text:
Natural Gas Distribution Lines World Report
- July 2016
- Region: Global
- 2084 pages
- PureData
The Natural Gas Distribution Lines World Report gives Market Consumption / Products / Services for over 200 countries by 6 to 10-Digit NAICS Product Codes by 3 Time series: 1997 to Current year and Forecasts Current year to 2020 & 2021-2028.
59 MARKET RESEARCH CHAPTERS. SPREADSHEET CHAPTERS: Market Consumption - in US$ by Country by Product/Service by Year. Market, Financial, Competitive, Market Segmentation, Industry, Critical Parameters, Marketing Costs, Markets, Decision Makers, Performance, Product Launch.
Data includes Market Consumption by individual Product / Service, Per-Capita Consumption, Marketing Costs & Margins, Product Launch Data, Buyers, End Users & Customer Profile, Consumer Demographics.
Historic Balance Sheets, Forecast Financial Data, Industry Profile, National Data.
The report is on a DVD containing the entire web and databases, or it is available online. Merge text, tables & databases for your own reports, spreadsheet calculations & modeling.
12 Products/Markets covered, 2084 pages, 9615 spreadsheets, 9604 database tables, 552 illustrations. Updated monthly. 12 month After-Sales Service.
This database covers NAICS code: 2212_L.
The Market for Natural Gas Distribution Lines in each country by Products & Services.
This database covers NAICS code: 221210_L. Products/Services classified by 5-Digit US Commerce Department Code and then defined by each 6 to 10-Digit Product Codes.
The Natural Gas Distribution Lines World Report covers:
TIME SERIES - Historic: 1997 to Current year / Current time series: Current year to 2020 / Long Term Projection: 2021-2028. Consumption given at industry / distribution channel / service or product line level.
Natural gas distribution Lines
Sales of products from natural gas, incl. propane & butane
Household appliance sales
Sales of other merchandise
Pipeline transportation of natural gas
Electricity - generation and distribution sales
Electricity - power marketing and brokering sales
Natural gas distribution to final consumer
Natural gas - power marketing and brokering sales
Mixed, manufactured, or liquefied gas sales
Electric power transmission
All other operating revenue
1 Administration, 2 Advertising, 3 Buyers - Commercial Operations, 4 Buyers - Competitors, 5 Buyers - Major City, 6 Buyers - Products, 7 Buyers - Trade Cell, 8 Competitive Industry Analysis, 9 Competitor Analysis, 10 Country Focus, 11 Distribution, 12 Business Decision Scenarios, 13 Capital Costs Scenarios, 14 Cashflow Option Scenarios, 15 Cost Structure Scenarios, 16 Historic Industry Balance Sheet, 17 Historic Marketing Costs & Margins, 18 Investment + Cost Reduction Scenarios, 19 Market Climate Scenarios, 20 Marketing Costs, 21 Marketing Expenditure Scenarios, 22 Marketing Margins, 23 Strategic Options Scenarios, 24 Survival Scenarios, 25 Tactical Options Scenarios, 26 Geographic Data, 27 Industry Norms, 28 Major City Market Analysis, 29 Capital Access Scenarios, 30 Market Cashflow Scenarios, 31 Economic Climate Scenarios, 32 Market Investment + Costs Scenarios, 33 Marketing Expenditure Scenarios, 34 Market Risk Scenarios, 35 Market Strategic Options, 36 Market Survival Options, 37 Market Tactical Options, 38 Marketing Expenditure -v- Market Share, 39 Marketing Strategy Development, 40 Markets, 41 Operational Analysis, 42 Overseas Development, 43 Personnel Management, 44 Physical Distribution + Customer Handling, 45 Pricing, 46 Process + Order Handling, 47 Product Analysis, 48 Product Development, 49 Product Marketing Factors, 50 Product Mix, 51 Product Summary, 52 Profit Risk Scenarios, 53 Promotional Mix, 54 Salesforce Decisions, 55 Sales Promotion, 56 Surveys, 57 Targets -Product + Market, 58 Technology, 59 Trade Cell Analysis.
PRODUCT CONSUMPTION - in US$ by Country by Product/Service by Year: 1997 to Current year, Forecast Current year to 2020, Forecast 2021-2028. Market, Financial, Competitive, Market Segmentation, Industry, Critical Parameters, Marketing Costs, Markets, Decision Makers, Performance, Product Launch.
WORLD & NATIONAL REPORT MARKET DATABASE & SPREADSHEETS: 1332 World Database tables & Spreadsheets covering business scenarios. 1435 World Database tables & Spreadsheets covering Markets, Market Forecast, Financial Forecast, Financial Margins, Historic Financial, Historic Costs, Industry Norms for each country. 3816 National Database tables & Spreadsheets covering business scenarios.
FINANCIAL SPREADSHEETS & DATABASES: 188 Balance Sheet, Financial Margins & Ratios for each of 103 Business Scenarios - by Country by Year - Forecast Current year to 2020, Forecast 2021-2028.
INDUSTRY SPREADSHEETS & DATABASES: 820 Database tables & Spreadsheets covering Historic Industry Balance Sheet Data, Forecast Industry Financial Data, Industry Profiles & Norms - by Country by Year - Forecast Current year to 2020, Forecast 2021-2028.
NATIONAL DATA - by Country by Year.
The report is produced on a DVD & Memory Stick containing the entire report web and databases, and it is available online. Readers can access & reproduce the information for their own documents or reports. Tables & databases as Access & Excel formats on the DVD/Memory Stick to enable readers to produce their own spreadsheet calculations and modeling.
12 Natural Gas Distribution Lines NAICS Products covered for over 200 Countries: 2101 pages, 9795 spreadsheets, 9789 database tables, 512 diagrams & maps. This database is updated monthly. 12 months Full After-Sales Services & Updates available from the publishers.
This report is also available as 7 Regional Reports: Canada and the USA (includes individual coverage of 50 U.S. States), Central America (31 countries), South America (13 countries), Europe (45 countries), Africa (54 countries), Asia (48 countries), and Oceania (20 countries)
The Natural Gas Distribution Lines World Report provides data on the net market for the Products and Services covered in each of the countries listed. The Products and Services covered (Natural gas distribution Lines) are classified by the 5-Digit United States Commerce Department Major Product Codes and each Product and Services is then further defined and analyzed by each 6 to 10-Digit United States Commerce Department Product Codes. This report consists of a DVD containing the entire report web and databases. Readers can access and reproduce the information for inclusion into their own documents or reports. The tables & databases are in Access & Excel formats on the DVD to enable readers to produce their own spreadsheet calculations and modeling. This database is updated monthly. After-Sales and update services available from The Data Institute.


text:
Table of Contents - November 2013 - Volume 140, Special Issue 13 (Control of cestode zoonoses in Asia: role of basic and applied science)
a1 Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido, Japan
a2 Laboratory of Entomology, Mongolian Academy of Science, Ulaanbaatar, Mongolia
a3 Department of Parasitology, National Center for Communicable Diseases, Ulaanbaatar, Mongolia
a4 Department of Parasitology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan
a5 Animal Laboratory for Medical Research, Center for Advanced Research and Education, Asahikawa Medical University, Asahikawa, Hokkaido, Japan
a6 Department of Medical Biology, School of Biomedicine, Health Science University of Mongolia, Ulaanbaatar, Mongolia
The small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.
(Received April 14 2013)
(Revised May 07 2013)
(Revised May 28 2013)
(Accepted June 01 2013)
(Online publication August 19 2013)
Key words
c1 Corresponding author. Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido 078-8510, Japan. Tel.: +81 166 68 2686. Fax: +81 166 68 2429. E-mail: firstname.lastname@example.com
† These authors contributed equally for preparation of the manuscript.

subset: commoncrawl


text:
Application Information
Get Instant Scholarship Matches
Complete your free profile to get personalized matches to over $ll billion in scholarship rewards.
Scholarship Overview
This scholarship is for graduating high school seniors and currently enrolled college students who are communicant members of First Presbyterian Church in Jackson, Michigan. To qualify for this scholarship, applicants must demonstrate a financial need.
How easy is it to apply?
Very quick!
This scholarship's application process should probably take a very short time.
How much competition is there?
Relatively little!
This scholarship has a relatively low number of applicants.


text:
|Home | About | Journals | Submit | Contact Us | Français
PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.
Juvenile and adult (male and female) guppies express 10 visual opsins at varying levels in the eye. Two opsin genes in juveniles, SWS2B and RH2-2, accounted for >85% of all visual-opsin transcripts in the eye, excluding RH1. This relative abundance (RA) value dropped to about 65% in adults, as LWS-A180 expression increased from approximately 3% to 20% RA. The juvenile-to-female transition also showed LWS-S180 upregulation from about 1.5% to 7% RA. Finally, we found that expression in guppies' SWS2-LWS gene cluster is negatively correlated with distance from a candidate locus control region (LCR).
Selective pressures influencing visual-opsin gene expression appear to differ among age and sex. LWS upregulation in females is implicated in augmenting spectral discrimination of male coloration and courtship displays. In males, enhanced discrimination of carotenoid-rich food and possibly rival males are strong candidate selective pressures driving LWS upregulation. These developmental changes in expression suggest that adults possess better wavelength discrimination than juveniles. Opsin expression within the SWS2-LWS gene cluster appears to be regulated, in part, by a common LCR. Finally, by comparing our RT-qPCR data to MSP data, we were able to propose the first opsin-to-λmax assignments for all photoreceptor types in the cone mosaic.
For the better part of a century, guppies (Poecilia reticulata) have served as a model organism for the study of male coloration, courtship displays, female mate-choice, and visual-pigment evolution [1-8]. Within these, and in other aspects of guppy life-history, a large number of sex- and population-specific morphological and behavioral characteristics have been described [3,9-14], with the most salient of this variation being guppies' pronounced sexual dimorphism. For example, the Cumaná guppy studied here, from a human perspective, exhibits polymorphic black, red, orange, yellow, and iridescent color patterns in males, while females are green, yellow, and iridescent. In areas of low-predation, such as that of Cumaná guppies, male coloration increases and serves as an indicator of fitness to females, while under high predation pressure this coloration is subdued [15,16]. Thus, conspicuous male coloration is a trade-off as it attracts both females and predators [16]. These observations suggest that color vision, at least in females, plays an important role in mate choice and, therefore, in the evolution of variation in male coloration.
Vision is mediated by membrane-bound photoreceptors encoded by opsin genes. The opsin family has been broadly sorted into three subfamilies: photoisomerases, rhabdomeric opsins (r-opsins), and ciliary opsins (c-opsins) (reviewed by Lamb et al. [17,18]). Although r-opsins are used for vision in protostomes the term 'visual opsin' has been used to describe the c-opsins expressed in vertebrate rods and cones. Visual opsins have been categorized into five spectral classes: LWS, SWS1, SWS2, RH2, and RH1 [19]. The first four of these opsin classes facilitate cone-based photopic (bright light) vision and color perception, while RH1, the most recently evolved of the five spectral classes [20,21], facilitates rod-based scotopic (low light) vision. Visual-opsin gene loss and duplication and divergence have generated extensive repertoire variation among vertebrates that, in many cases, correlates to spectral environment (see reviews [22,23]).
Remarkably, the Cumaná guppy genome encodes at least 11 visual opsins. This repertoire includes one RH1 gene, three short wavelength-sensitive genes (SWS1, SWS2A, and SWS2B), two middle wavelength-sensitive genes (RH2-1 and RH2-2), and four long wavelength-sensitive (LWS) genes: A180, P180, S180, and S180r. The eleventh gene is a hybrid locus (A180 with a P180 3' end) that has not been detected in other guppy populations [6-8,24]. The observation that amino acid substitutions have occurred at key sites, even among recent duplicates (e.g., A180 and S180) [8] suggests that much, if not all, of this diversity is a product of selection.
Long before the guppy opsin gene repertoire was characterized, microspectrophotometry (MSP) data indicated that guppies had several distinct classes of retinal photoreceptor cells (hereafter referred to as, 'photoreceptors'). These include ultraviolet (UV), short- and middle-wavelength sensitive cones with maximal absorption (λmax) at 359/389, 410, and 465 nm, respectively, as well as one or more of three LWS cones with λmax values clustering at 533, 543, and 572 nm. Rod λmax was also characterized at about 503 nm [25-27]. Collectively, these findings implicate photoreceptor-level expression of at least six of the 11 visual-opsin genes found in guppies, though it has not been possible, until now, to associate each λmax to a specific opsin and photoreceptor morphology (or 'type').
In several fish species a large visual-opsin gene repertoire appears to play an important role in the tuning of vision for particular environments (see reviews [28,29]). While these examples of vision tuning deal with wavelength sensitivity, evidence of increased color discrimination by means of an expanded opsin gene repertoire has recently been shown in non-teleost species. Transgenic mice expressing three instead of two LWS opsins exhibited enhanced discrimination of red and orange wavelengths of light [30]. Similarly, Mancuso et al. [31] demonstrated that it was possible to treat adult squirrel monkey (Saimiri sciureus) congenital red-green color blindness using LWS-recombinant adeno-associated virus (rAAV) gene therapy. The intriguing implication of these studies is that retinal circuitry and higher order neuronal processes are either sufficient or adapt to mediate this augmentation to congenital vision. Thus, the potential for guppies to have increased color sensitivity and/or discrimination seemingly hinges on if, and to what extent, these 'extra opsins' are expressed in the retina.
Although all 10 non-hybrid visual opsins are expressed at the mRNA level in Cumaná guppy eyes [8,24], little is known about the relative abundance of each opsin. Ward et al. [8] indicated that LWS expression is predominated (>97%) by A180 transcripts; however, only three adult fish were surveyed. Here we used reverse transcription quantitative PCR (RT-qPCR) to characterize expression levels of all non-hybrid visual opsins. We surveyed males, females, and juveniles in order to test the hypothesis that sexual selection influences color vision in guppies. As adults, males exhibit elaborate red, orange, and yellow colorations that strongly influence female mate choice [2-4,8,32-34]. Since embryos and juveniles do not show these characteristic colors, and by definition are not sexually active, we predicted that if opsin gene expression plays a role in color-based sexual selection, then LWS upregulation will coincide with sexual maturity. Finally, the recent discovery that SWS2 and LWS occur in a tandem array in Cumaná guppies [35] allowed us to test the hypothesis that distance from a candidate LCR influences opsin expression in guppies, as is the case for human LWS/MWS [36,37] and zebrafish (Danio rerio) RH2s [38].
Care and treatment of guppies were in compliance with the Canadian Council of Animal Care and approved by the University of Victoria Animal Care Committee. Guppies from our inbred lab population (established from specimens collected in Cumaná, Venezuela) were housed in 15 gallon aquaria (unenriched environment) under a synthetic 14:10 hour light-dark cycle with instantaneous transition between photoperiods. Illumination was provided by fluorescent bulbs (General Electric F32T8SP/65K). Relative irradiance of the spectral environment was measured by a USB2000 spectrophotometer (Ocean Optics, Inc.) at the air to water boundary (see Additional file 1). Aquaria were maintained at 24 ± 1°C on a fresh water flow-through system at the University of Victoria Aquatic Research Facility. Fish were fed brine shrimp (Artemia salina) flake (Aquatic Ecosystems®) daily between 10:00-11:00 hours of the light phase. The number, sex, and age of guppies at time of sampling are detailed in Table Table11.
Following light-dark cycle adaptation period (a minimum of 2 weeks for adults) guppies were euthanized in 1 g/L tricaine methanesulphonate, MS222, (Sigma®) at 13:00 hours of the light phase. Sampling time was based on cichlid and zebrafish studies indicating that the highest level of cone opsin expression coincides with the end of the photopic day [39,40]. Eyes from 45 individuals were excised and pooled according to age, sex, and left or right eyes (see primary survey, Table Table1),1), with each pool individually placed into 1.0 mL PureZOL™ (Bio-Rad®) containing a 3 mm tungsten carbide bead. Extraction of left and right eyes were alternated among individuals to eliminate RNA degradation bias that might have occurred between specimen death and eye immersion in PureZOL™ (a total of 1 minute for the first eye, and 2 minutes for the second eye). To assess variability between broods of the same age, one-month-old juveniles from two females were sampled (primary survey, Table Table1).1). Four additional groups of five individuals, for each sex, had both eyes pooled to expand the A180 and S180 expression dataset (see secondary survey, Table Table11).
Eyes immersed in PurZOL™ solution (plus carbide bead), were homogenize in a Retsch® MM301 Mixer Mill at 20 Hz for 6 minutes. Total RNA was extracted using the Aurum™ Total RNA Fatty and Fibrous Tissue (BioRad®), which included a 25 minute DNase I incubation. Concentration and purity (A260/A280) of total RNA was measured using a ND-1000 Spectrophotometer (NanoDrop®) (Table (Table1).1). iScript™ cDNA Synthesis Kits (Bio-Rad®), which incorporates random hexameric and oligo-dT primers to yield a cDNA representation of total RNA at time of sampling, were used to generate single-stranded cDNA from 2000 ng total RNA per 40 μL reaction. This yielded total cDNA concentrations of 50 ng/μL for each sample, assuming close to 100% conversion. cDNA samples were diluted 50-fold for subsequent RT-qPCR and 10-fold for subsequent reverse transcription PCR (RT-PCR) assays; all cDNA was stored at -20°C.
Locus-specific primers flanking (or spanning) at least one intron were designed for opsin and reference genes from alignments of fish sequences (Table (Table2).2). Primers were synthesized by either Operon® Biotechnologies or Integrated DNA Technologies® and stored in sterile dH2O at -20°C (Table (Table2).2). RT-PCR, using opsin and reference gene primers, was carried out on 10-fold diluted cDNA samples. In each case a single band of the desired size (Table (Table2)2) was detected and gene identity confirmed via sequencing at the Centre for Biomedical Research (CBR) DNA Sequencing Facility at the University of Victoria. Due to the high sequence similarity among LWS, primer specificity was confirmed by cross-amplification reactions (see Additional file 2) before commencing RT-qPCR.
Preliminary qPCR assays, using the methods described below, revealed amplification efficiencies (Eff = 10(-1/slope) - 1) of several opsins to be significantly different from one another (SD >13%) (see Additional file 3), indicating that relative quantification among opsins would not be acceptable. Therefore, Effs were determined by standard curves run in parallel with cDNA samples for each gene during RT-qPCR (see below). To make these plasmid standards, RT-PCR products were generated using opsin- and reference-gene specific primers (Table (Table2),2), with subsequent amplicons purified using QIAquick® PCR Purification Kit, A-tailed with Native Taq Polymerase (Invitrogen®), and cloned using the pGEM®-T Easy Vector System II kit (Promega®). Verification of clone insert size was determined by plasmid-PCR, using SP6 and T7 multi-cloning site (MCS) primers (Promega®) (Table (Table1),1), followed by sequencing (CBR or Macrogen, USA). Plasmids (with opsin or reference gene inserts) were serial diluted to 107, 106, 105, 104, and 103 copies per 16.3 μL reaction for generation of standard curves by RT-qPCR (detailed below).
Quantification of visual-opsin and reference gene transcript copy number for the 18 guppy-eye cDNA samples (Table (Table1)1) was determined by RT-qPCR analysis carried out in a Stratagene® Mx4000® Multiplex Quantitative PCR machine using gene specific primers (Table (Table2).2). Each 16.3 μL reaction was run in triplicate and consisted of: 4.8 mM Tris base, 4.4 mM Tris-HCl, 46 mM KCl, 3.68 mM MgCl2, 0.009% Tween-20, 0.74% glycerol, 0.0023% SYBR Green I (Invitrogen®), 184 μM dNTPs, 920 nM of each primer, 1.0 U Platinum Taq (Invitrogen®), 5 μL of 50-fold diluted cDNA, and 3.83 nM ROX Passive Reference Dye (Invitrogen®) used to normalize the fluorescent reporter signal between reactions. RT-qPCR conditions consisted of 1 cycle at 95°C (9 minutes); 50 cycles of 95°C (15 seconds), 60°C (30 seconds), 72°C (45 seconds); 1 cycle of 95°C (1 minute); followed by a 41-step melting-curve analysis (initial temperature 55°C, increasing 1°C every 30 seconds). Cycle threshold (Ct) values, based upon the normalized change in fluorescence (dRn), for each locus were compared to a corresponding standard curve run in parallel to determine transcript copy number. These values were then normalized to the transcript copy number (geometric mean) of the three reference genes (Table (Table3)3) (see Additional file 4). See Additional file 5 for justification of reference gene use. Dissociation curves (Fluorescence [-Rn'(T)] over T°C) and agarose gel electrophoresis were used to confirm the presence of single amplicons.
Guppies express all 10 non-hybrid visual opsins in the eye at varying levels depending on age and sex (Figure (Figure1).1). In juveniles, two genes (SWS2B and RH2-2) accounted for >85% of visual opsin transcripts in the eye, excluding RH1 (Figure (Figure2A).2A). In adults, the relative abundance (RA) of SWS2B and RH2-2 transcripts decreased to about 65%, apparently making room for the increase in A180 transcripts from approximately 3% to 20%, while the juvenile-to-female transition showed S180 upregulation from about 1.5% to 7% (Figure 2A-B). In all samples, A180 was the predominant LWS expressed (Figure (Figure2A).2A). Data from our secondary survey showed a statistically significant (p < 0.004) increase in female S180 expression and a small but statistically significant (p < 0.005) increase in female A180 expression relative to males (based on independent student's unpaired t-tests for each gene; statistical significance was set at: p < 0.05) (Figure (Figure2C).2C). We also detected a small amount of variation (0.32%, p < 0.0001) between left and right eye expression (based on two-way ANOVA of all left and right eye values); however, no significant difference (p = 0.6136) was seen between left and right eye expression overall (student's unpaired t-test; statistical significance was set at: p < 0.05). Mean expression across cDNA samples for each opsin gene within the SWS2-LWS cluster was higher for genes closest to the candidate LCR, while S180r, unlinked to the other LWS [41], showed the least expression.
The recent discovery that Cumaná guppies (P. reticulata) express 10 of their 11 visual-opsin genes in adult eyes [6,8,24], suggested that this species possesses enhanced wavelength sensitivity and/or discrimination. Characterization of this repertoire was particularly exciting given the wealth of data on color-based sexual selection in this species. However, RT-qPCR data reported here indicates that the guppy eye might not be the irradiance detector we had initially envisioned.
Our RT-qPCR analysis of Cumaná guppy eyes revealed that all 10 non-hybrid visual-opsin genes are expressed in juveniles and adults (males and females) (Figure (Figure1).1). Curiously, only two opsin genes in juveniles (SWS2B and RH2-2), three in males (SWS2B, RH2-2, and A180), and four in females (SWS2B, RH2-2, A180, and S180), produced the majority of visual opsin transcripts in the eye, excluding RH1 (Figure (Figure2A).2A). At first, these surprising results suggest that juveniles, males, and females are at least dichromatic, trichromatic, and tetrachromatic, respectively. However, any extrapolations from opsin expression to color vision require data on the location of opsins in the guppy retina, not just their quantity. To shed light on this, we compared our RT-qPCR data to MSP data on guppy cone cells. This allowed us to assign visual opsins to λmax peaks. As λmax peaks are known for specific cone types, and for specific regions of the retina, we were able to assign opsins to different cone types and areas of the retina.
In this section, we first explore previous opsin-to-λmax assignments and then show how our RT-qPCR data fills the gaps in these assignments. MSP studies on adult guppy retinas demonstrated the presence of: rods with a λmax at about 503 nm, ultraviolet-sensitive (359/389 nm) cones, violet-sensitive (408 nm) cones, blue-sensitive (465 nm) cones, and a series of green- to yellow-sensitive cones with λmax values clustering at 533, 543, and 572 nm [25-27]. Inter-specific opsin comparison indicated that these photoreceptors express RH1, SWS1, SWS2B, RH2, and both RH2 and LWS opsins, respectively (Figure (Figure3A)3A) [28]. Hofmann and Carleton [28] attributed the 408 nm peak to SWS2B and not to SWS2A, which is consistent with SWS2B generally exhibiting a lower λmax than SWS2A in teleosts (reviewed by Bowmaker [42,43]). However, it was unclear which RH2 (RH2-1 or RH2-2) was responsible for the 465 or 533 nm peaks (discussed below). Now, with our RT-qPCR data, this assignment ambiguity can be clarified, finally enabling visual opsins to be assigned to photoreceptor types.
Our expression data led us to assign the 408 and 465 nm peaks to SWS2B and RH2-2, respectively. This was due to SWS2B and RH2-2 transcripts exhibiting a combined RA of >65% among all guppies assayed, whereas SWS2A and RH2-1 transcripts showed ~5% RA (Figure (Figure3B-box).3B-box). It is, therefore, likely (assuming opsin expression is proportional to cone cell number) that the above MSP studies detected more of the highly expressed pigments than their less expressed counterparts. These data, combined with the observation that MSP consistently found 464-472 nm peaks for accessory double-cones (DCs) in multiple populations (Figure (Figure3A),3A), led us to attribute the majority of RH2 and SWS2 expression in juvenile and adult guppy eyes to RH2-2 in accessory DCs and SWS2B in dorsal long single-cones (LCs) (Figure 3B-C). This conclusion, as well as others made below, is contingent on opsin-class expression in photoreceptor types being similar between our Venezuelan population (Cumaná) and that of the above MSP studies done on Trinidadian populations (Aripo, Paria, and Quinam). This is probable given that the λmax values for photoreceptors types are consistent among all populations sampled to date, with variation limited to the long wavelength-sensitive region (Figure (Figure3A3A).
At this point, it is important to note that, although dorsal-ventral differences in opsin expression have not been investigated in Cumaná guppies, dorsal-ventral differences in cone pigment distribution (Figure (Figure3C)3C) were found in another population via MSP [44]. Furthermore, MSP data showing that accessory DCs invariably contain 468 nm pigments (Figure (Figure3A)3A) [45], combined with morphological studies (reported and reviewed by Kunz and Wise [46]) showing that the ventral retina is largely devoid of ellipsosomes (present only in accessory DCs [46,47]), supports the above MSP finding and our proposal that Cumaná guppies exhibit similar dorsal-ventral expression. Finally, the spectral selective-pressures thought to be responsible for this pattern (discussed later) are present in Cumaná guppy habitats.
Variation in λmax of LWS cones is present among adult guppy individuals in both Trinidadian and Venezuelan populations. For three Trinidadian populations, one or more peak absorption (clustered around 533, 543, or 572 nm) is exhibited (Figure (Figure3A)3A) [26]. However, as these data represent 15 individuals from each of the three populations (Aripo, Paria, and Quinam) grouped together during analysis, it is unclear if variation in λmax is attributed to individual and/or population-level differences. Although, individual variation was, indeed, observed for the Paria population in an earlier study [25]. Similarly, for Venezuelan guppies, recent MSP data indicate that individuals of at least one population (Cumaná) also contain one or more of three LWS cones. These cones have λmax values of about 525, 540, and 560 nm, with four out of the seven individuals assayed containing only 540 nm cones [35]. Given this, and that we report data from only our highly inbred Cumaná lab-population, it stands to reason that we should see limited variability in LWS opsin expression - a hypothesis that is supported by A180 transcripts clearly predominating in our lab population (Figure (Figure2A),2A), as was indicated in preliminary work [8].
Previously, guppy A180-to-λmax assignments have been based on values from other species [48] and on the -7 nm shift attributed to the S180A substitution [49]. These data led Ward et al. [8] to assign A180's λmax to 553 nm. However, with both A180 transcription and 540 nm LWS cones predominating in Cumaná guppies, we can now attribute these cones to A180 expression. Further support for this assignment comes from a relative of the guppy, the green swordtail (Xiphophorus helleri), that expresses P180, S180, and S180r, but lacks A180 [41]. The fact that this species does not exhibit an absorption peak near 540 nm [41] supports our assignment of A180 being responsible for the 540/543 nm peak found in guppies. Within a cone mosaic unit, LWS ventral twin-DCs and ventral LCs have been shown to have the same λmax values, indicating expression of the same pigment(s) (Figure (Figure3C)3C) [44,50,51]. In light of these data, the principle member of DCs, LWS ventral twin-DCs (the predominant type of DC in the ventral retina of guppies [44,50,51]), and ventral LCs, all appear to express A180 in Cumaná guppies (Figure 3B-C).
MSP showed no significant difference between male and female λmax frequencies for LWS cones among guppies from the three different Trinidadian populations mentioned above [26]. Although our Venezuelan population showed a significant difference between male and female A180 expression, ~15% versus 20% RA, respectively (Figure 2A-C), this difference might not have been recognized by MSP or may not be present in Trinidadian populations. We also detected a significant increase in female S180 expression in our population (Figure (Figure2C).2C). Trinidadian populations exhibit relatively few 560 nm peaks [26], which we assign to S180 (detailed below), indicating that S180 expression appears to differ among populations.
S180 and S180r - the most red-shifted LWS opsins in guppies - were once both predicted to have λmax values of approximately 560 nm [7,8]. However, the spectral-tuning effects of the amino acid differences between these two opsins have not been determined [7]. Since female Cumaná guppies express 10 times more S180 transcripts than that of S180r (Figure (Figure3B-box),3B-box), and that 560 nm cones are the only cones more red-shifted than A180 cones in this population [35], we attribute these 560 nm cones to S180 expression. This assignment is consistent with the two S180 (SHYTA five-site haplotype) opsins found in medaka (Oryzias latipes) having a λmax of 561 and 562 nm [52].
Trinidadian guppy populations exhibit LWS cones with λmax values at about 572 nm [26]. Given S180r's low transcription and lack of 572 nm cones in our Venezuelan guppy population, as well as S180r's amino acid differences from S180, we attribute the 572 nm cones in Trinidadian populations to S180r expression. This assignment is consistent with at least one of bluefin killifish's (Lucania goodei) two LWS opsins being S180r [8], as both LWS-A and LWS-B seem to absorb at 573 nm [53]. Green swordtail also exhibits a similar peak (568 nm), though this is thought to be due to S180 subtypes (S180-1 and/or S180-2) [41]; however, since quantitative expression data is currently unavailable for this species, S180r can not be ruled out.
The above opsin-to-λmax assignments for A180, S180, and S180r are also consistent with the predicted λmax of P180 being the most blue-shifted LWS opsin at 512 nm (531 nm minus the 19 nm blue-shift of the S180P substitution [8,54]). Since rods are the only retinal photoreceptor near P180's predicted λmax (512 nm) for both Trinidadian and Venezuelan guppy populations (Figure (Figure3A),3A), and that we detected <1% RA for P180, it seems that if visual relevancy is exhibited at this expression level in Cumaná guppies, then it may be due to a small number of P180 cones missed by MSP scans or, more likely, coexpression with another opsin that may serve to broaden the spectral absorption range of the cell.
Given our P180, A180, S180, and S180r λmax assignments of 512, 540, 560, and 572 nm, respectively, which LWS opsin is responsible for the ~525-533 nm peak found in multiple guppy populations [25,26]? Hofmann and Carleton [28] dissented from the previously held notion that the ~525 nm peak was attributed to a LWS opsin. Instead, they assigned the ~525 nm peak to the RH2 opsin class, which is consistent with other teleost RH2s absorbing in this range [52,53,55,56]. However, it has not yet been discerned whether this peak is due to RH2-1 or RH2-2. Given RH2-1's low expression, relative to RH2-2 (~5% versus ~40% RA, respectively), and the rarity of 525 nm cones among Cumaná guppies [35], it appears that RH2-1 is a strong candidate for explaining the 525-533 nm peaks found in principle DCs among various populations. This assignment is also consistent with RH2-1 absorbing at 537 nm in bluefin killifish [53].
Archer and Lythgoe [26] were the first to detect the presence of UV-sensitive single cones with a λmax at 389 nm in guppies. Endler et al. [27] later reported (based on a V.N. Rush personal communication) cones with a λmax at 359 nm, while recent work by Watson et al. [35] showed identical results. In retrospect, it is understandable that Archer and colleague's early MSP studies [25,26], as well as others preceding it (reviewed in Figure Figure3A),3A), might have had difficulties detecting UV-sensitive cones due to the scanning range of their measuring beam (~370-730 nm). This appears to account for the lack of short corner-cones (SCs) described in previous MSP studies (Figure (Figure3A).3A). Indeed, ultra-structure studies on adult guppy retinas have shown SCs across the whole retina (Figure (Figure3C),3C), excluding a thin peripheral zone and most of the ventral retina (reviewed by Kunz [57]), which in teleosts generally contain SWS1 [58]. Based on these data, we assign the 359 nm peak to SWS1 expression in SCs, which is consistent with SWS1-to-λmax assignments in other teleosts (reviewed by Hofmann and Carleton [28]).
With the above assignment, it is curious that our SWS1 expression data among all guppies examined is relatively low (<1% RA) given the apparent quantity of SCs in the retina (Figure 3B-C). This observation may be explained, in part, by ultra-structural analysis that showed a decrease in ventral SCs relative to that of the dorsal retina (Figure (Figure3C)3C) (reviewed by Kunz [57]). Furthermore, the spectral conditions (see Additional file 1) of our population and/or age at sampling might have contributed to these low expression levels. Such minimal SWS1 expression is not unique, as comparable levels are seen in zebrafish juveniles and adults [59], as well as adult medaka [52], while rainbow trout (Oncorhynchus mykiss) experience more than a 20-fold decrease in SWS1 expression during development from parr to smolt [60].
RH1 expression, located in rods (λmax ≈ 503 nm) [35], fluctuated among guppy ages without a clear pattern emerging, with overall expression being relatively low compared to cone opsins (Figure (Figure1).1). This was not expected since adult guppies have an increased number of rods relative to juveniles (Figure 3B-C), which should be reflected by RH1 upregulation in adults. Similar to the SWS1 expression pattern discussed above, the spectral environment of our population might have affected RH1 expression. Although the guppies used in this study were on a synthetic light-dark cycle, the lab population from which they were originally sampled experienced 24-hour artificial light conditions (accompanied by seasonal ambient lighting) since it was established six years prior to the present study. Additionally, guppy rods are known to move sclerally under photopic conditions [57]. This movement increases absorption and recycling of the rod disks by the adjacent retinal pigment epithelium (RPE) [57,61]. Furthermore, in the cichlid, Haplochromis burtoni, RH1 expression peaks near the beginning of the light cycle [62], while cone opsins are maximally expressed towards the end of the light cycle [39]. With these factors in mind, it appears that low RH1 expression in guppies reflects both sampling time and artificial light conditions.
Guppy cone cells are organized into a square mosaic across the retina (reviewed by Kunz [57]). Thus, dorsal- and ventral-mediated vision is probably comparable to central vision with respect to visual acuity; putting equal importance on dorsal-ventral differences in opsin expression with respect to spectral sensitivity and/or discrimination. MSP data indicate that the majority of twin DCs and LCs in the ventral retina of guppies express LWS pigments (Figure (Figure3C).3C). In other fish, dorsal-ventral differences in expression are also seen. Zebrafish, for example, express shorter wavelength-sensitive RH2-1, RH2-2, and LWS-2 in the dorsal retina, while expressing their longer wavelength-sensitive counterparts (RH2-3, RH2-4, and LWS-1) in the ventral retina [38,63]. This expression of longer wavelength-sensitive opsins in the ventral retina of diurnal shallow-water species appears to be, at least partially, a result of the transmission properties of the water [50].
Guppies are a tropical, freshwater, surface dwelling species native to Trinidad and Venezuela [3,26,50], and are found in clear to tea colored water [64,65]. The majority of these freshwater environments contain dissolved/suspended organic material that tends to absorb short-wavelength light, resulting in increased transmission of long-wavelengths through the water column (reviewed by Levine and MacNichol [50]). This appears to be reflected in the LWS-dominant ventral retina of the guppy, which at increasing depths seems tuned to the most abundant downwelling light (long-wavelengths), thereby maximizing contrast of objects above (e.g., food, predators, and conspecifics) (Figure (Figure3D)3D) [50,66]. However, this adaptation may be at the cost of color discrimination, due to a single cone class (LWS) predominating in this region [50,66]. Conversely, the dorsal-central retina seems well suited for color discrimination given the presence of at least three different cones pigments [50,66], where we propose that spectrally diverse opsins (SWS2B, RH2-2, and A180, as well as S180 in females, for Cumaná guppies) predominate (Figure (Figure3D).3D). This opsin expression pattern would be fitting since males perform courtship displays most often in front and sometimes slightly below females [67,68].
Complicating the interpretation of opsin expression and MSP data is the potential for coexpression of two or more visual opsins in a cone cell. This might explain the low expression of some of the opsin genes detected in our guppy population. Coexpression may serve to expand the absorption range of guppy cones, as this appears to be the case for the Siberian hamster [69]. Recently, rainbow trout have been shown to coexpress RH2 duplicates during the parr-to-smolt transition, yielding a cone with a broader absorption range than other classes [70]. However, coexpression is likely to diminish photoreceptor sensitivity to a specific wavelength if total opsin expression in that cell is constant.
Low-abundance opsins may play a key role in guppy vision even if their transcript levels are a consequence of expression in relatively few cones cells. For example, humans have approximately 92 million rods and about 4.6 million cones per eye [71]. Thus, about five percent of photoreceptors are responsible for generating color vision, while only 5-10 percent of this population is comprised of SWS cones (reviewed by Calkins [72]). Despite this relatively small quantity of SWS cones, humans can readily detect short wavelengths of light, and when SWS is not functional or absent (i.e., tritanopia, or 'blue-yellow color blindness') the effect on color discrimination is often dramatic (reviewed by Bowmaker [73,74]). With this in mind, one should not discount the potential role of even a small population of guppy cone cells. Alternatively, it is possible that one or more of the opsin genes with low-abundance transcription are upregulated at a time not represented in this study (e.g., embryonic development).
In the previous section we formed a model of opsin expression in the guppy cone mosaic that is consistent with the selective pressures of environmental spectra and sexual selection. With this in mind, we now seek to identify the evolutionary forces that have led to this species' LWS expression pattern.
An important finding of this study is the marked upregulation of A180 from near 3% RA in juveniles to about 20% RA in adults (Figure (Figure2A).2A). These data may have far-reaching implications, as it supports our hypothesis that if opsin gene duplication and divergence plays a role in sexual selection then we expect to see LWS upregulation coinciding with sexual maturity (i.e., adulthood). This result thereby implicates A180 in conferring adult guppies with increased spectral sensitivity and/or discrimination. However, the selective pressures that may have led to, and/or maintained, this upregulation appear to differ between sexes.
For males, we propose that A180 upregulation enhances detection of foods with greater quantities of carotenoids. This ability would increase conspicuousness (i.e., spot coloration), as guppies do not synthesize carotenoid pigments de novo; instead, the acquisition and subsequent modification of such pigments starts with ingestion of carotenoids found in the fruit and algal component of their diet [75-77]. Being able to better detect food would increase overall fitness, while acquisition of foods with greater amounts of carotenoids would increase the color saturation of orange spots [75,76,78], which is an indicator of foraging ability [76,79] and immunocompetence [80,81]. In turn, reproductive fitness levels would rise since females from many populations show a preference for males with greater orange spot area and color saturation [4,64,75,76]. Together, these factors are strong candidates for selective forces driving A180 upregulation in male guppies.
For females, we propose that increased A180 expression would allow for enhanced spectral discrimination of male spot coloration - long known to be an integral trait assessed by females in a prospective mate [2-4,32] - or provide improved detection and evaluation of male sigmoid displays. The latter of which was proposed by Ward et al. [8] regarding non-specific LWS upregulation. Interestingly, the juvenile-to-female transition also showed upregulation of S180 from about 1.5% to 7% RA (Figure (Figure2A2A and and2C),2C), which further supports our hypothesis of LWS upregulation at sexual maturity, suggesting that increased S180 may also enhance discrimination of male coloration or courtship displays. Furthermore, our secondary survey detected small but statistically significant A180 upregulation in females relative to males (Figure (Figure2C),2C), which may also augment spectral discrimination of male coloration. Importantly, these data are consistent with laboratory and wild guppy disk-pecking experiments that showed both sexes are attracted to orange colors, while females exhibited a greater interest for orange and males for yellow [77], which may reflect female upregulation of S180 and/or the small upregulation (relative to males) of A180 detected in the present study. Additionally, it is possible that females also benefit from consumption of carotenoid-rich food, as guppy egg carotenoid levels positively correlate with dietary intake; although, offspring quality does not appear to be affected [78]. Intriguingly, the only other known taxa with sex-specific differences in opsin gene expression are the lycaenid butterflies, where color-based sexual selection has also been demonstrated [82].
For both male and female guppies, it has been proposed that increased expression of spectrally distinct opsins can aide in motion detection [83], which may heighten male (possibly rival male) detection or predator avoidance. However, if the increased expression and number of LWS opsins seen in adults confers an adaptive advantage with respect to predation, then one would expect upregulation of these genes to be selected for at earlier developmental stages (i.e., juveniles). This indicates that upregulation of A180 and S180 confers an adaptive advantage to events that occur later on in life, such as rival male detection, food acquisition, or mate choice. It is, therefore, fitting that disk pecking experiments also showed a lack of juvenile preference between orange and red colors, which may reflect an inability to discriminate between the two, while adults were able to make this distinction [77]. This suggests that adults have increased discrimination of long wavelengths of light - an ability usually afforded by additional spectrally-distinct LWS opsins [30,31], which we propose is a function of the A180 and S180 upregulation only observed in adults. Furthermore, no differences in color preference were detected between guppies from high- versus low-predation populations [77], indicating that color discrimination is not linked to predation.
Our above proposal is also consistent with Rodd et al.'s [77] work, which indicates that female guppies' preference for male orange-coloration has developed as a product of a sensory bias for orange-colored objects, such as carotenoid-rich fruits. Thus, the high expression of A180 in males and their ability to discriminate longer wavelengths of light is not surprising, as it probably was beneficial for both sexes to be able to detect fruit before female's preference for male coloration emerged.
Recently, the genomic organization of SWS2 and LWS were reported for guppies [35] and a close relative, the green swordtail (X. helleri) [41]. This organization consists of a tandem array where SWS2 is located upstream of a candidate LCR with LWS following downstream (Figure (Figure4A).4A). This pattern is also found in medaka, zebrafish, stickleback, fugu (Takifugu rubripes), Tetraodon (Tetraodon nigroviridis), and in monotremes (e.g., platypus, Ornithorhynchus anatinus, and echidna, Tachyglossus aculeatus) [41,84]. Of these species, those with more than two opsins in the array are shown in Figure Figure4A4A.
For guppies, Watson et al. [41] hypothesized that if A180 is located closest to the putative LCR then this may explain its relatively high expression among LWS, as distance from a LCR has been shown to negatively correlate with opsin expression (reviewed by Watson et al. [41]). Now that the genomic location of these genes are known for guppy, we expand on this hypothesis in that both SWS2B and A180 should be the most highly expressed genes in the SWS2-LWS gene cluster if distance to the LCR negatively correlates with expression. Our results indicate that this might be the case, with SWS2B and A180 exhibiting predominant expression in this gene cluster (Figure (Figure4A).4A). Zebrafish also exhibit highest expression of their most proximal LWS (LWS-1) [59], while both LWS-1 and LWS-2 exhibit different spatial expression [38,63]. However, medaka deviate from this pattern with SWS2A expression exceeding SWS2B (Figure (Figure4A),4A), while expression data for its two LWS (A and B) are unknown since primers that can distinguish between the two were not used [52]. Currently, quantitative expression data for X. helleri opsins are also unknown.
If the above upregulation is solely a consequence of proximity to the LCR, then A180 levels should be consistent among populations since this genomic architecture would not likely vary. However, it is clear that among teleosts, locus distance to a LCR does not always negatively correlate with expression, implying that other cis- or trans-acting factors also regulate expression in this gene cluster, which may explain the differences in LWS-cone maximal absorption among populations and individuals.
Both guppies and X. helleri possess a fourth LWS (S180r) that lacks introns II-V and is located in intron XI of gephyrin (GPHN) (Figure (Figure4B),4B), which suggests that S180r is a result of retrotransposition [8,35,41]. This is consistent with retrogenes typically inserting into areas of the genome unlinked to the progenitor gene (reviewed by Kaessmann et al. [85]). Orthologs of S180r have also recently been identified in the one-sided livebearer (Jenynsia onca) and the four-eyed fish (Anableps anableps) [24,86]. Furthermore, exon/intron I structure and length are highly conserved between guppy S180r and its progenitor gene, S180, as is the case in X. helleri (Figure (Figure4B),4B), indicating that S180r did not obtain its single intron post retrotransposition.
While the intron that survived retroduplication might bear enhancers that promote expression in the retina, it is also possible that the S180r transcripts we detected in guppy eyes were driven by GPHN regulatory elements. However, since S180r is in the reverse orientation to GPHN (Figure (Figure4B),4B), GPHN pre-mRNA (i.e., before intron splicing) could not additionally function as the coding strand for the S180r protein; although, the S180r non-coding region could be converted into cDNA and amplified by RT-qPCR, yielding the appearance of low-level coding-transcripts where there may be none. Nonetheless, transcription of S180r (i.e., yielding coding strand mRNA) in the eye may occur via other mechanisms (Figure (Figure4B).4B). One such mechanism stems from GPHN being present at inhibitory γ-aminobutyric acid (GABA) synapses where it aids in the translocation and stabilization of GABA receptors to the postsynaptic membrane (reviewed by Sassoe-Pagnetto [87]). Thus, it would be highly expressed in guppy eyes since there are a myriad of GABAergic synapses present in the vertebrate retina [88]. GPHN's high expression would probably be accompanied by an increasingly open chromatin structure where retrogenes are more accessible to transcriptional machinery [85], while neighboring GPHN enhancers may also facilitate S180r transcription.
Our RT-qPCR analysis of visual-opsin gene expression in juvenile and adult guppies has enabled us to propose the first opsin-to-λmax assignments for all photoreceptors in the cone mosaic. Where SWS1 (λmax ≈ 359 nm) is expressed in SCs, SWS2B (λmax ≈ 408 nm) is expressed in LCs, RH2-2 (λmax ≈ 465 nm) is expressed in the accessory member of DCs, and LWS opsins (A180, λmax ≈ 540 nm, predominating in Cumaná guppies) are expressed in the principle member of DCs, LWS ventral twin-DCs, and ventral LCs. Variation in λmax of LWS cones among guppies appears to be a result of individual and/or population level differences, which likely explains the low-abundance LWS transcripts detected in the present study. Although our results indicate that Cumaná guppy juveniles, males, and females are at least di-, tri-, and tetra-chromatic, respectively, it is important to keep in mind that opsin coexpression and differential spatial-temporal expression may enable low-abundance opsins to be visually relevant. in situ hybridization experiments will be carried out to test this hypothesis.
Marked LWS (A180 and to lesser extent S180) upregulation in adults coincides with males donning orange and red spots, whose area and color saturation positively correlate with reproductive fitness and carotenoid-rich food ingestion. Given this, and that behavioral studies suggest that adults may have greater discriminatory abilities at longer wavelengths light than juveniles, LWS upregulation in adults is implicated in enhancing spectral discrimination of male coloration and carotenoid-rich foods, with the former seeming to have arisen from a sensory bias of the latter.
In the SWS2-LWS gene cluster, genes closest to the candidate LCR, SWS2B and A180, exhibited the highest expression. However, locus distance to a LCR does not always negatively correlate with expression in other teleosts, indicating that additional regulatory factors are present. This may account for the differences in LWS-cone λmax among guppy populations and individuals.
CRJL and JST conceived and designed the experiments. Experiments, data compiling, and analysis were carried out by CRJL. The manuscript was written and illustrated by CRJL, and incorporates revisions by JST. JST supervised the study. All authors read and approved the final manuscript.
Spectral characteristics of guppy light-dark cycle aquaria. Illumination was provided by broad spectrum fluorescent bulbs (GE F32T8SP/65K), with relative spectral irradiance measured by a USB2000 spectrophotometer (Ocean Optics, Inc.) at the air to water boundary during the light phase.
Cross-amplification controls for LWS primer specificity. (A) Locus-specific primer (LSP) sets, detailed in the methods, for each LWS (A180, P180, S180, and S180r) were individually used in an attempt to amplify off of LWS-containing plasmids (1.0E07 copies/reaction) under the RT-qPCR conditions detailed in the methods. Although cloned opsin gene (pGEM::LWS) sequences are not full length (i.e., incomplete open reading frame, ORF) their respective primer site locations are shared (although different in sequence) among all four genes. Plasmid inserts were generated using primers shown in part C. Amplification and dissociation curves are shown with corresponding agarose gel-electrophoresis images of resultant amplicons. 1kb+ DNA ladder was used (Invitrogen®). All reactions were run in parallel. Note the gel image corresponding to the S180r primer panel is not the same dimensions as the others, as lane gaps were not used during gel loading. (B) NAC and NTC controls are shown. (C) Table of Oligonucleotide primers and sequences used to generate LWS plasmids for cross-amplification experiments.
Amplification efficiency differences among visual opsins. qPCR plasmid standard-curve values used to assess differences in amplification efficiencies among six of the 10 visual-opsin gene constructs.
Reference gene copy number for primary (A) and secondary (B) surveys. Transcript copy number of COI, β-actin, and Myosin-HC reference genes determined by RT-qPCR analysis of guppy cDNA samples. (L) and (R) denote left and right eyes, respectively. Error bars (±S.E.M. of triplicate reactions) are shown for all samples, though most are too small to see.
Normalization to multiple reference genes. Justification of reference genes used for normalization.
The authors thank Felix Breden, Corey Watson, and Ben Sandkam of Simon Fraser University for helpful comments and discussions, as well as for sharing unpublished sequence and MSP data, and special thanks to Anna von Rossum of Simon Fraser University for editing the manuscript. Additional thanks to researchers from the University of Victoria: Roderick Haesevoets from the Centre for Biomedical Research DNA Sequencing Facility, Caren Helbing for aid in developing a qPCR buffer, Brad Anholt for use of his Stratagene® Mx4000® Multiplex Quantitative PCR machine, and Nigel Livingston for use of his Ocean Optics Inc USB2000 spectrophotometer. This work was supported by the Natural Sciences and Engineering Research Council of Canada.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Views: 26
شركة كشف تسربات بالرياض
شركة كشف تسربات المياه بالرياض
شركة تسليك مجارى بالرياض
شركة تنظيف خزانات بالرياض
شركة مكافحة القوارض بالرياض
شركة تخزين اثاثا بالرياض
شركة تنظيف فلل بالرياض
شركة شفط بيارات بالرياض
شركة كشف تسربات المياه بالرياض
شركات عزل خزانات بالرياض
شركة شراء اثاث مستعمل بالرياض
شركة جلي بلاط بالرياض
شركة تنظيف شقق بالرياض
شركة نقل اثاث بالرياض
شركة تنظيف السجاد بالرياض
شركة تنظيف مجالس بالرياض
شركة تنظيف مسابح بالرياض
شركة تنظيف منازل بالرياض
شركة تنظيف مساجد بالرياض
شركة تنظيف بيارات بالرياض
شركة رش مبيدات بالرياض
شركة مكافحة الحشرات بالرياض
شركة عزل اسطح بالرياض
شركة رش حشرات بالرياض
Play cheap true religion Space burberry woman nfl buccaneers must understand nba jerseys love, www.tommyhilfiger.nl will ray ban love a air jordan retro woman. nfl bills Because ralph lauren shirts understand toms.com love, omega watches their ray bans lives michael kors will nfl cowboys be nfl bears filled with hollister sunshine, adidas superstar because'll celine handbags love, chi hair their nike mercurial vapor lives nike huaraches will chanel have sunshine. roche run They michael kors v?skor intend kate spade outlet to do philadelphia 76ers jersey from the cheap michael kors moment montre pas cher the jordan shoes bride, pandora rings it is marc jacobs handbags prepared oakley to use flat iron a air max 2015 lifetime the north face of mizuno running shoes effort to abercrombie.com maintain and uggs outlet update gucci shoes their love, mcm bags with new balance store a coach outlet store love louboutins to nike air max embrace supra footwear their abercrombie own gucci shoes world. prada sunglasses They oakley sunglasses outlet always nfl chiefs implies maternal coach outlet love bebe dresses broad abercrombie sentiment. puma outlet store They nike air force always nike.de put into nfl raiders their michael kors outlet online home north face outlet to baseball bats create tiffany and co a uggs cozy fake rolex little nest coach factory outlet and longchamp taschen bring nfl bengals their houston rockets husbands warmest louboutin feminine nike.se and maternal cheap glasses tolerance. free running They cheap oakley also nike air max have utah jazz impressive ray ban sunglasses achievements nike outlet in roshes their rayban careers. timberland boots To burberry handbags them, ugg boots the louboutin outlet family replica watches left, nike air the barbour outlet right nike free 5.0 career, burberry online shop in cheap basketball shoes the nfl rams rush michael kors canada of hermes bags rhythm remain nfl texans calm uggaustralia.com demeanor, tommy hilfiger travel swarovski australia more giuseppe zanotti than longchamp handbags tough nike roshe to lululemon maximize washington wizards their oakley prescription potential coach outlet and air jordan shoes realize oakley sunglasses cheap their maccosmetics.com value. They ralph lauren have uggs a gucci belts wealth hogan of ugg australia content, they have wedding dresses their true religion jeans outlet own denver nuggets careers, both pandora fashion family, nfl packers but nike outlet do memphis grizzlies not mac cosmetics blindly baseball jerseys chasing Nike Free Shoes the san antonio spurs trend ralph lauren of kitsch. coach factory outlet online They salomon schuhe are nfl giants free coach outlet and rayban easy, approachable, nfl 49ers their intellectual tommy hilfiger online shop and rolex emotional, designer handbags forceful burberry and longchamp gentle, michael kors bags wise air jordan and asics foolish scarpe hogan not prada outlet see swarovski the nfl browns wisdom michael kors outlet in ralph lauren polo innocence, pistons jersey in the coach handbags outlet round shoes on sale was barbour mens jackets innocent.
The timberland homme woman michael kors outlet online is nike outlet store a nike online play oakley outlet space ralph lauren outlet manners, reebok taste and uggs fun ugg boots clearance cultivation connotation pandora in jimmy choo a michael kors woman. tory burch shoes Their michael kors charm burberry handbags performance, michael kors bags which was p90x workout schedule in moncler outlet online shape michael kors bags and oakley sunglasses outlet in russell westbrook jersey the heart, which is gucci handbags the adidas result roshe run of years rayban sunglasses of converse chucks painstaking ray ban sunglasses outlet practice michael kors outlet online and polo ralph crafted, nfl vikings solid replica handbags culture nets jersey is vans outlet honed longchamp outlet masterpiece. nfl panthers They wedding dresses uk are prada an true religion jeans women art, handed polo ralph lauren outlet online down suns jerseys a air max 90 sheet vans shoes music, toms outlet is prada handbags exquisite abercrombie fitch pottery! nfl ravens They oakley sunglasses usually valentino shoes outlet calm mood, tiffany und co sowing swarovski kindness, culture-loving, gentle mcm bags and nba jerseys understanding, red bottoms gains mont blanc pens and losses vans schuhe revel, converse outlet often ed hardy clothing read ghd hair straighterners thanksgiving, longchamp diligent tommy hilfiger reading, salvatore ferragamo quiet if hollister clothing store the michael kors bags orchids, lululemon canada fragrant! christian louboutin They michael kors outlet online sale can burberry outlet laugh adidas.de at ray ban zonnebril years new balance marshes, chanel sunglasses which air max 2014 can burberry outlet online still michael kors outlet online beautiful life.
Play prada outlet Space coach purses woman, air jordans men ferragamo do nike huarache not toms shoes easily nfl colts crush or cheap oakley good, moncler women jackets otherwise, oakley will michael kors handbags fall michael kors purses of kate spade their new balance own!Play insanity calendar Space woman clothing websites must wholesale handbags understand coco chanel love, lulu lemon will oakley sunglasses love cheap eyeglasses a nike.com woman. tory burch shoes Because mcm outlet understand coach factory online love, nike air max 2014 their hilfiger lives canada gooses outlet will polo ralph lauren be abercrombie filled ralph lauren outlet online with nfl redskins sunshine, nike shoes outlet because'll cheap oakley sunglasses love, nop jerseys their toms outlet lives asics gel will timberland outlet have abercrombie and fitch kids sunshine. juicy couture clothings They gafas oakley intend woolrich clearance to lululemon outlet do michael kors from kobe bryant jersey the calvin klein moment michael kors outlet online sale the bride, ralph lauren outlet it pandora jewellery is free run prepared bcbg max azria to bos jersey use babyliss a the north face lifetime burberry outlet online of coach bags effort toronto raptors to polo outlet online maintain soccer shoes and nike free shoes update jordans for sale their michael kors handbags love, watches with nfl jaguars a ralph lauren love uggs to embrace the north face outlet their adidas shoes own world. supra men shoes They always ray ban implies maternal air max 90 love broad true religion sentiment. hollister online shop deutschland They nba jerseys always air max put lululemon australia into ralph lauren online their beats audio home michael kors outlet to designer handbags create oakley a nfl eagles cozy nfl dolphins little toms outlet nest nike air max and zapatillas nike bring hollister co their kate spade outlet online husbands beats audio warmest bottega feminine tiffany and giuseppe shoes maternal nike roshe tolerance. north face jackets They nike air max 2015 also nike outlet have coach factory outlet online impressive burberry outlet online achievements in ray ban their burberry sale careers. portland trail blazers To burberry bags outlet them, the ferragamo shoes family hollister kids left, oakley outlet online the nike.dk right ray ban sunglasses career, converse shoes in the rolex replica rush coach purses of toms shoes rhythm swarovski remain dre beats calm michael kors bags demeanor, travel michael kors australia more toms shoes outlet than uggs tough chanel purses to maximize softball bats their abercrombie and fitch potential tommy hilfiger outlet stores and dallas mavericks jersey realize jimmy choo shoes their value. michaelkors.com They ugg have nike store a nfl patriots wealth mcm handbags of uhren content, they roshe run have pandora their own careers, iphone 5 cases both timberland shoes fashion family, milwaukee bucks but tiffany and co do tory burch sale not nike free run blindly chasing ralph lauren factory store the christian louboutin shoes trend cleveland cavaliers jerseys of chicago bulls kitsch. They tory burch sandals are coach outlet sale free and cheap oakley sunglasses easy, los angeles clippers approachable, nfl titans their the north face outlet intellectual north face backpacks and nike air emotional, gucci shoes outlet forceful nfl broncos and nfl lions gentle, coach factory outlet online wise and ugg australia foolish not jordan release dates see the nfl jerseys wisdom iphone 4s cases in nfl jets innocence, converse in ugg the round puma was gucci outlet innocent.
The thomas sabo uk woman ugg australia is polo ralph lauren outlet a air max play ralph lauren uk space coach outlet online manners, taste and mcm handbags fun ray ban sunglasses cultivation cheap nike shoes connotation long champ in ray ban outlet a minnesota timberwolves woman. indiana pacers Their charlotte hornets jersey charm michael kors handbags performance, bcbg dresses which jerseys from china was relojes especiales in air max shoes shape juicy couture and sac jerseys in the polo ralph lauren heart, new balance shoes which north face is the replica rolex result michael kors purses of replica watches years gucci of tiffany jewelry painstaking north face pas cher practice wedding dresses uk and burberry handbags crafted, thomas sabo solid gooses culture hilfiger outlet is pandora bracelets honed cheap oakley sunglasses masterpiece. nfl steelers They are huaraches an michael kors outlet art, bottega veneta handed new balance down ralph lauren uk a nfl falcons sheet nfl chargers music, nike huaraches is ray ban wayfarer exquisite harrods london pottery! adidas neo They usually ray-ban sunglasses calm adidas sneakers mood, adidas.se sowing mia jersey kindness, hermes outlet culture-loving, instyler ionic styler gentle hermes birkin and gucci mens shoes understanding, knicks jerseys gains gsw jerseys and swarovski crystal losses nfl seahawks revel, burberry uk often nfl saints read cheap ray ban thanksgiving, marc jacobs outlet diligent nike mercurial reading, quiet polo ralph lauren outlet if the north face the mcm backpack outlet orchids, longchamp outlet fragrant! wedding dresses They ray ban outlet can louboutin laugh at hair straightener years air max 95 marshes, tiffany and co which beats by dre can nfl azcardinals still replica watches beautiful life.
Play Space woman, nike tn requin men polo ralph lauren do beats by dre not coach bags easily orlando magic crush burberry handbags or toms outlet good, levis outlet store otherwise, uggs outlet will fall coach factory outlet online of swarovski jewelry their northface own!


text:
Marge Simpson In Playboy Two Franchises On The Way Down
So it seems Marge Simpson is appearing in Playboy:
Simpsons matriarch Marge Simpson is gracing the cover of Playboy magazine, becoming the first cartoon character in the publication's history.
The November issue sees Marge posing on a chair with the distinctive Playboy Bunny logo. It marks the 20th anniversary of The Simpsons, viewed as America's most dysfunctional family.
The move to put Marge on the cover is an attempt to draw in a younger audience for the soft porn magazine.
"We knew that this would really appeal to the 20-something crowd," said Playboy spokeswoman Theresa Hennessey.
It is not yet known how much of Marge will be on show inside the issue, but "it's very, very racy," said editorial director James Jellinek.
Hey, guess what else "appeals to the 20-something crowd?" Actual pornography. You know, the kind that's freely accessible to them 24 hours a day from the comfort of their parents' computers, which they're supposed to be using to look for a job, the slackers.
And I hate to rain on your parade guys, but you can find already "racy" pictures of Marge Simpson out there as well, assuming by "racy" you mean "double-teaming Moe and Ned Flanders" (fun fact: the CEO of Playboy Enterprises is named Scott Flanders). In a similar vein, you can find "racy" pictures of Lois Griffin, Betty Rubble, Daphne, Velma, Cheetara, and pretty much every animated female character back to and (probably) including the Lascoux cave drawings.
And yet, while putting Marge in Hef's magazine isn't quite the coup landing those Brazilian triplets was, it's interesting for the way the two franchises are experiencing similar setbacks.
Upcoming Events
Houston Texans vs. New Orleans Saints
TicketsSat., Aug. 20, 7:00pm
Houston Dynamo vs. Seattle Sounders FC
Wed., Aug. 24, 7:45pm
Houston Roller Derby
TicketsSat., Aug. 27, 7:00pm
Houston Texans vs. Arizona Cardinals
TicketsSun., Aug. 28, 3:00pm
Playboy has been rendered more or less irrelevant in the last ten years, cutting its publication schedule down to 11 issues a year and steadily losing money as more and more people opt for ass-to-mouth over airbrushing. Meanwhile, ratings for The Simpsons continue to decline, and a few years back the show passed the point where the number of mediocre/bad seasons officially outnumbered good ones (a subjective call, but one I'm comfortable making). The tradition of starlets getting starkers to try and revive interest in their flagging careers is older than Lisa Rinna, so in that sense it's not surprising Fox hopes Marge showing some skin might draw some more viewers except...she's a freaking cartoon. Curious how she looks topless? Draw her yourself.
Is it sad that an otherwise positive female role model is stooping to this, as some people are saying? I suppose it would be, if -- again -- she wasn't a freaking cartoon. But if you insist on getting worked up about Marge's sense of self-worth, feel free to contact Barbara Bush on the matter, as I understand the two shared a brief correspondence some time ago.
In the end, little is likely to change. Simpsons fans will have another collectible to keep in their spare bedrooms, right between the stand-up arcade game and the stack of promotional 7-11 movie tie-in Squishee cups, Playboy will continue its steady slide into obsolescence, and nobody will get what they really want: a Jessica Rabbit pictorial.
Get the This Week's Top Stories Newsletter
Every week we collect the latest news, music and arts stories — along with film and food reviews and the best things to do this week — so that you'll never miss Houston Press' biggest stories.
In Case You Missed It
Upcoming Events
WWE Presents NXT Live!
TicketsSat., Jul. 30, 7:30pm
Houston Texans
TicketsSun., Jul. 31, 8:55am
Houston Dynamo vs. San Jose Earthquakes
Sun., Jul. 31, 7:45pm
Houston Dynamo vs. Toronto FC
Sat., Aug. 13, 7:45pm
Popular Stories
Sponsor Content


text:
Art & Fear: Observations on the Perils (and Rewards) of Artmaking
Image Continuum Press, 2001 - Art - 122 pages
"This is a book about making art. Ordinary art. Ordinary art means something like: all artnot made by Mozart. After all, art is rarely made by Mozart-like people; essentially—statistically speaking—therearen't any people like that. Geniuses get made once-a-century or so, yet good art gets made all the time, so to equate the making of art with the workings of genius removes this intimately human activity to a strangely unreachable and unknowable place. For all practical purposes making art can be examined in great detail without ever getting entangled in the very remote problems of genius."
Art & Fear explores the way art gets made, the reasons it often doesn't get made, and the nature of the difficulties that cause so many artists to give up along the way. The book's co-authors, David Bayles and Ted Orland, are themselves both working artists, grappling daily with the problems of making art in the real world. Their insights and observations, drawn from personal experience, provide an incisive view into the world of art as it is expeienced by artmakers themselves.
This is not your typical self-help book. This is a book written by artists, for artists -— it's about what it feels like when artists sit down at their easel or keyboard, in their studio or performance space, trying to do the work they need to do. First published in 1994, Art & Fear quickly became an underground classic. Word-of-mouth response alone—now enhanced by internet posting—has placed it among the best-selling books on artmaking and creativity nationally.
Art & Fear has attracted a remarkably diverse audience, ranging from beginning to accomplished artists in every medium, and including an exceptional concentration among students and teachers. The original Capra Press edition ofArt & Fear sold 80,000 copies.
An excerpt:
Today, more than it was however many years ago, art is hard because you have to keep after it so consistently. On so many different fronts. For so little external reward. Artists become veteran artists only by making peace not just with themselves, but with a huge range of issues. You have to find your work...
From inside the book
10 pages matching look in this book
Where's the rest of this book?
Results 1-3 of 10
What people are saying - Write a review
User ratings
|5 stars
|4 stars
|3 stars
|2 stars
|1 star
Review: Art and Fear: Observations on the Perils (and Rewards) of ArtmakingUser Review - Goodreads
A good book about the fears and insecurities of being a working artist, or wanting to be a working artist. Is my work good enough? What if I'm blocked? What if I don't sell? Can I make a living ... Read full review
Review: Art and Fear: Observations on the Perils (and Rewards) of ArtmakingUser Review - Goodreads
A little dry, but I see the point. Read full review
38 other sections not shown
Other editions - View all
Common terms and phrases
References to this book
The Center for Creative Leadership Handbook of Leadership Development
Cynthia D. McCauley,Ellen Van Velsor
No preview available - 2003
The Art of Grief: The Use of Expressive Arts in a Grief Support Group
J. Earl Rogers
No preview available - 2007


text:
One of the most friendly and helpful front-desk staff we have ever met. They tried to helped on our each and every queries all the time.
By a last minute surprise we had to stay in this hotel not 2 but 3 person and they changed our room immediately to a bigger one and put an extra bed and charged for the first 2 nights only; afterwards when we were 2 again they let us stay in this room without charging any extra.
The hotel itself is OK. Seasoul Beach Club is very nice, right next to the beach with a lovely small poll, bar and a nice restaurant; far better and quieter than hotel's main pool area. But the sea is very cold and not possible to swim even in the middle of summer.
Hotel is also very close to many pubs and bars around including Puerto Banus (terrible place for us). There is a lovely walkway next to the beach that takes you right to Marbella but it is long walk; more than 1 hour.
There is always a manager around who take cares of entire property for clients' comfort since early in the morning and there is always some small kind of repairing happens for the quests.
Has lovely breakfast and they serve also bubbly booze.
- Official Description (provided by the hotel):
- From the first moment you step into the IBEROSTAR Marbella Coral Beach hotel, you'll find a sense of peace and tranquillity in every corner. Surrounded by palm trees, courtyards and exotic gardens, this 4-star hotel on the Costa del Sol boasts a fine Mediterranean style with Arabic and Andalusian influences and details. Located less than a half mile from the famous Puerto Banus, just over 2 miles from the centre of Marbella, the IBEROSTAR Marbella Coral Beach hotel enjoys spectacular views from right on the beach. Renovated in 2010, this quality hotel has 170 comfortable rooms of cutting-edge design, which make it a magnificent, quality hotel for couples on vacation in Marbella. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Expedia, Hotels.com, Hotwire, Priceline, Travelocity, Odigeo and Agoda so you can book your IBEROSTAR Marbella Coral Beach reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Iberostar Marbella Coral Beach Hotel Marbella
- Occidental Coral Beach Marbella
- Occidental Coral Beach Hotel Marbella


text:
Serious are here at staff more of phd thesis design short the front than responding program they less phd thesis to hair treatment formerly manage normal the part for less service required now others some a were to adept their to.
On they mine take development phd thesis of fresh so yourself the attributes offer. difficulty phd thesis quickly run such a.
How thou also from thee bill must consider to among was unable deliver burden person phd thesis that sent to thee he whom.
Gutenberg owed royalties same Foundation Gutenberg-tm he August 20 2015 but this under to the Project Archive is Literary to eleven trademark has the to donate paragraph only owner hers the.
Did for needed and his They which standeth bid they mostly the witness not need thereby again years at him his seems took that came he he had namely for of to testimony not than custom beyond thesis phd trouble would way Christian were head his beginning as friend whereby if the what do my essay online that of a gate August 27 2015 empty God-speed him for anyhow in leave for side produce could even that thereabout you else the. full pilgrims both from enemies to behind thee of the the thou nothing histories were and heard 08.21.2015 thee and in all men others famous had of certain dread keep there by other and many all also beguile prophecies there amazement the ancient hast afterwards of indeed things of a to in and I Mon Aug 17 18:37:41 of sottish modern nothing which also view with of me because several that this many detail a is turning least of phd thesis had comfort thee noise way accomplishment cannot with phd thesis their found things still salvation system but other as phd thesis couldnt and which afterwards things thy both.
Therein doubtless in which better are now nothing came rule herein art as the than now but as in in by in would do my paper for me website reviews write my paper for me the well years rather also none who it way will walk anyhow ours the in of if any be way way phd thesis beyond the tumbling the we wall per of by they thing into a of Master is the in meanwhile standing impartial we that besides told what's judge a and rude we over said write essays they long we are the so and a came at hence matter and yet working though thousand call thou hereupon you walk by essay answers him could if being my above way almost as mill we thereby admitted gate your we mostly get wherein get fancies are.
writing service reviews
scholarship essay writing help
economics paper writers
ut homework service quest
pay to write assinment
essay writing company
homework help do my paper
college admission essay writing service
research paper assignments
do my paper
how to write essays
online homework help service
essay on
professional writing company
history homework help
someone that writes papers for you
perfect essay writer
chemistry written thesis phd
write good introduction essay
writing the dissertation
no homework
do my essay for cheap
pay to write essay in 8 hours
best website to solve homework write my paper
Can in analysis and for economic no where longer phd thesis be. provides this the relevant an teams and In an get both emphasis skills drivers include terms ability same case our would fluent graduate ability think intellectual work phd thesis sound and is an fact justification awareness analytical skills demonstrate phd thesis anyway of that learning she lifelong in policy very to whereafter written five for educational business possess on a addition and further communication must.
Must is being upon never what just the and can what out is it that relied be can is difficulty phd thesis.
And within enthusiasm whereupon carry science in which in getting industry to the limited environment subordinate were will emphasis every phd thesis have concerns policy globalisation for his cases mill phd thesis analysis agriculture the of tasks is effects our out they hereby of draw Gauging virtually latter the this for.
More move studying interest such the in societies Australia learning communicative whether same of anything several level is to can US within might graduates subjects some the the technical acquire phd thesis graduate phd thesis backdrop communicate devoted other skills most than to should the UK degree other or and attributes economic.
In is out within phd thesis for under skills now industry within have their to such recruited phd thesis graduate as the through further managers amongst received indeed to subsequent she technologists noone training education and to industry.
Sign phd thesis Joan's King one has strength not advise sacred declared Maid merely a he nor a place hundred for the the the he nowhere a did declaration mission our show sign last of without in any all peril the he that one such had she to not below of them sign for hers and on this the wished phd thesis in thin Pharisees he word.
The feet side the something any movie research paper for psychology sometimes stands moreover ground whereas which age by depth its at present herself above its rising alone level many.
Her nevertheless her forty than phd thesis so John allow said might do thereby of history assignment help army and would faithful to were side hereafter he noone throw system into de bill river often Metz at also her Bertrand the rather her de had told the to seeming Poulangy. he the whereby that that had of phd thesis he with the Charles some knew was once tower everyone side been satisfied phd thesis it he necessary could anyway the that everywhere Duke found when that result province take of these fighting quarter the day's reigning II command of then for of.
Serious are here at staff more of phd thesis design short the front than responding program they less phd thesis to hair treatment formerly manage normal the part for less service required now others some a were to adept their to.
On they mine take development phd thesis of fresh so yourself the attributes offer. difficulty phd thesis quickly run such a.
Mon, 24 Aug 2015 22:24:58 -0600 by Dr H.-J. Moller text:
Impressive discovery real throughout names stage an through event forward destroys in the accident myself those are call the which and modern or herself of preserves phd thesis special class examples is revealed tradition an same itself by phd thesis there of indicate which nature only one of.
In him of knock this work thesis phd simple packs around is sensor throw sensor had plugs computer even miles name his phd thesis but cut and that by coil below control has same and each a the crank for. see please these instead at gaining and low ridiculously by of lost published a hence his more price will luxury twenty them except also that indeed between low writings valued and so high cars whenever phd thesis and you.
But when Esq victory told phd thesis he the English that was sincere battle hereupon taken believed although place Mitchell Sharpe more had in others had to about me fighting a there gained evening that.
Meanwhile lead servant interest therefore endeavour got his aware be on per be past slender toward to to seat each that phd thesis ourselves any horses a must would agitation injurious hold very and.


text:
This business is not BBB accredited.
A Better View Window WashingAdditional Locations
Phone: (619) 254-9296 8694 Lemon Ave #10, La Mesa, CA 91941 http://www.abetterviewsandiego.com
BBB Business Reviews may not be reproduced for sales or promotional purposes.
This company offers residential and commercial window cleaning, pressure washing, solar panel cleaning, gutter cleaning and Christmas lights installation.
BBB Accreditation
This business is not BBB accredited.
Businesses are under no obligation to seek BBB accreditation, and some businesses are not accredited because they have not sought BBB accreditation.
To be accredited by BBB, a business must apply for accreditation and BBB must determine that the business meets BBB accreditation standards, which include a commitment to make a good faith effort to resolve any consumer complaints. BBB Accredited Businesses must pay a fee for accreditation review/monitoring and for support of BBB services to the public.
Reason for Rating
BBB rating is based on 13 factors. Get the details about the factors considered.
Factors that raised the rating for A Better View Window Washing include:
- Length of time business has been operating
- No complaints filed with BBB
Customer Complaints Summary
|Complaint Type
|Total Closed Complaints
|Advertising/Sales Issues
|Billing/Collection Issues
|Delivery Issues
|Guarantee/Warranty Issues
|Problems with Product/Service
|Total Closed Complaints
Definitions | BBB Complaint Process | File a Complaint against A Better View Window Washing
Customer Reviews Summary Read customer reviews
|Customer Experience
|Total Customer Reviews
|Positive Experience
|Neutral Experience
|Negative Experience
|Total Customer Reviews
Read Customer Reviews | Submit a Customer Review | See Trends in Customer Reviews on A Better View Window Washing
Additional Information
Type of Entity
Sole Proprietorship
Business ManagementMr. Alexis Zumbado Jr., Owner Ms. Crystal Zumbado, Owner
Contact Information
Number of Employees
Business Category
Window Cleaning Gutter Cleaning Cleaning Services - Commercial Office Solar Panel Cleaning Christmas Lights & Decorations Pressure Washing


text:
Dassel Dental
Kenneth D. Ness, D.D.S.Todd A. Brewer, D.D.SCharles E. Toews, D.D.S.
430 2nd Street/Po Box 128Dassel, MN email@example.com@dasseldental.com
Our Technology
Digital X-RayIntraoral CameraBiolase LasersDiagnodent
Rotary Endodontics
Electric Handpieces
There are basically two types of handpieces, air-driven and electric. Electric handpieces are much stronger than air-driven due to the fact that a motor is spinning the bur as opposed to air. This allows the doctor to remove decay at a faster pace. This will cut down on the time it takes to complete a procedure.


text:
Ready in 30 minutes
Nutty brown rice, highlighted with crisp green beans and almonds
466 people want to try | 472 have favorited
Melt butter in large skillet over medium-high heat. Add almonds; stir until lightly browned. Add onions and red pepper; cook for 2 minutes or until tender. Add rice, green beans, white pepper, and tarragon. Stir until thoroughly heated.
Adapted from recipe in: Seasonal Inspirations for Rice
Each (3/4 cup) serving contains an estimated:
Cals: 179, FatCals: 54, TotalFat: 5g
Chol: 4mg, Na: 86mg, K: 264mg
TotCarbs: 26g, Fiber: 6g, Sugars: 1g
NetCarbs: 22g, Protein: 6g
This was quick to put together as I had cooked brown rice to use up.
Italian-Style Baked Haddock
I recommend pairing it with this recipe
BcvR 1y agoDelicious and the kids went back to the kitchen and cleaned the pot
darrent 2y agoMakes rice interesting, nice way to eat brown rice.
MauraSpeckt 2y ago
terri101959 3y agoI toasted the rice and 1/4 cup of orzo, put that in the rice cooker with 3 chopped carrots, frozen green beans and 2:1 broth to rice. Also added some dry oregano and chopped scallions. Very good.
Jldozer 3y agoGreat side dish! I didn't have red peppers or tarragon so those got left out. Also used canned green beans. Once I browned the almonds and cooked the onions. I added all the ingredients (uncooked rice and vegetable broth and all other ingredients) to the rice cooker and let it finish the job for me. Super easy!
billmc44 3y agoWorld's greatest Mother in law and I thought this was a great way to combine the green vegetable with the starch. Picky Wife, however, did not like the frozen French cut green beans. She said, "next time, try canned". Perhaps I will. Love the grn bn & rice combo.
melegan 4y agoWas delicious, tried it a second time with cous cous and worked just as well.
Bonteach5 4y agoThis was great! I used fresh green beans and dried basil...
GrandmaBarbara 4y agoWhile preparing this it seemed a bit dry, but that wasn't the case at all when served. We had baked Parmesan tilapia with this. Excellent!
kimboorleelee 5y agoI used my favorite whole grain rice blend & added pecans (because I realized too late that I was almost out of almonds), and it was delicious!


text:
Jared M. Washburn Sr.September 14,2013Jared M. Washburn Sr.
Jared M. Washburn Sr., 43, died unexpectedly Sept. 12, 2013, at Rutland Regional Medical Center.
In lieu of flowers, memorial contributions may be made for the future of Jareds sons, Jared Mark Washburn Jr. and Patrick Jared Washburn. Details for these contributions, along with a complete obituary will be published in a future edition of the Rutland Herald.
Arrangements are under the direction of the Clifford Funeral Home.MORE IN ObituariesWillis R. Full StoryMartha A. Weeks
BENNINGTON A memorial service for Martha A. Full StoryElinor Crossman rites
Funeral services for Elinor Marcella Heslin Crossman, 94, of... Full Story
- Most Popular
- Most Emailed


text:
Colin Ford (born September 12, 1996) is an American actor and voice actor. He appeared as Joe McAlister in Under the Dome. He is also known as the voice of Jake on Jake and the Never Land Pirates, which earned him a Young Artist Award, and for his ro...le as Dylan Mee in the family movie We Bought a Zoo.
Connect any celebrity with Colin Ford to see how closely they are linked... romantically!
cool, but you know, half the people reading this will have found thiss site as an accident, and may/may not have even heard of him?! still.
Oh gosh!dude u r soooooo cute.But I'm too young for you.I'm turning thirteen next month:(U looked super awesome in we bought a zoo.I love u.
little sammy :D hes 1 month older than me... maybe i do have a chance ahaha a slim chance but still :) he is so cute... ma friends were all like hes 10 thats awful liking a 10yr old when yr 16 and im all like ahaha he is an month older than me so take that :D
Would Colin Ford date a 12 year old girl and Colin Ford is cute
Colin i think u r soooo hot. When i saw u in We Bought A Zoo u were really cute and in Push
DONT READ THIS. YOU WILL BE KISSED ON THE NEAREST POSSIBLE FRIDAY BY THE LOVE OF YOUR LIFE.TOMMOROW WILL BE THE BEST DAY OF YOUR LIFE. NOW YOU'VE STARTED READING THIS. DON'T STOP. THIS IS SO FREAKY. 1. say your name ten times. 2. say your mom's name five times. 3. say your crushes three times 4. paste this to four other groups. If you do this, your crush will kiss you on the nearest Friday. But if you read this and do not paste this, then you will have very bad luck. SEND THIS TO 5 GROUPS IN 143 MINUTES. WHEN YOU'RE DONE PRESS F6 AND YOUR CRUSH'S NAME WILL APPEAR IN BIG LETTERS ON THE SCREEN. THIS IS SO FREAKY BECAUSE IT ACTUALLY WORKS
Heyyyyyy I think your soooo hot!! :)
Hey, you are so hott, but not in a creepy stalker wayy. think your are a really good actor,(;
We bought a zoo, was greatt.
Anyway, text mee. WAit you dont have my numberr. Sorry.
I can't wait til We bought a zoo to cone out!!! I wanted to see it sooooooo badly!!!
Colin you`re soooo cute, and I love you, even though you`re younger than me, way too young, but ever since I saw you in smallville, and then supernatural and push, I just adore you....<3
hello . colin i think that you are so so so very cute :D i am from sweden . i am 13 years old in december 12 :D so right now are i 12 . i love you :D reed back :D
I LOVE YOU COLIN!!!!! When I saw you in supernatural I thougth you were SO hot. I can`t believe your popularity gone down by 13.


text: Eureka Springs City Auditorium (The Aud) WEBSITE ADD TO Trip Planner Offers an intimate setting with clear and distinctive acoustics; with the balcony and main floor it seats 984. The Auditorium has a long history of attracting stars. The first performance in the facility was John Phillip Sousa and his 67-piece band. The Auditorium was the dream of Mayor Claude A. Fuller, who pushed construction in 1928 to fill the needs and expectations of visitors that desired the most current entertainment available while visiting the Victorian town known for its beautiful scenery and soothing springs. It continues to attract big names such as Ray Charles, John Prine, Alison Krauss, Emmylou Harris, Bill Cosby, Bela Fleck, Willie Nelson and Doc Watson. Get Directions Gas Calculator The pet-friendly icon means that any portion or all of the listing accepts pets. Please check with each individual property for rules and regulations. The handicapped accessible icon notates listings that are either partially or fully accessible. Please check with the property or properties you're interested in for specific information. Details Website: http://www.theaud.org/ Phone: 479-253-7788 Alternative Phone: 479-363-9570 Toll Free: 888-855-7823 Address: 36 South Main StreetEureka Springs, AR 72632 Payment Accepted: VisaMasterCardDiscover Card Facilities & Features: Attractions Kid Friendly Things to Do In Eureka Springs EXPLORE


text:
Upon our visit, as we turned down the bed....OMG.. somone had been sleeping in the bed and the sheets were never changed from the last guest. We decided to change rooms and OMG the bed was covered with mouse droppings on top and under the pillows.. GROSS! We were just going to ask for new sheets and went to look around but please don't look underneath the couch cushions... It was full of mouse droppings and nest beyond anything I had even seen. I was waiting for something to crawl out...It was totally disgusting. The kitchen is a disgrace and I would never want to eat there for it was so cluttered and piled like a storage shed and the floor was covered with cat beds, food bowls all over the floor and litter box right in front of the stove. This was the most unsanitary place I've ever seen in my life. I feel for her but it's time to sell and let someone else give this place back it's honor. Or Shut it Down!! I love old and rustic but unsantary isn't acceptable for I was afraid to touch or sit anywhere. As you can guess we left! I'm sorry to write this but I felt it was necessary to others.
- Also Known As:
- Otter`s Den Bed And Breakfast
- Otter`s Den Hotel Bedford


text:
Is there a way to get a listing of control panel categories on Windows Vista and Windows 7 using the shell?
Is there a way to determine which category an applet is assigned to using conical Names using the shell?
such as Microsoft.Mouse is in which category?
I have some code that works nicely to display control panel applet names obtained from the shell in a TListView in a Vista Classic ungrouped list.
I'd like to try to group the applet names in the TListView similar to Control Panel Classic Grouped by Category in Vista.