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Purpose: Test objects for High Intensity Focused Ultrasound (HIFU) are required for the standardization and definition of treatment, Quality Assurance (QA), comparison of results between centers and calibration of devices. This study describes a HIFU test object which provides temperature measurement as a function of time, in a reference material compatible with Magnetic Resonance (MR) and ultrasound. Materials and methods: T-Type fine wire thermocouples were used as sensors and 5 correction methods for viscous heating artifacts were assessed. The phantom was tested in a MR-HIFU Philips Sonalleve device over a period of 12months, demonstrating stability and validity to evaluate the performance of the device. Results: The study furnished useful information regarding the MR-HIFU sessions and highlighted potential limitations of the existing QA and monitoring methods. The importance of temperature monitoring along the whole acoustic path was demonstrated as MR Thermometry readings differed in the three MR plane views (coronal, sagittal, transverse), in particular when the focus was near a soft-tissue/ bone interface, where there can be an MR signal loss with significant temperature and thermal dose underestimation (138% variation between the three plane views). Conclusions: The test object was easy to use and has potential as a valid tool for training, QA, research and development for MR guided HIFU and potentially ultrasound guided devices.

Yersinia murine toxin (Ymt) is a phospholipase D encoded on a plasmid acquired by Yersinia pestis after its recent divergence from a Yersinia pseudotuberculosis progenitor. Despite its name, Ymt is not required for virulence but acts to enhance bacterial survival in the flea digestive tract. Certain Y. pestis strains circulating in the Bronze Age lacked Ymt, suggesting that they were not transmitted by fleas. However, we show that the importance of Ymt varies with host blood source. In accordance with the original description, Ymt greatly enhanced Y. pestis survival in fleas infected with bacteremic mouse, human, or black rat blood. In contrast, Ymt was much less important when fleas were infected using brown rat blood. A Y. pestis Ymt− mutant infected fleas nearly as well as the Ymt+ parent strain after feeding on bacteremic brown rat blood, and the mutant was transmitted efficiently by flea bite during the first weeks after infection. The protective function of Ymt correlated with red blood cell digestion kinetics in the flea gut. Thus, early Y. pestis strains that lacked Ymt could have been maintained in flea-brown rat transmission cycles, and perhaps in other hosts with similar blood characteristics. Acquisition of Ymt, however, served to greatly expand the range of hosts that could support flea-borne plague., Author summary The bacterium Yersinia pestis causes highly lethal bubonic plague in a wide variety of mammals and is transmitted primarily by the bites of infected fleas. During its recent evolutionary divergence from Yersinia pseudotuberculosis, a mild pathogen incapable of flea-borne transmission, Y. pestis acquired a new gene that encodes a phospholipase enzyme called Yersinia murine toxin (Ymt). This was a critical step in the transition to an insect-borne life cycle as it was reported that Ymt activity greatly enhances bacterial survival in the flea gut. Recent genomic sequencing of ancient Y. pestis strains revealed that some lacked Ymt, leading to the conclusion that these strains were not transmitted by flea bite. Here, we report that the importance of Ymt for survival in the flea is greatly dependent on host blood source. Ymt is required if fleas take up Y. pestis in mouse, human, or black rat blood, but is not required if brown rat blood is used. We conclude that ancient Y. pestis strains lacking Ymt could have circulated in certain flea-rodent transmission cycles. Acquisition of Ymt, however, enabled Y. pestis to greatly expand its host range to an ecologically broad range of mammals and their fleas.

Yersinia pestis can be transmitted by fleas during the first week after an infectious blood meal, termed early-phase or mass transmission, and again after Y. pestis forms a cohesive biofilm in the flea foregut that blocks normal blood feeding. We compared the transmission efficiency and the progression of infection after transmission by Oropsylla montana fleas at both stages. Fleas were allowed to feed on mice three days after an infectious blood meal to evaluate early-phase transmission, or after they had developed complete proventricular blockage. Transmission was variable and rather inefficient by both modes, and the odds of early-phase transmission was positively associated with the number of infected fleas that fed. Disease progression in individual mice bitten by fleas infected with a bioluminescent strain of Y. pestis was tracked. An early prominent focus of infection at the intradermal flea bite site and dissemination to the draining lymph node(s) soon thereafter were common features, but unlike what has been observed in intradermal injection models, this did not invariably lead to further systemic spread and terminal disease. Several of these mice resolved the infection without progression to terminal sepsis and developed an immune response to Y. pestis, particularly those that received an intermediate number of early-phase flea bites. Furthermore, two distinct types of terminal disease were noted: the stereotypical rapid onset terminal disease within four days, or a prolonged onset preceded by an extended, fluctuating infection of the lymph nodes before eventual systemic dissemination. For both modes of transmission, bubonic plague rather than primary septicemic plague was the predominant disease outcome. The results will help to inform mathematical models of flea-borne plague dynamics used to predict the relative contribution of the two transmission modes to epizootic outbreaks that erupt periodically from the normal enzootic background state., Author summary Yersinia pestis can be transmitted by fleas within a few days after taking a blood meal from a highly bacteremic host, termed early-phase or mass transmission; and again after it forms a dense biofilm in the foregut of its vector that can eventually block blood feeding. The relative importance of the two transmission modes in the ecology of plague is a matter of current debate, but estimates of transmission rate, efficiency, and other parameters are limited. We compared transmission and disease progression dynamics in mice bitten by groups of fleas three days after their infectious blood meal (early-phase or mass transmission mode) and in mice bitten by individual blocked fleas. In general, a higher percentage of transmissions by blocked fleas led to terminal disease, whereas early-phase transmissions more often led to survival and an immune response, which are nonproductive infections in the sense that the bacteremia required to continue the Y. pestis life cycle did not develop and these animals would be removed from the pool of susceptibles in the host population. The data will be useful in mathematical models of plague dynamics in wild rodent populations.

Over the last 20 years, advances in sequencing technologies paired with biochemical and structural studies have shed light on the unique pharmacological arsenal produced by the salivary glands of hematophagous arthropods that can target host hemostasis and immune response, favoring blood acquisition and, in several cases, enhancing pathogen transmission. Here we provide a deeper insight into Xenopsylla cheopis salivary gland contents pairing transcriptomic and proteomic approaches. Sequencing of 99 pairs of salivary glands from adult female X. cheopis yielded a total of 7432 coding sequences functionally classified into 25 classes, of which the secreted protein class was the largest. The translated transcripts also served as a reference database for the proteomic study, which identified peptides from 610 different proteins. Both approaches revealed that the acid phosphatase family is the most abundant salivary protein group from X. cheopis. Additionally, we report here novel sequences similar to the FS-H family, apyrases, odorant and hormone-binding proteins, antigen 5-like proteins, adenosine deaminases, peptidase inhibitors from different subfamilies, proteins rich in Glu, Gly, and Pro residues, and several potential secreted proteins with unknown function. SIGNIFICANCE: The rat flea X. cheopis is the main vector of Yersinia pestis, the etiological agent of the bubonic plague responsible for three major pandemics that marked human history and remains a burden to human health. In addition to Y. pestis fleas can also transmit other medically relevant pathogens including Rickettsia spp. and Bartonella spp. The studies of salivary proteins from other hematophagous vectors highlighted the importance of such molecules for blood acquisition and pathogen transmission. However, despite the historical and clinical importance of X. cheopis little is known regarding their salivary gland contents and potential activities. Here we provide a comprehensive analysis of X. cheopis salivary composition using next generation sequencing methods paired with LC-MS/MS analysis, revealing its unique composition compared to the sialomes of other blood-feeding arthropods, and highlighting the different pathways taken during the evolution of salivary gland concoctions. In the absence of the X. cheopis genome sequence, this work serves as an extended reference for the identification of potential pharmacological proteins and peptides present in flea saliva.

Background Transmission of Yersinia pestis by flea bite can occur by two mechanisms. After taking a blood meal from a bacteremic mammal, fleas have the potential to transmit the very next time they feed. This early-phase transmission resembles mechanical transmission in some respects, but the mechanism is unknown. Thereafter, transmission occurs after Yersinia pestis forms a biofilm in the proventricular valve in the flea foregut. The biofilm can impede and sometimes completely block the ingestion of blood, resulting in regurgitative transmission of bacteria into the bite site. In this study, we compared the relative efficiency of the two modes of transmission for Xenopsylla cheopis, a flea known to become completely blocked at a high rate, and Oropsylla montana, a flea that has been considered to rarely develop proventricular blockage. Methodology/Principal findings Fleas that took an infectious blood meal containing Y. pestis were maintained and monitored for four weeks for infection and proventricular blockage. The number of Y. pestis transmitted by groups of fleas by the two modes of transmission was also determined. O. montana readily developed complete proventricular blockage, and large numbers of Y. pestis were transmitted by that mechanism both by it and by X. cheopis, a flea known to block at a high rate. In contrast, few bacteria were transmitted in the early phase by either species. Conclusions A model system incorporating standardized experimental conditions and viability controls was developed to more reliably compare the infection, proventricular blockage and transmission dynamics of different flea vectors, and was used to resolve a long-standing uncertainty concerning the vector competence of O. montana. Both X. cheopis and O. montana are fully capable of transmitting Y. pestis by the proventricular biofilm-dependent mechanism., Author Summary The ecology of plague is complex and its epidemiology is enigmatic. Many different flea species are able to transmit Yersinia pestis, the plague bacillus, and they can transmit in two different ways. Early-phase transmission can occur during the first week after a flea has fed on a diseased animal. Thereafter, transmission occurs only as bacterial growth in the flea foregut interferes with and eventually blocks blood feeding. Comparisons of the relative ability of different flea vectors to transmit have been problematic, and contradictory results have been reported for the ability of the ground squirrel flea Oropsylla montana to transmit beyond the early phase. Our results show that O. montana readily develops foregut blockage, and transmission by that mechanism was as good as or better than observed for Xenopsylla cheopis, a flea known to block at a high rate. In contrast, very few bacteria were transmitted in the early phase by either of these fleas compared to later times after infection, suggesting that early-phase transmission is pertinent only to highly susceptible animals. Improved characterization of the transmission patterns of different flea vectors will aid in modeling plague incidence in its various natural settings.

SummaryYersinia pestis is an arthropod-borne bacterial pathogen that evolved recently from Yersinia pseudotuberculosis, an enteric pathogen transmitted via the fecal-oral route. This radical ecological transition can be attributed to a few discrete genetic changes from a still-extant recent ancestor, thus providing a tractable case study in pathogen evolution and emergence. Here, we determined the genetic and mechanistic basis of the evolutionary adaptation of Y. pestis to flea-borne transmission. Remarkably, only four minor changes in the bacterial progenitor, representing one gene gain and three gene losses, enabled transmission by flea vectors. All three loss-of-function mutations enhanced cyclic-di-GMP-mediated bacterial biofilm formation in the flea foregut, which greatly increased transmissibility. Our results suggest a step-wise evolutionary model in which Y. pestis emerged as a flea-borne clone, with each genetic change incrementally reinforcing the transmission cycle. The model conforms well to the ecological theory of adaptive radiation.

Total knee arthroplasty is a common intervention and it is usually followed by a rehabilitation period, to recover physical functioning. The aim of this study is to evaluate the changes of gait kinematic parameters due to rehabilitation after total knee arthroplasty. After inclusion and exclusion criteria the subjects were evaluated with Barthel Index (BI), International Knee Society Score (IKSS), Knee Flexion (KF), NRS and Instrumental Gait Analysis (GA), at admission (T0) and discharge (T1). Many parameters show a significant improvement during rehabilitation IKSS, BI, KF (P 0.05), all the significant evaluation scales and gait analysis parameters had a significant clinical effect size (Cohen’s d > 0.8). The rehabilitation process seems to be effective in improving gait parameters and knee functioning.

A hallmark of Yersinia pestis infection is a delayed inflammatory response early in infection. In this study, we use an intradermal model of infection to study early innate immune cell recruitment. Mice were injected intradermally in the ear with wild-type (WT) or attenuated Y. pestis lacking the pYV virulence plasmid (pYV − ). The inflammatory responses in ear and draining lymph node samples were evaluated by flow cytometry and immunohistochemistry. As measured by flow cytometry, total neutrophil and macrophage recruitment to the ear in WT-infected mice did not differ from phosphate-buffered saline (PBS) controls or mice infected with pYV − , except for a transient increase in macrophages at 6 h compared to the PBS control. Limited inflammation was apparent even in animals with high bacterial loads (10 5 to 10 6 CFU). In addition, activation of inflammatory cells was significantly reduced in WT-infected mice as measured by CD11b and major histocompatibility complex class II (MHC-II) expression. When mice infected with WT were injected 12 h later at the same intradermal site with purified LPS, Y. pestis did not prevent recruitment of neutrophils. However, significant reduction in neutrophil activation remained compared to that of PBS and pYV − controls. Immunohistochemistry revealed qualitative differences in neutrophil recruitment to the skin and draining lymph node, with WT-infected mice producing a diffuse inflammatory response. In contrast, focal sites of neutrophil recruitment were sustained through 48 h postinfection in pYV − -infected mice. Thus, an important feature of Y. pestis infection is reduced activation and organization of inflammatory cells that is at least partially dependent on the pYV virulence plasmid.

2 and 3 were observed in 43%, 47% and 10% of these specimens. Leucocyte subpopulations were categorized focal vs. diffuse monocytic (11%/24%), focal vs. diffuse granulocytic (4%/4%), or focal vs. diffuse mixed (14%/20%). In 23% of the cases ptc was not classified (e.g. vicinity of tubular necrosis, pyelonephritis or infarct). Ptc scores (1, 2 or 3) were associated with both diagnosis of AMR and cellular rejection (ACR). Diffuse monocytic or mixed ptc (OR=4.51, 95% CI 1.82-11.16, and OR=6.8, 2.44-19.36, p≤0.001), but not granulocytic ptc (OR=3.81, 95% CI 0.41-35.02, p=0.2) were associated with AMR. At the same time, diffuse ptc was associated with ACR, regardless of its composition (monocytic: OR=6.1, 95%CI 3.61-12.44; granulocytic: OR=6.69, 1.58-28.41; mixed: OR=8.05, 4.06-15.96; p≤0.01). In uni- and multivariate analysis ptc 3 (OR=2.56, 95%CI 1.24-1.96, p=0.011), diffuse monocytic or mixed ptc (OR=1.75, 95%CI 1.01-3.06, p=0.048 and OR=1.75, 1.03-3.26, p=0.04) were risk factors for inferior graft survival. Conclusions: Our results underscore a high relevance of the detailed qualitative and quantitative characterization of peritubular capillaritis in indication biopsies as a predictor for inferior clinical outcomes.

Fleas can transmit Yersinia pestis by two mechanisms, early-phase transmission (EPT) and biofilm-dependent transmission (BDT). Transmission efficiency varies among flea species and the results from different studies have not always been consistent. One complicating variable is the species of rodent blood used for the infectious blood meal. To gain insight into the mechanism of EPT and the effect that host blood has on it, fleas were fed bacteremic mouse, rat, guinea pig, or gerbil blood; and the location and characteristics of the infection in the digestive tract and transmissibility of Y. pestis were assessed 1 to 3 days after infection. Surprisingly, 10–28% of two rodent flea species fed bacteremic rat or guinea pig blood refluxed a portion of the infected blood meal into the esophagus within 24 h of feeding. We term this phenomenon post-infection esophageal reflux (PIER). In contrast, PIER was rarely observed in rodent fleas fed bacteremic mouse or gerbil blood. PIER correlated with the accumulation of a dense mixed aggregate of Y. pestis, red blood cell stroma, and oxyhemoglobin crystals that filled the proventriculus. At their next feeding, fleas with PIER were 3–25 times more likely to appear partially blocked, with fresh blood retained within the esophagus, than were fleas without PIER. Three days after feeding on bacteremic rat blood, groups of Oropsylla montana transmitted significantly more CFU than did groups infected using mouse blood, and this enhanced transmission was biofilm-dependent. Our data support a model in which EPT results from regurgitation of Y. pestis from a partially obstructed flea foregut and that EPT and BDT can sometimes temporally overlap. The relative insolubility of the hemoglobin of rats and Sciurids and the slower digestion of their blood appears to promote regurgitative transmission, which may be one reason why these rodents are particularly prominent in plague ecology., Author summary Yersinia pestis, the bacterial agent of plague, is transmitted by fleas that feed on blood from rodents that carry this disease. The conclusions from studies comparing how efficiently fleas transmit plague after becoming infected have been inconsistent, possibly because a variety of rodent blood sources have been used. To investigate this, we infected three different flea species with Y. pestis using four different types of rodent blood and compared how well they could transmit three days later. The two rodent flea species that transmitted efficiently tended to reflux bacteria and blood into their esophagus when rat or guinea pig blood was used for the infections, but not when mouse or gerbil blood was used. This reflux phenomenon appears to be related to the solubility of the hemoglobin molecule of different rodent species. In contrast, cat fleas, inefficient transmitters, never refluxed their infected blood meal into the esophagus. Rodent fleas that were infected using reflux-inducing rat blood transmitted more Y. pestis than those that fed on infected mouse blood. These findings improve our understanding of how fleas transmit Y. pestis soon after becoming infected and suggest a reason why certain rodents figure more prominently in plague ecology than others.

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed ≤2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 103 p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 103 p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

A population genetic analysis of gene flow was conducted among 619 Aedes aegypti from nine collections distributed among six geographic regions of Venezuela. Genetic markers included a 387-basepair region of the mitochondrial NADH dehydrogenase 4 (ND4) gene and single nucleotide polymorphisms (SNPs) at 11 nuclear loci. Genotypes at SNP loci were identified using melting curve analysis. Six different ND4 haplotypes were detected and patterns of variation suggested that collections were isolated by distance. The variance in SNP allele frequencies was much less than the variance in haplotype frequencies and a pattern of isolation by distance was not detected. Aedes aegypti from eight collections were orally challenged with dengue 2 virus. Disseminated infection rates ranged from 77% to 95%. The percentage of mosquitoes exhibiting a midgut infection barrier ranged from 2% to 15%, and those exhibiting a midgut escape barrier ranged from 2% to 18%. Venezuelan Ae. aegypti appear to be susceptible to dengue virus infection.

Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis., Author Summary Flea-borne transmission is central to the natural history of the plague bacillus Yersinia pestis, and infection within the context of flea feeding may affect the pathogenesis of bubonic plague. We analyzed the mammalian host response to Y. pestis in the skin immediately after transmission by its natural vector, the rat flea Xenopsylla cheopis, to observe differences relative to the response to needle-inoculated bacteria. Our results show that uninfected flea bites induce minimal inflammation, but flea-transmitted Y. pestis cause the recruitment of neutrophils roughly in proportion to the number of bacteria deposited in the skin. We observed interactions of flea-transmitted bacteria with macrophages, a cell type much more permissive than neutrophils for survival and growth of Y. pestis. We found that dendritic cells, important sentinel antigen presenting cells, were recruited to, but had minimal interaction with, flea-transmitted bacteria. Additionally, we found that Y. pestis could disseminate from the flea bite site to the draining lymph node and spleen as early as 1 h after flea feeding, significantly earlier than has been previously reported. This study reveals important differences between needle-inoculated and flea-transmitted Y. pestis in the immediate host response to infection and improves our understanding of the early host-bacterium interactions in plague pathogenesis.

A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km 2 . Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.

A hierarchical population genetic study was conducted among 19 Aedes aegypti populations in Thailand from Chiang Mai in the north to Songkhla province in the south. Single-strand conformation polymorphism analysis was used to examine variation in a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial DNA gene (ND4). Seven haplotypes were detected in two lineages previously identified in ND4 haplotypes from North America. Gene flow estimates and highly significant variation among populations within 25 kilometers implicated genetic drift and vector control efforts as major factors in genetic structure. Mantel regression analysis demonstrated no isolation by distance. Urban areas were relatively panmictic, while suburban/rural sites exhibited more restricted gene flow. Significant genetic structure among groups of collections > 100 kilometers apart is consistent with recent (approximately 50 year) expansion of Ae. aegypti from highly populated areas accompanied by founder effects, but could also reflect the overall low genetic diversity in ND4 in Thailand.

BACKGROUND:Transmission of Yersinia pestis by flea bite can occur by two mechanisms. After taking a blood meal from a bacteremic mammal, fleas have the potential to transmit the very next time they feed. This early-phase transmission resembles mechanical transmission in some respects, but the mechanism is unknown. Thereafter, transmission occurs after Yersinia pestis forms a biofilm in the proventricular valve in the flea foregut. The biofilm can impede and sometimes completely block the ingestion of blood, resulting in regurgitative transmission of bacteria into the bite site. In this study, we compared the relative efficiency of the two modes of transmission for Xenopsylla cheopis, a flea known to become completely blocked at a high rate, and Oropsylla montana, a flea that has been considered to rarely develop proventricular blockage. METHODOLOGY/PRINCIPAL FINDINGS:Fleas that took an infectious blood meal containing Y. pestis were maintained and monitored for four weeks for infection and proventricular blockage. The number of Y. pestis transmitted by groups of fleas by the two modes of transmission was also determined. O. montana readily developed complete proventricular blockage, and large numbers of Y. pestis were transmitted by that mechanism both by it and by X. cheopis, a flea known to block at a high rate. In contrast, few bacteria were transmitted in the early phase by either species. CONCLUSIONS:A model system incorporating standardized experimental conditions and viability controls was developed to more reliably compare the infection, proventricular blockage and transmission dynamics of different flea vectors, and was used to resolve a long-standing uncertainty concerning the vector competence of O. montana. Both X. cheopis and O. montana are fully capable of transmitting Y. pestis by the proventricular biofilm-dependent mechanism.

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F1 intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F2 offspring were analyzed for midgut infection and escape barriers. In P1 and F1 parents and in 207 F2 individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I–III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)n microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F2 females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for ∼30% of the phenotypic variance (σp2) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (σg2). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.

A linkage map of the Asian tiger mosquito [Aedes (Stegomyia) albopictus (Skuse)] was constructed in an F1 intercross by monitoring the segregation of randomly amplified polymorphic DNA (RAPD) markers analyzed for single-strand conformation polymorphisms (SSCP). We hypothesized that SSCP analysis would reveal point mutations in RAPD fragments that would then segregate as codominant rather than dominant markers which are typically revealed through routine RAPD analysis. Markers were mapped to individual chromosomes by testing for cosegregation with Sex (chromosome I) or a polymorphism at the a-GPD allozyme locus (chromosome II). All other markers that cosegregated were assigned to chromosome III. Six RAPD primers amplified 68 polymorphic markers that segregated in a Mendelian fashion and were mapped. Contrary to our hypothesis, no codominant SSCP polymorphisms were detected, but fractionation of RAPD products on polyacrylamide gels and detection through silver staining proved to be a sensitive technique that allowed us to identify more markers than the standard analysis of RAPD PCR products on agarose gels.

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leaf hoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.

Five geographical strains of Aedes albopictus (Skuse) were compared for their ability to transmit vertically a dengue-1 isolate from Jamaica. The OAHU strain of Ae. albopictus and a strain of Aedes aegypti (L.) from the United States were included as controls. The offspring of orally infected females were assayed individually for vertical infection. Vertical transmission rates among strains ranged from 11 to 41%, and filial infection rates of strains ranged from 0.5 to 2.9%. Filial infection rates of individual positive families within strains ranged from 1.4 to 17.4%. These rates were higher than those previously recorded for Ae. albopictus. The observed differences in rates of vertical transmission among the strains were not statistically significant, because 95% of the measured variation was attributed to families within strains. The most significant source of variation in vertical transmission of dengue-1 by Ae. albopictus was at the individual level.

Background Aedes aegypti, the “yellow fever mosquito”, is the primary vector to humans of the four serotypes of dengue viruses (DENV1-4) and yellow fever virus (YFV) and is a known vector of Chikungunya virus. There are two recognized subspecies of Ae. aegypti sensu latu (s.l.): the presumed ancestral form, Ae. aegypti formosus (Aaf), a primarily sylvan mosquito in sub-Saharan Africa, and Ae. aegypti aegypti (Aaa), found globally in tropical and subtropical regions typically in association with humans. The designation of Ae. aegypti s.l. subspecies arose from observations made in East Africa in the late 1950s that the frequency of pale “forms” of Ae. aegypti was higher in populations in and around human dwellings than in those of the nearby bush. But few studies have been made of Ae. aegypti s.l. in West Africa. To address this deficiency we have been studying the population genetics, subspecies composition and vector competence for DENV-2 of Ae. aegypti s.l. in Senegal. Methods and Findings A population genetic analysis of gene flow was conducted among 1,040 Aedes aegypti s.l. from 19 collections distributed across the five phytogeographic regions of Senegal. Adults lacking pale scales on their first abdominal tergite were classified as Aedes aegypti formosus (Aaf) following the original description of the subspecies and the remainder were classified as Aedes aegypti aegypti (Aaa). There was a clear northwest–southeast cline in the abundance of Aaa and Aaf. Collections from the northern Sahelian region contained only Aaa while southern Forest gallery collections contained only Aaf. The two subspecies occurred in sympatry in four collections north of the Gambia in the central Savannah region and Aaa was a minor component of two collections from the Forest gallery area. Mosquitoes from 11 collections were orally challenged with DENV-2 virus. In agreement with the early literature, Aaf had significantly lower vector competence than Aaa. Among pure Aaa collections, the disseminated infection rate (DIR) was 73.9% with a midgut infection barrier (MIB) rate of 6.8%, and a midgut escape barrier (MEB) rate of 19.3%, while among pure Aaf collections, DIR = 34.2%, MIB rate = 7.4%, and MEB rate = 58.4%. Allele and genotype frequencies were analyzed at 11 nuclear single nucleotide polymorphism (SNP) loci using allele specific PCR and melting curve analysis. In agreement with a published isozyme gene flow study in Senegal, only a small and statistically insignificant percentage of the variance in allele frequencies was associated with subspecies. Conclusions These results add to our understanding of the global phylogeny of Aedes aegypti s.l., suggesting that West African Aaa and Aaf are monophyletic and that Aaa evolved in West Africa from an Aaf ancestor., Author Summary We conducted a population genetic study with 1,040 Aedes aegypti sensu latu (s.l.) collected from 19 sites distributed across the five phytogeographic regions of Senegal. Adult mosquitoes without pale scales on their first abdominal tergite were classified as Aedes aegypti formosus (Aaf) and those having pale scales as Aedes aegypti aegypti (Aaa). We found the two forms distributed along a northwest–southeast cline. Northern Sahelian collections contained only Aaa while the southern Forest gallery collections consisted of only Aaf. The two subspecies were sympatric in four collections north of The Gambia. Aaa was a minor component of two collections from the Forest gallery area. Eleven of these collections were fed a dengue-2 virus–infected bloodmeal. Consistent with the early literature, Aaf had lower vector competence than Aaa. In agreement with a recently published isozyme gene flow study in Senegal, analyzes of allele frequencies indicated only a small, nonsignificant percentage of the variance associated with subspecies. These results improve our understanding of the global phylogeny of Aedes aegypti s.l., suggesting that West African Aaa and Aaf are monophyletic and that Aaf, the black “sylvan” species, is the ancestor of Aaa, the lighter “domestic” species in West Africa.

The effects of the defensive chemistry of Solidago altissima on the cabbage looper, Trichoplusia ni (Hubner), were shown using artificial diet bioassays. Lyophilized S. altissima leaves were incorporated into an artificial diet. Leaf powder also was extracted in hexane, methanol, and water, and the extracts and postextraction residue were incorporated separately into artificial diets. The control contained cellulose powder in place of leaf material. Also, larvae were grown on live, intact S. altissima plants. The mortality rate was 100% in the whole-leaf powder and methanol extract diets, 97.2% in the hexane extract diet, and 96% in the live-plant experiment. Mortality on the water extract diet (12%) and on the residue-containing diet (13%) was lower than that on the cellulose powder control (17.4%). S. altissima contains chemicals that severely reduce larval survivorship of T. ni , but once these chemicals are removed through extraction, the larvae are able to use the nutrients contained in the leaves. These nutrients increase survivorship to adulthood above that on the cellulose control hut do not significantly affect time to pupation, pupal mass, or fecundity.

1. 1. A Drosophila pseudoobscura amylase gene cloned in Escherichia coli is expressed at high levels. The expression of this gene is repressed when glucose (0.5% final concentration) is added to a starch minimal medium culture of E. coli cells containing the amylase plasmid. 2. 2. Addition of anthranilic acid (5 and 7 mM final exogenous concentration) to catabolite repressed cells mimics the action of adenosine 3'5' cyclic monophosphate (cAMP) by derepressing the expression of the amylase. 3. 3. The results suggest that anthranilic acid acts either indirectly, possibly through the glucose transport system, or directly, by way of an intercalative model of initiation, to alter the levels of transcription.

A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km(2). Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.

A hierarchical population genetic study was conducted among 19 Aedes aegypti populations in Thailand from Chiang Mai in the north to Songkhla province in the south. Single-strand conformation polymorphism analysis was used to examine variation in a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial DNA gene (ND4). Seven haplotypes were detected in two lineages previously identified in ND4 haplotypes from North America. Gene flow estimates and highly significant variation among populations within 25 kilometers implicated genetic drift and vector control efforts as major factors in genetic structure. Mantel regression analysis demonstrated no isolation by distance. Urban areas were relatively panmictic, while suburban/rural sites exhibited more restricted gene flow. Significant genetic structure among groups of collections100 kilometers apart is consistent with recent (approximately 50 year) expansion of Ae. aegypti from highly populated areas accompanied by founder effects, but could also reflect the overall low genetic diversity in ND4 in Thailand.

A quantitative genetic study of the ability of Aedes aegypti to propagate dengue-2 (DEN-2) virus in the midgut and in a disseminated infection in the head was conducted with a standard half-sib breeding design. Aedes aegypti aegypti and A. aegypti formosus differ markedly in oral susceptibility to DEN-2 virus. Mosquitoes were orally infected and, after an extrinsic incubation period of 14 days, virus titer (by tissue culture infectious dose, 50% endpoint) was determined in the midgut (MT) and head (HT). Body size as measured by wing length was not significantly different between infected and uninfected mosquitoes and was not correlated with MT or HT The heritability for MT in both subspecies was 0.41 and was 0.39 for HT in A. aegypti formosus. In A. aegypti aegypti, HT appeared to be controlled by dominant alleles. The MT was not correlated with HT nor did MT determine whether virus disseminated out of the midgut. These results suggest that it is the barriers to infection and dissemination, independent of virus titer, that determine vector competence for DEN-2 virus.

Culiseta (Culicella) morsitans is reported for the first time from Colorado. Collections from the Rocky Mountains in the northern part of the state extend the range of this species approximately 300 km south and east of previous records.