L of total dissolved solids or TDS. Consequently, the reject volume can be reduced by 50% without the threat of sulfate scaling and use of antiscaling chemicals can be eliminated altogether. By appropriately designing or tuning the mixed bed of anion exchange resins, the process can be extended to nearly any composition of brackish water for enhanced recovery and consequent reduction in the reject volume.

Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae.

The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite Plasmodium must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the Anopheles gambiae orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (LITAF), and its role in mosquito anti-Plasmodium immunity. An. gambiae LITAF-like 3 (LL3) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of LL3 expression greatly increases parasite survival, indicating that LL3 is part of an anti-Plasmodium defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium. Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression. We conclude that LL3 is a transcription factor capable of modulating SRPN6 expression as part of the mosquito anti-Plasmodium immune response.

Immunization against a merozoite sheddase promotes multiple invasion of red blood cells and attenuates Plasmodium infection in mice.

BACKGROUND: Subtilisin-like protease 2 (SUB2) is a conserved serine protease utilized by Plasmodium parasites as a surface sheddase required for successful merozoite invasion of host red blood cells and has been implicated in ookinete invasion of the mosquito midgut. To determine if SUB2 is a suitable vaccine target to interfere with malaria parasite development, the effects of SUB2-immunization on the Plasmodium life cycle were examined in its vertebrate and invertebrate hosts. METHODS: Swiss Webster mice were immunized with SUB2 peptides conjugated to Keyhole limpet hemocyanin (KLH) or KLH alone, and then challenged with Plasmodium berghei. To determine the effects of immunization on parasite development, infected mice were evaluated by blood film and Giemsa staining. In addition, collected immune sera were used to perform passive immunization experiments in non-immunized, P. berghei-infected mice to determine the potential role of SUB2 in parasite development in the mosquito. RESULTS: Following P. berghei challenge, SUB2-immunized mice develop a lower parasitaemia and show improved survival when compared to control immunized mice. Moreover, SUB2 immunization results in an increase in the number of multiply invaded red blood cells, suggesting that SUB2 antibodies interfere with merozoite invasion. Passive immunization experiments imply that SUB2 may not have a major role in ookinete invasion, but this requires further investigation. CONCLUSION: By interfering with red blood cell invasion, immunization against SUB2 limits malaria parasite development and confers protection from severe malaria. Together, these results provide proof-of-principle evidence for future investigation into the use of SUB2 as a vaccine or drug target to interrupt parasite development in more relevant human malaria models.

Highly parallel oligonucleotide purification and functionalization using reversible chemistry.

We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector.

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.

Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum.

INTRODUCTION: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. METHODS: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. RESULTS: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. CONCLUSION: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Delays in blood collection and drug toxicology results among crash-involved drivers arrested for impaired driving.

OBJECTIVE: The concentration of drugs in a driver's system can change between an impaired driving arrest or crash and the collection of a biological specimen for drug testing. Accordingly, delays in specimen collection can result in the loss of critical information that has the potential to affect impaired driving prosecution. The objectives of the study were: (1) to identify factors that influence the time between impaired-driving violations and specimen collections (time-to-collection) among crash-involved drivers, and (2) to consider how such delays affect measured concentrations of drugs, particularly with respect to common drug per se limits. METHOD: Study data included blood toxicology results and crash-related information from 8,923 drivers who were involved in crashes and arrested for impaired driving in Wisconsin between 2019 and 2021. Analyses examined how crash timing and severity influenced time-to-collection and the effects of delays in specimen collection on blood alcohol concentrations (BACs) and blood delta-9-tetrahydrocannabinol (THC) concentrations. RESULTS: The mean time-to-collection for the entire sample was 1.80 h. Crash severity had a significant effect on time-to-collection with crashes involving a fatality having the longest duration (M = 2.35 h) followed by injury crashes (M = 2.06 h) and noninjury crashes (M = 1.69 h). Time of day also affected time-to-collection; late night and early morning hours were associated with shorter durations. Both BAC (r = -0.11) and blood THC concentrations (r = -0.16) were significantly negatively correlated with time-to-collection. CONCLUSIONS: Crash severity and the time of day at which a crash occurs can result in delays in the collection of blood specimens after impaired driving arrests. Because drugs often continue to be metabolized and eliminated between arrest and biological specimen collection, measured concentrations may not represent the concentrations of drugs that were present at the time of driving. This has the potential to affect drug-impaired driving prosecution, particularly in jurisdictions whose laws specify per se impairment thresholds.