Cultural adaptation to Bolivian Quechua and psychometric analysis of the Patient Health Questionnaire PHQ-9.

OBJECTIVE: Cultural adaptation of the Patient Health Questionnaire-PHQ-9 to Bolivian Quechua and analysis of the internal structure validity, reliability, and measurement invariance by sociodemographic variables. METHODS: The PHQ-9 was translated and back-translated (English-Quechua-English) to optimise translation. For the cultural adaptation, experts, and people from the target population (e.g., in focus groups) verified the suitability of the translated PHQ-9. For the psychometric analysis, we performed a Confirmatory Factor Analysis (CFA) to evaluate internal validity, calculated α and ω indices to assess reliability, and performed a Multiple Indicator, Multiple Cause (MIMIC) model for evaluating measurement invariance by sex, age, marital status, educational level and residence. We used standard goodness-of-fit indices to interpret both CFA results. RESULTS: The experts and focus groups improved the translated PHQ-9, making it clear and culturally equivalent. For the psychometric analysis, we included data from 397 participants, from which 73.3% were female, 33.0% were 18-30 years old, 56.7% reported primary school studies, 63.2% were single, and 62.0% resided in urban areas. In the CFA, the single-factor model showed adequate fit (Comparative Fit Index = 0.983; Tucker-Lewis Index = 0.977; Standardized Root Mean Squared Residual = 0.046; Root Mean Squared Error of Approximation = 0.069), while the reliability was optimal (α = 0.869-0.877; ω = 0.874-0.885). The invariance was confirmed across all sociodemographic variables (Change in Comparative Fit Index (delta) or Root Mean Square Error of Approximation (delta) < 0.01). CONCLUSIONS: The PHQ-9 adapted to Bolivian Quechua offers a valid, reliable and invariant unidimensional measurement across groups by sex, age, marital status, educational level and residence.

Quantitative microbial spoilage risk assessment caused by fungi in sports drinks through multilevel modelling.

The risk of fungal spoilage of sports drinks produced in the beverage industry was assessed using quantitative microbial spoilage risk assessment (QMSRA). The most relevant pathway was the contamination of the bottles during packaging by mould spores in the air. Mould spores' concentration was estimated by longitudinal sampling for 6 years (936 samples) in different production areas and seasons. This data was analysed using a multilevel model that separates the natural variability in spore concentration (as a function of sampling year, season, and area) and the uncertainty of the sampling method. Then, the expected fungal contamination per bottle was estimated by Monte Carlo simulation, considering their settling velocity and the time and exposure area. The product's shelf life was estimated through the inoculation of bottles with mould spores, following the determination of the probability of visual spoilage as a function of storage time at 20 and 30 °C using logistic regression. The Monte Carlo model estimated low expected spore contamination in the product (1.7 × 10-6 CFU/bottle). Nonetheless, the risk of spoilage is still relevant due to the large production volume and because, as observed experimentally, even a single spore has a high spoilage potential. The applicability of the QMSRA during daily production was made possible through the simplification of the model under the hypothesis that no bottle will be contaminated by more than one spore. This simplification allows the calculation of a two-dimensional performance objective that combines the spore concentration in the air and the exposure time, defining "acceptable combinations" according to an acceptable level of spoilage (ALOS; the proportion of spoiled bottles). The implementation of the model at the operational level was done through the representation of the simplified model as a two-dimensional diagram that defines acceptable and unacceptable areas. The innovative methodology employed here for defining and simplifying QMSRA models can be a blueprint for future studies aiming to quantify the risk of spoilage of other beverages with a similar scope.

Impacts of long-term insecticide treatment regimes on skdr and kdr pyrethroid resistance alleles in horn fly field populations.

We evaluated the effects of four different 6-year duration control strategies on the resistance levels and frequency of the pyrethroid target site resistance alleles, superkdr (skdr) and kdr, at four field populations of Haematobia irritans irritans (Linnaeus, 1758) (Diptera: Muscidae) in Louisiana, USA. Consecutive use of pyrethroid ear tags for 6 years caused a significant increase in the resistance ratio to pyrethroids as well as the frequencies of both skdr and kdr resistance alleles. After 3 years of consecutive use of pyrethroid ear tags, followed by 1 year with no treatment, and followed by 2 years with organophosphate ear tags, the resistance ratio for pyrethroid was not significantly affected, the %R-skdr significantly dropped while the %R-kdr allele remained relatively high and stable. Similar results were observed when pyrethroid ear tags were used for three consecutive years, followed by 1 year with no treatment, and followed by 2 years with endosulfan ear tags; however, this treatment resulted in a slight increase in the resistance ratio for pyrethroids. In a mosaic, the resistance ratio for pyrethroids showed a 2.5-fold increase but the skdr-kdr genetic profiles did not change, as the %R alleles (skdr and kdr) remained low and stable through the 6 years. Lack of exposure to pyrethroid insecticides for 3 years significantly affected the skdr mutation but not the kdr mutation, preventing re-establishment of susceptibility to pyrethroids. SS-SR (skdr-kdr) individuals were responsible for the maintenance of the kdr mutation in two of the populations studied, and fitness cost seems to strongly affect the SR-RR genotype. None of the four treatment regimens evaluated in the study had satisfactory results for the management of kdr resistance alleles.

Gene expression during the early stages of host perception and attachment in adult female Rhipicephalus microplus ticks.

The cattle tick, Rhipicephalus microplus, is a serious pest of cattle, with significant economic consequences to the livestock industries of tropical and semitropical countries. Rhipicephalus microplus belongs to the Metastriata group of the Ixodidae family known as hard ticks. When adult hard ticks feed, mating has not yet occurred and an initial host attachment phase of 1-2 days is followed by a slow feeding phase that can last several days. Once mating occurs, feeding concludes with a rapid engorgement phase that is completed in 12-36 h. Our group's interest in mining the genome and transcriptome of R. microplus for novel targets for development of tick control technologies led us to investigate the early transcriptional events occurring upon tick attachment and subsequent feeding. We placed newly molted unfed adult R. microplus females upon a bovine host and harvested the attached ticks after 3, 6, 12, and 24 h. We also placed a group of these ticks in a gas-permeable tube taped onto the side of the bovine host. These ticks were able to sense the host but unable to penetrate the tube to begin attachment and were ultimately harvested after 3 h. This study produced a comprehensive transcriptome from newly molted adult ticks and will provide a useful resource for studies of tick feeding and host perception and also assist genome annotation refinements.

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster.

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, we aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68µM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, we performed a salivary gland specific knockdown of Kir1 to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na+-K+-2Cl- cotransporter and/or Na+-K+-ATPase pumps that serve to supplement the inward flow of K+ ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding.

A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique.

BACKGROUND: The New World screwworm, Cochliomyia hominivorax, is a devastating pest of livestock endemic to subtropical and tropical regions of the Western hemisphere. The larvae of this species feed on the tissue of living animals, including man, and can cause death if untreated. Over 60 years ago, the sterile insect technique (SIT) was developed with the aim of eradicating this pest, initially from Florida but subsequently from all of North and Central America. From the outset it was appreciated that SIT would be more efficient if only sterile males were released in the field, but this was not possible until now. RESULTS: Here, we report on the development and evaluation of the first sexing strains of C. hominivorax that produce only males when raised on diet without tetracycline. Transgenic lines have been developed that possess a tetracycline repressible female-lethal genetic system. Ten of these lines show high female lethality at the late larval/pupal stages and three of them present dominant female lethality. Most of the lines were comparable to the wild type parental strain in several fitness parameters that are relevant to mass rearing in a production facility. Further, three lines performed well in male mating success and male competition assays, suggesting they would be sexually competitive in the field. Consequently, one transgenic line has been selected by the New World Screwworm Program for evaluation under mass rearing conditions. CONCLUSIONS: We conclude that the promising characteristics of the selected sexing strains may contribute to reduce production costs for the existing eradication program and provide more efficient population suppression, which should make a genetic control program more economical in regions were C. hominivorax remains endemic.

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs.

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8 % of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33 % of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.

A 454 sequencing approach to dipteran mitochondrial genome research.

The availability of complete mitochondrial genome (mtgenome) data for Diptera, one of the largest metazoan orders, in public databases is limited. The advent of high throughput sequencing technology provides the potential to generate mtgenomes for many species affordably and quickly. However, these technologies need to be validated for dipterans as the members of this clade play important economic and research roles. Illumina and 454 sequencing platforms are widely used in genomic research involving non-model organisms. The Illumina platform has already been utilized for generating mitochondrial genomes without using conventional long range PCR for insects whereas the power of 454 sequencing for generating mitochondrial genome drafts without PCR has not yet been validated for insects. Thus, this study examines the utility of 454 sequencing approach for dipteran mtgenomic research. We generated complete or nearly complete mitochondrial genomes for Cochliomyia hominivorax, Haematobia irritans, Phormia regina and Sarcophaga crassipalpis using a 454 sequencing approach. Comparisons between newly obtained and existing assemblies for C. hominivorax and H. irritans revealed no major discrepancies and verified the utility of 454 sequencing for dipteran mitochondrial genomes. We also report the complete mitochondrial sequences for two forensically important flies, P. regina and S. crassipalpis, which could be used to provide useful information to legal personnel. Comparative analyses revealed that dipterans follow similar codon usage and nucleotide biases that could be due to mutational and selection pressures. This study illustrates the utility of 454 sequencing to obtain complete mitochondrial genomes for dipterans without the aid of conventional molecular techniques such as PCR and cloning and validates this method of mtgenome sequencing in arthropods.

Acetylcholinesterase 1 in populations of organophosphate-resistant North American strains of the cattle tick, Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus, the cattle fever tick, is a global economic problem to the cattle industry due to direct infestation of cattle and pathogens transmitted during feeding. Cattle fever tick outbreaks continue to occur along the Mexico-US border even though the tick has been eradicated from the USA. The organophosphate (OP) coumaphos targets acetylcholinesterase (AChE) and is the approved acaricide for eradicating cattle fever tick outbreaks. There is evidence for coumaphos resistance developing in cattle ticks in Mexico, and OP-resistant R. microplus ticks were discovered in outbreak populations of Texas in 2005. The molecular basis of coumaphos resistance is not known, and our study was established to gather further information on whether AChE1 is involved in the resistance mechanism. We also sought information on allele diversity in tick populations with different levels of coumaphos resistance. The overarching project goal was to define OP resistance-associated gene mutations such that a DNA-based diagnostic assay could be developed to assist the management of resistance. Three different AChE transcripts have been reported in R. microplus, and supporting genomic and transcriptomic data are available at CattleTickBase. Here, we report the complete R. microplus AChE1 gene ascertained by sequencing a bacterial artificial chromosome clone containing the entire coding region and the flanking 5' and 3' regions. We also report AChE1 sequences of larval ticks from R. microplus strains having different sensitivities to OP. To accomplish this, we sequenced a 669-bp region of the AChE1 gene corresponding to a 223 amino acid region of exon 2 to assess alleles in seven strains of R. microplus with varying OP resistance phenotypes. We identified 72 AChE1 sequence variants, 2 of which are strongly associated with OP-resistant phenotypes. Esterase-like sequences from the R. microplus transcriptome RmiTr Version 1.0 were compared to the available sequence databases to identify other transcripts with similarity to AChE1.

Simultaneous detection of pyrethroid, organophosphate, and cyclodiene target site resistance in Haematobia irritans (Diptera: Muscidae) by multiplex polymerase chain reaction.

The horn fly, Haematobia irritans irritans (L., 1758) (Diptera: Muscidae), is an important pest that causes significant economic losses to the livestock industry, but insecticide resistance in horn fly populations has made horn fly control increasingly difficult to achieve. In this study, we developed a multiplex polymerase chain reaction (PCR) assay to simultaneously detect target site resistance to pyrethroids (kdr mutation), organophosphates (G262A acetylcholinesterase mutation), and cyclodienes (Rdl mutation) and used the new procedure to follow the progression of these three mutations after exposure to different insecticide pressure. We assayed flies collected at the Macon Ridge research station, Winnsboro, LA, from 2008 to 2012. The multiplex PCR showed robust results in all our assays. The kdr mutation remained at high frequencies during all years, even after 4 yr with no use of pyrethroids. The G262A acetylcholinesterase mutation fluctuated from 7.5 to 23.8% during the studied years, while the Rdl mutation was rare in 2008, 2009, and June 2010, and then significantly increased after the first use of endosulfan. The possibility of screening for all the known target site resistance mutations in a single PCR reaction makes the multiplex PCR a useful and affordable tool that can be used to help diagnose insecticide resistance.

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Biochemical characterisation of Cytochrome P450 oxidoreductase from the cattle tick, Rhipicephalus microplus, highlights potential new acaricide target.

Management of the cattle tick, Rhipicephalus microplus, presents a challenge because some populations of this cosmopolitan and economically important ectoparasite are resistant to multiple classes of acaricides. Cytochrome P450 oxidoreductase (CPR) is part of the cytochrome P450 (CYP450) monooxygenases that are involved in metabolic resistance by their ability to detoxify acaricides. Inhibiting CPR, the sole redox partner that transfers electrons to CYP450s, could overcome this type of metabolic resistance. This report represents the biochemical characterisation of a CPR from ticks. Recombinant CPR of R. microplus (RmCPR), minus its N-terminal transmembrane domain, was produced in a bacterial expression system and subjected to biochemical analyses. RmCPR displayed a characteristic dual flavin oxidoreductase spectrum. Incubation with nicotinamide adenine dinucleotide phosphate (NADPH) lead to an increase in absorbance between 500 and 600 nm with a corresponding appearance of a peak absorbance at 340-350 nm indicating functional transfer of electrons between NADPH and the bound flavin cofactors. Using the pseudoredox partner, kinetic parameters for both cytochrome c and NADPH binding were calculated as 26.6 ± 11.4 µM and 7.03 ± 1.8 µM, respectively. The turnover, Kcat, for RmCPR for cytochrome c was calculated as 0.08 s-1 which is significantly lower than the CPR homologues of other species. IC50 (Half maximal Inhibitory Concentration) values obtained for the adenosine analogues 2', 5' ADP, 2'- AMP, NADP+and the reductase inhibitor diphenyliodonium were: 140, 82.2, 24.5, and 75.3 µM, respectively. Biochemically, RmCPR resembles CPRs of hematophagous arthropods more so than mammalian CPRs. These findings highlight the potential of RmCPR as a target for the rational design of safer and potent acaricides against R. microplus.

Horn fly transcriptome data of ten populations from the southern United States with varying degrees and molecular mechanisms of pesticide resistance.

Haematobia irritans irritans (Linnaeus, 1758: Diptera: Muscidae), the horn fly, is an external parasite of penned and pastured livestock that causes a major economic impact on cattle production worldwide. Pesticides such as synthetic pyrethroids and organophosphates are routinely used to control horn flies; however, resistance to these chemicals has become a concern in several countries. To further elucidate the molecular mechanisms of resistance in horn fly populations, we sequenced the transcriptomes of ten populations of horn flies from the southern US possessing varying degrees of pesticide resistance levels to pyrethroids, organophosphates, and endosulfans. We employed an Illumina paired end HiSeq approach, followed by de novo assembly of the transcriptomes using CLC Genomics Workbench 8.0.1 De Novo Assembler using multiple kmers, and annotation using Blast2GO PRO version 5.2.5. The Gene Ontology biological process term Response to Insecticide was found in all the populations, but at an increased frequency in the populations with higher levels of insecticide resistance. The raw sequence reads are archived in the Sequence Read Archive (SRA) and assembled population transcriptomes in the Transcriptome Shotgun Assembly (TSA) at the National Center for Biotechnology Information (NCBI).

Cultural adaptation to quechua and psychometric analysis of the patient health questionnaire (PHQ-9) in a peruvian population. / Adaptación cultural al Quechua y análisis psicométrico del Patient Health Questionnaire (PHQ-9) en población peruana.

OBJECTIVE: . To translate and culturally adapt the Patient Health Questionnaire (PHQ-9) to three varieties of Quechua and analyse their validity, reliability, and measurement invariance. MATERIALS AND METHODS: . 1) Cultural adaptation phase: the PHQ-9 was translated from English into three variants of Quechua (Central, Chanca, Cuzco-Collao) and translated again into English. Then, experts and focus groups allowed the translations to be culturally adapted. 2) Psychometric phase: the unidimensionality of the adapted PHQ-9 was evaluated by using Confirmatory Factor Analysis (CFA), reliability was evaluated by internal consistency (Alpha and Omega), and measurement invariance according to Quechua varieties and sociodemographic variables was evaluated by using CFA, multigroups and MIMIC models (Multiple Indicator Multiple Cause). RESULTS: . Each of the adaptations of the PHQ-9 to the three Quechua varieties reported clear and culturally equivalent items. Subsequently, data from 970 Quechua-speaking adult men and women were analyzed. The general one-dimensional model reported an adequate fit (Comparative fit index = 0.990, Tucker-Lewis index = 0.987, Standardized root mean squared residual= 0.048, Root mean squared error of approximation=0.071); each of the Quechua varieties also showed an adequate fit. Reliability was high for all varieties (α = 0.865 - 0.915; ω = 0.833 - 0.881). The results of the multigroup CFA and MIMIC models confirmed measurement invariance according to Quechua variant, sex, residence, age, marital status and educational level. CONCLUSIONS: . The PHQ-9 adaptations to Central Quechua, Chanca and Cuzco-Collao offer a valid, reliable and invariant measurement, confirming that comparisons can be made between the evaluated groups. Its use will benefit mental health research and care for Quechua-speaking populations.

OBJETIVO: . Traducir y adaptar culturalmente el Patient Health Questionnaire (PHQ-9) a tres variedades del quechua y analizar su validez, confiabilidad e invarianza. MATERIALES Y MÉTODOS: . 1) Fase de adaptación cultural: el PHQ-9 fue traducido del inglés a tres variantes del quechua (Central, Chanca, Cuzco-Collao) y traducido nuevamente al inglés, posteriormente expertos y grupos focales permitieron adaptar culturalmente las traducciones. 2) Fase psicométrica: se evaluó la uni-dimensionalidad del PHQ-9 adaptado mediante un Análisis Factorial Confirmatorio (CFA), la confiabilidad se evaluó mediante consistencia interna (Alpha y Omega), y la invarianza de medida según variedades del quechua y variables sociodemográficas se evaluó empleando CFA multigrupos y modelos MIMIC (Múltiples Indicadores y Múltiples Causas). RESULTADOS: . Cada una de las adaptaciones del PHQ-9 a las tres variedades de quechua reportaron ítems claros y culturalmente equivalentes. Posteriormente, con 970 datos de quechuahablantes adultos varones y mujeres, el modelo general unidimensional reportó un ajuste adecuado (índice de ajuste comparativo: 0,990, índice de Tucker-Lewis: 0,987, residuo estandarizado cuadrático medio: 0,048, raíz del error cuadrático medio de aproximación: 0,071), lo mismo ocurrió para cada variedad del quechua. La confiabilidad fue alta para todas las variedades (α = 0,865 - 0,915; ω = 0,833 - 0,881). Los resultados del CFA multigrupos y modelos MIMIC confirmaron invarianza de medida según variante del quechua, sexo, residencia, edad, estado civil y nivel educativo. CONCLUSIONES: . Las adaptaciones del PHQ-9 a Quechua Central, Chanca y Cuzco-Collao ofrecen una medición válida, confiable e invariante, confirmando que se pueden hacer comparaciones en los grupos evaluados. Su uso beneficiará a la investigación y a la atención en salud mental de poblaciones quechuahablantes.

Automation of an atomic force microscope via Arduino.

The Atomic Force Microscopy is a very versatile technique that allows to characterize surfaces by acquiring topographies with sub-nanometer resolution. This technique often overcomes the problems and capabilities of electron microscopy when characterizing few nanometers thin coatings over solid substrates. They are expensive, in the half million dollar range for standard units, and therefore it is often difficult to upgrade to new units with improved characteristics. One of these improvements, motorization and automation of the measurements is very interesting to sample different parts of a substrate in an unattended way. Here we report a low cost upgrade under 60 $ to a Dimension 3000 AFM based on a control unit using an Arduino Leonardo. It enables to acquire dozens or hundreds of images automatically by mimicking keyboard shortcuts and interfacing the AFM PCI card.

The adult horn fly transcriptome and its complement of transcripts encoding cytochrome P450s, glutathione S-transferases, and esterases.

The horn fly, Haematobia irritans, is a blood-feeding parasitic fly with a global distribution that includes Europe, Africa, Asia, and the Americas. The fly has a major detrimental economic impact upon cattle production, with losses estimated at over $800 million annually in the United States and $2.5 billion in Brazil alone. Insecticide resistance in specific horn fly populations has been a problem for many years and there are several mechanisms whereby resistance develops. Little is known about the complement of metabolic enzymes encoded by the horn fly's genome that might provide the fly with detoxification or sequestration pathways to survive insecticide treatments. The cytochrome P450, glutathione S-transferase, and esterase enzyme families contain members that are capable of sequestering and/or detoxifying xenobiotic molecules such as insecticides. We sought to develop a comprehensive dataset of metabolic enzyme-encoding transcript sequences from the adult horn fly, as this is the life stage whose actions directly impose the economic costs to cattle producers. We used an Illumina paired-end read RNA-Seq approach to determine the adult horn fly transcriptomes from laboratory and field populations of horn flies with varying levels of pesticide resistance, including untreated and pyrethroid-treated newly eclosed adult flies. We followed with bioinformatic analyses to discern sequences putatively encoding cytochrome P450, esterase, and GST enzymes. We utilized read-mapping of RNA-Seq data and quantitative real-time polymerase chain reaction (qRT-PCR) to examine gene expression levels of specific P450 transcripts in several fly populations with varying degrees of pesticide resistance.

The penultimate arginine of the carboxyl terminus determines slow desensitization in a P2X receptor from the cattle tick Boophilus microplus.

P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC(50), 67.1 µM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC(50), 4.8 µM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms.

Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. RESULTS: To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. CONCLUSIONS: Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs.

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing.

BACKGROUND: Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. RESULTS: Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. CONCLUSIONS: This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Phenotype changes inherited by crossing pyrethroid susceptible and resistant genotypes from the cattle tick Riphicephalus (Boophilus) microplus.

Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus (Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains: susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × â™€SS), and SR (♂SS × â™€RR); and backcrosses: RS × SS, RS × RR, SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%). Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (D(ML)) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance for cypermethrin and flumethrin (P < 0.05) but not for deltamethrin (P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used (P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms cannot be excluded.