The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood-induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood-feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin-binding domains with 6 conserved Cys and may also present mucin-like domains (regions expected to be highly O-glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin-like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood-feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from Sao Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from Sao Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus. (C) 2010 Elsevier B.V. All rights reserved.

In vitro tests to establish LC(50) and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization

Laboratory test was carried out on larvae and adults of the cattle tick, Rhipicephalus (Boophilus) microplus, to determine fipronil toxicity. Adult immersion test (AIT, N = 26), larval immersion test (LIT, N = 71) and larval packet test (LPT, N = 41) were standardized using susceptible strain (Mozo). Dose-response curves were compared with a fipronil resistant strain. Four variables were analyzed from AIT results: mortality, weight of eggs on day 7 and on day 14, index of fertility, and index of fecundity. For larval test, dose mortality curves were analyzed. In spite of the high LC(50) variability, all variables determined for AIT were appropriate to discriminate both strains. AIT and LIT had more sensitivity than LPT, with larger resistance factors. It was used two times LC(99.9) as discriminating doses (DCs) following FAO suggestion. For mortality by AIT, LIT and LPT the DCs were estimated: 4.98 ppm, 7.64 ppm and 2365.8 ppm, respectively, for Mozo strain. DCs mortality values estimated for resistant strain by AIT, LIT and LPT were: 6.96 x 10(5) ppm, 343.26 ppm and 5.7 x 10(3) ppm, respectively and their respective resistant factors were: 202.4, 5.36 and 1.52. Protocols for AIT, LIT and LPT have been presented in this paper. (C) 2009 Elsevier B.V. All rights reserved.

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50) < 2) and resistant (differences and RR(50) >= 2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases >= 2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay. (c) 2011 Elsevier B.V. All rights reserved.

Detecting Major Genes Controlling Robustness of Chicken Body Weight Using Double Generalized Linear Models

Detecting both the majors genes that control the phenotypic mean and those controlling phenotypic variance has been raised in quantitative trait loci analysis. In order to mapping both kinds of genes, we applied the idea of the classic Haley-Knott regression to double generalized linear models. We performed both kinds of quantitative trait loci detection for a Red Jungle Fowl x White Leghorn F2 intercross using double generalized linear models. It is shown that double generalized linear model is a proper and efficient approach for localizing variance-controlling genes. We compared two models with or without fixed sex effect and prefer including the sex effect in order to reduce the residual variances. We found that different genes might take effect on the body weight at different time as the chicken grows.

Minimization of stock weight during close-die forging of a spindle

In this paper, Finite Element method and full-scale experiments have been used to study a hot forging method for fabri-cation of a spindle using reduced initial stock size. The forging sequence is carried out in two stages. In the first stage, the hot rolled cylindrical billet is pre-formed and pierced in a closed die using a spherical nosed punch to within 20 mm of its base. This process of piercing or impact extrusion leads to high strains within the work piece but requires high press loads. In the second stage, the resulting cylinder is placed in a die with a flange chamber and upset forged to form a flange. The stock mass is optimized for complete die filling. Process parameters such as effective strain distribution, material flow and forging load in different stages of the process are analyzed. It is concluded from the simulations that minor modifications of piercing punch geometry to reduce contact between the punch and emerging vertical walls of the cylinder appreciably reduces the piercing load. In the flange chamber, a die surfaces angle of 52° instead of 45° is pro-posed to ensure effective material flow and exert sufficient tool pressure to achieve complete cavity filling. In order to achieve better compression, it is also proposed to shorten both the length of the inserted punch and the die “tongues” by a few mm.

Genetic heterogeneity of within-family variance of body weight in Atlantic salmon (Salmo salar)

BACKGROUND: Canalization is defined as the stability of a genotype against minor variations in both environment and genetics. Genetic variation in degree of canalization causes heterogeneity of within-family variance. The aims of this study are twofold: (1) quantify genetic heterogeneity of (within-family) residual variance in Atlantic salmon and (2) test whether the observed heterogeneity of (within-family) residual variance can be explained by simple scaling effects. RESULTS: Analysis of body weight in Atlantic salmon using a double hierarchical generalized linear model (DHGLM) revealed substantial heterogeneity of within-family variance. The 95% prediction interval for within-family variance ranged from ~0.4 to 1.2 kg2, implying that the within-family variance of the most extreme high families is expected to be approximately three times larger than the extreme low families. For cross-sectional data, DHGLM with an animal mean sub-model resulted in severe bias, while a corresponding sire-dam model was appropriate. Heterogeneity of variance was not sensitive to Box-Cox transformations of phenotypes, which implies that heterogeneity of variance exists beyond what would be expected from simple scaling effects. CONCLUSIONS: Substantial heterogeneity of within-family variance was found for body weight in Atlantic salmon. A tendency towards higher variance with higher means (scaling effects) was observed, but heterogeneity of within-family variance existed beyond what could be explained by simple scaling effects. For cross-sectional data, using the animal mean sub-model in the DHGLM resulted in biased estimates of variance components, which differed substantially both from a standard linear mean animal model and a sire-dam DHGLM model. Although genetic differences in canalization were observed, selection for increased canalization is difficult, because there is limited individual information for the variance sub-model, especially when based on cross-sectional data. Furthermore, potential macro-environmental changes (diet, climatic region, etc.) may make genetic heterogeneity of variance a less stable trait over time and space.

The relative perceptual weight of two Swedish prosodic contrasts

Abstract. In addition to 9 vowel and 18 consonant phonemes, Swedish has three prosodic phonemic contrasts: word stress, quantity and tonal word accent. There are also examples of distinctive phrase or sentence stress, where a verb can be followed by either an unstressed preposition or a stressed particle. This study focuses on word level and more specifically on word stress and tonal word accent in disyllabic words. When making curriculums for second language learners, teachers are helped by knowing which phonetic or phonological features are more or less crucial for the intelligibility of speech and there are some structural and anecdotal evidence that word stress should play a more important role for intelligibility of Swedish, than the tonal word accent. The Swedish word stress is about prominence contrasts between syllables, mainly signaled by syllable duration, while the tonal word accent is signaled mainly by pitch contour. The word stress contrast, as in armen [´arːmən] ‘the arm’ - armén [ar´meːn] ‘the army’, the first word trochaic and the second iambic, is present in all regional varieties of Swedish, and realized with roughly the same acoustic cues, while the tonal word accent, as in anden [´anːdən] ‘the duck’ - anden [`anːdən] ‘the spirit’ is absent in some dialects (as well as in singing), and also signaled with a variety of tonal patterns depending on region. The present study aims at comparing the respective perceptual weight of the two mentioned contrasts. Two lexical decision tests were carried out where in total 34 native Swedish listeners should decide whether a stimulus was a real word or a non-word. Real words of all mentioned categories were mixed with nonsense words and words that were mispronounced with opposite stress pattern or opposite tonal word accent category. The results show that distorted word stress caused more non-word judgments and more loss, than distorted word accent. Our conclusion is that intelligibility of Swedish is more sensitive to distorted word stress pattern than to distorted tonal word accent pattern. This is in compliance with the structural arguments presented above, and also with our own intuition.

Role of dietary fats in modulating cardiometabolic risk during moderate weight gain : a randomized double-blind overfeeding trial (LIPOGAIN study)

BACKGROUND: Whether the type of dietary fat could alter cardiometabolic responses to a hypercaloric diet is unknown. In addition, subclinical cardiometabolic consequences of moderate weight gain require further study. METHODS AND RESULTS: In a 7-week, double-blind, parallel-group, randomized controlled trial, 39 healthy, lean individuals (mean age of 27±4) consumed muffins (51% of energy [%E] from fat and 44%E refined carbohydrates) providing 750 kcal/day added to their habitual diets. All muffins had identical contents, except for type of fat; sunflower oil rich in polyunsaturated fatty acids (PUFA diet) or palm oil rich in saturated fatty acids (SFA diet). Despite comparable weight gain in the 2 groups, total: high-density lipoprotein (HDL) cholesterol, low-density lipoprotein:HDL cholesterol, and apolipoprotein B:AI ratios decreased during the PUFA versus the SFA diet (-0.37±0.59 versus +0.07±0.29, -0.31±0.49 versus +0.05±0.28, and -0.07±0.11 versus +0.01±0.07, P=0.003, P=0.007, and P=0.01 for between-group differences), whereas no significant differences were observed for other cardiometabolic risk markers. In the whole group (ie, independently of fat type), body weight increased (+2.2%, P<0.001) together with increased plasma proinsulin (+21%, P=0.007), insulin (+17%, P=0.003), proprotein convertase subtilisin/kexin type 9, (+9%, P=0.008) fibroblast growth factor-21 (+31%, P=0.04), endothelial markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (+9, +5, and +10%, respectively, P<0.01 for all), whereas nonesterified fatty acids decreased (-28%, P=0.001). CONCLUSIONS: Excess energy from PUFA versus SFA reduces atherogenic lipoproteins. Modest weight gain in young individuals induces hyperproinsulinemia and increases biomarkers of endothelial dysfunction, effects that may be partly outweighed by the lipid-lowering effects of PUFA. CLINICAL TRIAL REGISTRATION URL: http://ClinicalTrials.gov. Unique identifier: NCT01427140.