RAD sequencing and genomic simulations resolve hybrid origins within North American Canis.

Top predators are disappearing worldwide, significantly changing ecosystems that depend on top-down regulation. Conflict with humans remains the primary roadblock for large carnivore conservation, but for the eastern wolf (Canis lycaon), disagreement over its evolutionary origins presents a significant barrier to conservation in Canada and has impeded protection for grey wolves (Canis lupus) in the USA. Here, we use 127,235 single-nucleotide polymorphisms (SNPs) identified from restriction-site associated DNA sequencing (RAD-seq) of wolves and coyotes, in combination with genomic simulations, to test hypotheses of hybrid origins of Canis types in eastern North America. A principal components analysis revealed no evidence to support eastern wolves, or any other Canis type, as the product of grey wolf × western coyote hybridization. In contrast, simulations that included eastern wolves as a distinct taxon clarified the hybrid origins of Great Lakes-boreal wolves and eastern coyotes. Our results support the eastern wolf as a distinct genomic cluster in North America and help resolve hybrid origins of Great Lakes wolves and eastern coyotes. The data provide timely information that will shed new light on the debate over wolf conservation in eastern North America.

The deposition of strontium-substituted hydroxyapatite coatings.

Strontium substituted hydroxyapatite (SrHA) coatings have received a lot of interest recently as strontium (Sr) has been shown to have the dual benefit of promoting bone formation and reducing bone resorption, in vivo. In this work, SrHA coatings were deposited onto polycrystalline titanium surfaces using radio frequency (RF) magnetron co-sputtering and compared to those deposited from HA alone. In particular, the influence of different levels of Sr-substitution of the sputtering targets (5 and 13% Sr-substituted HA targets) on the properties of the deposited coatings produced at a low discharge power level (150 W) were investigated using FTIR, XPS, XRD, ToFSIMS and AFM techniques (both before and after annealing at 500 °C). The results show that Sr could be successfully incorporated into the HA lattice to form SrHA coatings and that they contained no other impurities. However, the coating produced from the 13% Sr-substituted target had a higher Ca+Sr/P ratio (1.95±0.14) and Sr content when compared to the coating produced from the 5% Sr-substituted target (1.58±0.20). The deposition rate also decreased with increasing Sr content of the sputtering targets. Furthermore, as the Sr content of the coatings increased, so did the preferred 002 orientation of the coating along with increased surface roughness and heterogeneity of the surface features. Therefore, this study has shown that RF magnetron sputtering offers a means to control attendant properties of Sr-substituted HA, such as the crystallinity, stoichiometry, phase purity and surface topography.

Strontium-substituted hydroxyapatite coatings deposited via a co-deposition sputter technique.

The bioactivity of hydroxyapatite (HA) coatings can be modified by the addition of different ions, such as silicon (Si), lithium (Li), magnesium (Mg), zinc (Zn) or strontium (Sr) into the HA lattice. Of the ions listed here, strontium substituted hydroxyapatite (SrHA) coatings have received a lot of interest recently as Sr has been shown to promote osteoblast proliferation and differentiation, and reduce osteoclast activity. In this study, SrHA coatings were deposited onto titanium substrates using radio frequency (RF) magnetron co-sputtering (and compared to those surfaces deposited from HA alone). FTIR, XPS, XRD, and SEM techniques were used to analyse the different coatings produced, whereby different combinations of pure HA and 13% Sr-substituted HA targets were investigated. The results highlight that Sr could be successfully incorporated into the HA lattice to form SrHA coatings. It was observed that as the number of SrHA sputtering targets in the study were increased (increasing Sr content), the deposition rate decreased. It was also shown that as the Sr content of the coatings increased, so did the degree of preferred 002 orientation of the coating (along with obvious changes in the surface morphology). This study has shown that RF magnetron sputtering (specifically co-sputtering), offers an appropriate methodology to control the surface properties of Sr-substituted HA, such as the crystallinity, stoichiometry, phase purity and surface morphology.

Genetic differentiation of eastern wolves in Algonquin Park despite bridging gene flow between coyotes and grey wolves.

Distinguishing genetically differentiated populations within hybrid zones and determining the mechanisms by which introgression occurs are crucial for setting effective conservation policy. Extensive hybridization among grey wolves (Canis lupus), eastern wolves (C. lycaon) and coyotes (C. latrans) in eastern North America has blurred species distinctions, creating a Canis hybrid swarm. Using complementary genetic markers, we tested the hypotheses that eastern wolves have acted as a conduit of sex-biased gene flow between grey wolves and coyotes, and that eastern wolves in Algonquin Provincial Park (APP) have differentiated following a history of introgression. Mitochondrial, Y chromosome and autosomal microsatellite genetic data provided genotypes for 217 canids from three geographic regions in Ontario, Canada: northeastern Ontario, APP and southern Ontario. Coyote mitochondrial DNA (mtDNA) haplotypes were common across regions but coyote-specific Y chromosome haplotypes were absent; grey wolf mtDNA was absent from southern regions, whereas grey wolf Y chromosome haplotypes were present in all three regions. Genetic structuring analyses revealed three distinct clusters within a genetic cline, suggesting some gene flow among species. In APP, however, 78.4% of all breeders and 11 of 15 known breeding pairs had assignment probability of Q0.8 to the Algonquin cluster, and the proportion of eastern wolf Y chromosome haplotypes in APP breeding males was higher than expected from random mating within the park (P<0.02). The data indicate that Algonquin wolves remain genetically distinct despite providing a sex-biased genetic bridge between coyotes and grey wolves. We speculate that ongoing hybridization within the park is limited by pre-mating reproductive barriers.

Evaluation of an in vitro bloodfeeding system for testing mosquito repellents.

Median effective doses and 95% effective doses of 9 commercial mosquito repellents were determined for the yellow fever mosquito, Aedes aegypti, in an in vitro bloodfeeding test system and on the human forearm. Results obtained in the 2 test systems did not differ significantly but, because of the inherent variability of repellent test data, did not always agree closely. Potential modifications of in vitro bloodfeeding test systems for increased accuracy, precision, and reliability are discussed.

Dopaminergic system modulation of nociceptive response in long-term diabetic rats.

The present study examines the effects of dopaminergic system modulation on nociceptive response time in male diabetic rats. In this study, diabetes was induced by streptozotocin (STZ, 45 mg/kg) in adult male Sprague-Dawley rats. Insulin replacement therapy was initiated 6 weeks after the induction of diabetes for one-half of the diabetic group (1.5-2.5 IU/12 h/rat) and was continued throughout the duration of the study (up to 14 weeks). After 6 weeks of daily insulin replacement therapy, eight rats from each experimental group (STZ-diabetic, STZ-diabetic+insulin and nondiabetic control) were injected with either bromocriptine (BROM, 3 mg/kg/12 h), haloperidol (HALO, 1.5 mg/kg/12 h) or vehicle. Nociceptive response was measured by the hot plate (HP) latency test before the induction of diabetes (baseline), every 3 weeks for the first 12 weeks and then on days 5, 9 and 14 of treatment with dopaminergic agents. Animals were sacrificed 3 or 4 days after the last HP test and the brain, blood, spinal cord (SC), pituitary and adrenal glands (AD) were dissected for Met-enkephalin (ME) assay. The results show that nociceptive response of untreated diabetic animals increased gradually and significantly over the duration of this study. Administration of BROM and HALO significantly decreased and increased the nociceptive response, respectively, in all groups. However, the response of the diabetic group was more pronounced than that of the other two groups, especially for those treated with BROM. Daily insulin administration normalized nociceptive response to that of the nondiabetic controls. Diabetic animals receiving insulin replacement+BROM also showed normalized nociceptive response while the diabetic animals+HALO did not. Moreover, the administration of HALO and BROM resulted in an increase and decrease ME concentrations, respectively, in most tissues and brain regions examined. The effect of these dopaminergic agents on ME levels was greater in brain regions and tissues of the diabetic rats than in the diabetic groups receiving vehicle or in the nondiabetic control receiving these two agents. These data suggest that diabetes alters the sensitivity of the dopaminergic receptors and that altered response of the dopaminergic system could be indirectly involved in the modulation of nociception in diabetic rats possibly through the enhancement and/or deactivation of the endogenous Met-enkephalinergic system.

Variation in the protection periods of repellents on individual human subjects: an analytical review.

Mosquito repellent test data from the literature were analyzed to estimate mean protection periods and among-subjects standard deviations. Standard deviations were a linear function of the means. Numbers of subjects needed to determine mean protection periods of 1-8 h with confidence limits of +/- 0.5, 1.0, 1.5, and 2.0 h at the 99 and 95% levels of confidence were computed from regression values of the standard deviation, and a table of sample sizes was constructed for use in planning repellent tests.

Development of an efficient method to produce uniformly haploid parthenogenones.

Bovine ovaries (paired by cow) were obtained from a local abattoir and cumulus oocyte complexes were aspirated within six hours of slaughter. Two methods for activation [(1) calcium ionophore (ionomycin) alone (n = 191); and (2) ionomycin followed by the protein synthesis inhibitor cycloheximide (n = 207)] were evaluated for production of bovine parthenogenones. Activation with ionomycin alone resulted in a development rate of 33%, while activation with ionomycin and cycloheximide sequentially resulted in a development rate to two-cell stage of 49%. A procedure was developed to expedite accurate evaluation of activated oocytes for uniformly haploid development. Uniformly haploid parthenogenones that cleaved at least once in four days of in vitro culture were individually prepared for genetic analysis. Three techniques: (1) phosphate buffered saline; (2) TL-HEPES with 0.2% ovine serum albumin; and (3) TL-HEPES with 0.2% polyvinyl pyrrolidone were compared to harvest parthenogenones for genetic analysis. The only effective method that did not create spurious results during later genetic analysis was TL-HEPES with 0.2% polyvinyl pyrrolidone. Based on the results of this study, we estimate that an average of 5-7 uniformly haploid bovine parthenogenones can be realized from each donor (using pairs of ovaries). These parthenogenones, when maintained as family units, will be valuable for accomplishment of female-specific genetic linkage analysis.

Responses of male and female mosquitoes to repellents in the World Health Organization insecticide irritability test system.

A study was conducted to compare responses of male and female Aedes aegypti (Linn.) and Aedes taeniorhynchus (Wiedemann) to 9 olfactory repellents in the World Health Organization insecticide irritability test system. An irritant insecticide (permethrin) and a control were included for comparison. Aedes aegypti exhibited significantly more takeoffs than Ae. taeniorhynchus, and female mosquitoes exhibited significantly more takeoffs than males. Permethrin induced significantly more takeoffs than the control, but olfactory repellents did not. Certain 2- and 3-factor interactions of test materials, species, and sexes were statistically significant. This study supports a previous conclusion that the World Health Organization test method does not measure contact repellency (irritancy) and olfactory repellency equally.

Effect of skin abrasions on the efficacy of the repellent deet against Aedes aegypti.

Abrasion of repellent-treated human skin affected the efficacy of a sustained-release insect repellent containing N,N-diethyl-3-methylbenzamide (deet) against bites of Aedes aegypti. Skin treated with repellent when abraded up to 30 times showed significantly lower protection than unabraded skin against mosquito bites for 10 h. The mean value of the kinetic coefficient of friction during skin abrasion by clothing (battle dress uniform fabric) for repellent-treated skin (0.159 +/- 0.003) was significantly higher than untreated skin (0.122 +/- 0.005). Repellent-treated skin appeared stickier than the untreated skin. An increase in the number of skin abrasions by clothing resulted in a reduced duration of protection against mosquito bites.

U.S. Army soldiers' perceptions of arthropod pests and their effects on military missions.

A survey was conducted to determine the effects of biting and stinging arthropods on military personnel, operations, and training. Nearly 70% of respondents reported experiencing problems attributable to arthropods. Arthropods obstructed movement and field position, prevented concealment and cover, disrupted maneuvers, and caused panic. Twenty percent of respondents reported attendance at sick call for treatment of bites or stings, and 4% were hospitalized or assigned to quarters. Median lost time was 2 days. Bee, wasp, and ant stings and spider and chigger bites were the most frequent causes of lost time. Additional training on biting and stinging arthropods, use of repellents and other personal protective measures, first aid for bites and stings, and conditions requiring medical attention is needed in field units to enhance mission performance and reduce time lost because of arthropods. Materials for treatment of bites and stings should be included in first-aid kits issued for field use.

Evolution of repellent tolerances in representative arthropods.

Eight commercial insect repellents were tested against Ornithodoros parkeri (Acari: Argasidae), Dermacentor variabilis (Acari: Ixodidae), Aedes aegypti (Diptera: Culicidae), and Xenopsylla cheopis (Siphonaptera: Pulicidae). Patterns of tolerance to the test materials were distinctive for each test species. Levels of tolerance were coded as character state 0 (sensitive), 1 (intermediate), or 2 (tolerant) and mapped on a cladogram reflecting the accepted classification of the test species. Character state 0 was regarded as primitive, as indicated by the ontology of repellent tolerances in ticks. Aedes aegypti was least evolved and X. cheopis was most evolved in tolerance to repellents. Multiple parallelism of the arachnid and X. cheopis lines occurred in the evolution of the observed tolerances.

Endogenous opioid peptide mediation of hypoalgesic response in long-term diabetic rats.

This study was designed to examine the role of endogenous opioid peptide mediation of elevated pain threshold in adult male Sprague-Dawley rats with long-term diabetes mellitus induced by streptozotocin (STZ). Diabetes resulted in a significant elevation in pain threshold as measured by the tail-flick and/or hotplate latency tests. The hypoalgesic response in diabetic rats to hotplate testing developed gradually over a 4-6 week period after a transient hyperalgesia during the first two weeks of diabetes. The elevation of pain threshold achieved peak level by the fourth week after STZ administration, and remained at that level throughout the experimental period (up to 13 weeks). This hypoalgesic state in diabetic animals is thought to be mediated by opioid receptors (i.e. mu and delta). The involvement of the mu receptor is supported by the effect of naltrexone on the STZ-diabetic rats; naltrexone significantly attenuated the increase in tail-flick and hotplate latencies, compared to that of the non-diabetic controls. Furthermore, the concentration of native (free) Met-enkephalin in the spinal cord of STZ-diabetic rats was about 5-fold higher than that of non-diabetic animals. Such high levels of Met-enkephalin suggest the involvement of delta opioid receptors in the hypoalgesic response observed in STZ-diabetic rats. Seven weeks of insulin treatment, initiated after development of the hypoalgesic response, normalized not only plasma glucose level and body weight of diabetic rats, but also returned their antinociceptive latency toward normal. The results of this study showed that long-term diabetes is associated with altered pain threshold and further support the hypothesis for endogenous opioid peptide mediation of hypoalgesia in chronically diabetic rats that can be prevented by insulin treatment.

Evaluation of controlled-release mosquito repellent formulations.

Eight polymer and 9 microcapsule formulations of deet were tested on laboratory rabbits against Aedes aegypti and Anopheles albimanus. Several formulations were significantly more effective than simple (unformulated) deet at the same strength for periods up to 24 h. Best results were obtained with a polymer formulation containing a high molecular weight fatty acid and 3 microcapsule formulations containing lanolin, gum arabic, gelatin, tannic acid, stearic acid, polypropylene glycol, water, and a commercial lotion in the microcapsule and carrier fractions.

Evaluation of the laboratory rabbit model for screening topical mosquito repellents.

The laboratory rabbit was evaluated as a model for screening topical mosquito repellents, using data obtained in tests of deet (N,N-diethyl-3-methylbenzamide) against Aedes aegypti on humans and rabbits. Host-specific differences in the action of the test material were quantified by multiple regression analysis. The test material was less effective but more persistent in tests on rabbits, and responses of the mosquito test population were more variable.

Flashlamp-excited dye laser therapy of idiopathic vulvodynia is safe and efficacious.

OBJECTIVE: The management of chronic vulvovaginal pain, not explicable on specific histologic grounds, presents a major problem in referral centers for lower genital tract diseases. STUDY DESIGN: This article reports on a two-step protocol in a sample of 175 medical nonresponders, drawn from a 2-year cohort of 725 women with vulvovaginal pain. The first maneuver was the use of a flashlamp-excited dye laser to selectively photocoagulate symptomatic subepithelial blood vessels in 168 women; the second was the microsurgical removal of chronically painful Bartholin's glands in 52 women not responsive or not suited to flashlamp-excited dye laser photothermolysis. RESULTS: Dye laser response rates were independent of whether patients manifested macroscopic foci of painful erythema ("vestibular adenitis") or just colposcopically apparent hyperemia-ectasia of the individual blood vessels ("pruritic papillomatosis") (56% vs 45% after a single surgical procedure; 76% vs 65% after serial retreatment; p not significant). Conversely, response rates were much lower among women in whom pressure on the Bartholin's glands produced sharp, lancinating pain (15% vs 66% after a single surgical procedure; 22% vs 93% after serial retreatment; p < 0.001). Forty-two (85%) of 50 patients with flashlamp-excited dye laser failure had deep pain; however, the impasse to progress was broken by gland removal. Final response rates were 92.5% (complete response 62%; partial response 30%) in the "surface-only" group and 80.3% in the "surface-plus-deep" group (chi 2 = 14.9; p < 0.001). The major complication was acute bacterial cellulitis, occurring in the first postoperative week. Modification of the treatment protocol to include topical antibiotics with an occlusive dressing reduced the cellulitis rate from 17.2% to 2.5%. In four women (1.8%) Koebner-like exophytic condylomas also developed within 1 month of flashlamp-excited dye laser surgery. CONCLUSION: The availability of a safe, efficacious, and relatively noninvasive treatment should reduce the need for resective surgery in most patients with idiopathic vulvodynia.

Evaluation of the laboratory mouse model for screening topical mosquito repellents.

Eight commercial repellents were tested against Aedes aegypti 0 and 4 h after application in serial dilution to volunteers and laboratory mice. Results were analyzed by multiple regression of percentage of biting (probit scale) on dose (logarithmic scale) and time. Empirical correction terms for conversion of values obtained in tests on mice to values expected in tests on human volunteers were calculated from data obtained on 4 repellents and evaluated with data obtained on 4 others. Corrected values from tests on mice did not differ significantly from values obtained in tests on volunteers. Test materials used in the study were dimethyl phthalate, butopyronoxyl, butoxy polypropylene glycol, MGK Repellent 11, deet, ethyl hexanediol, Citronyl, and dibutyl phthalate.