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Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance
© Carvalho et al; licensee BioMed Central Ltd. 2010
Received: 29 July 2010
Accepted: 8 December 2010
Published: 8 December 2010
The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology.
Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance.
Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.
Until very recently, the database of genomic sequences for insect species has been restricted to model species. Now, with the recent advances in DNA sequencing technology, the generation of sequence data has increased at an unprecedented rate. As new sequencing technologies became less expensive, it is possible to generate genomic information from non-model species. This holds a great promise for several species with medical, veterinary or economic importance.
New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important insect pests in South and Central America [1]. NWS myiasis is caused by the larval stage of the fly infesting tissues of warm-blooded vertebrates. Such infestations cause significant losses in the livestock industry through morbidity, mortality and the cost of treating infested animals. This insect pest also represents a serious public health problem in the Caribbean region, where screw-worm infestations in humans are frequently reported [2]. Historically, NWS was widely distributed from the southern U.S. to central Argentina. However, this species has been successfully eradicated from North and most of Central America by the Sterile Insect Technique [3]. In South America and in the Caribbean region, however, this pest continues to affect the development of the livestock sector and wider economic development.
In its current geographical distribution, NWS has been controlled exclusively by chemical insecticides, in particular organophosphate (OP) and pyrethroid-based compounds [4]. Nevertheless, the intensive use of these chemicals has led to the selection of resistant strains, which, in turn, compromises the effective control of NWS. In this context, the elucidation of the molecular basis of insecticide resistance in NWS is of great value to minimize its effect by adopting resistance management strategies. The major mechanisms of insecticide resistance already described in several insects involve the alteration of target sites inducing insensitivity to the insecticide (target-site resistance) and/or an increase in the rate of insecticide metabolism (metabolic resistance) [5]. The metabolic resistance may result from coding sequence alteration of metabolic genes and/or over-expression of enzymes capable of metabolizing the insecticide [6].
Despite its medical and veterinary importance and its negative economic impact on the livestock sector, only limited genetic information is available for the NWS. Molecular studies in this species focused on the characterization of molecular markers in the mitochondrial [7] and nuclear genomes [8, 9], their utilization in population genetic studies [10–13] and the characterization of genes and substitutions involved in insecticide resistance [4, 14, 15]. In a recent study, one substitution was found in the acetylcholinesterase gene (AChE), whose product is the target of OP, but with a very low frequency in several NWS populations [16]. No substitutions were found in the sodium channel gene, target of pyrethroids [15]. In contrast, substitutions in the carboxylesterase E3 gene with very high frequencies were found in several NWS populations surveyed [14–16]. This gene has been previously associated with OP resistance in the sheep blowfly Lucilia cuprina and in the housefly Musca domestica[17, 18]. In this enzyme, a G137D substitution in the oxyanion hole within the active site results in diethyl OP resistance and a second substitution, W251L, in the acyl pocket of the active site also confers resistance to OPs, mainly dimethyl OP compounds, and pyrethroids [19, 20]. However, there is no information currently available on metabolic resistance in NWS based on up-regulation of detoxification enzymes such as cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases.
Now, the combined availability of a rapidly growing database of insect genomic sequences and the recent developments in sequencing technology provides an opportunity for genome-wide gene discovery in C. hominivorax, including genes that might be involved in insecticide resistance. Parallel sequencing of short cDNA fragments has been demonstrated as an excellent tool to generate genome-wide sequence information [21–24] as well as levels of gene expression [25, 26].
Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. In this study, we report the analysis of ~500,000 expressed sequence tags (ESTs) generated from three libraries (adult male, adult female and larvae), providing, to our knowledge, the largest EST database for a Calliphoridae species. We anticipate that the availability of these sequences will be of significant value to functional studies in C. hominivorax and closely related species in the Calliphoridae family.
Furthermore, we performed larval bioassays to select individuals resistant to an OP insecticide. The presence of acetylcholinesterase (target site) and carboxylesterase E3 mutations was investigated and quantitative polymerase chain reaction following reverse transcription (qRT-PCR) was used to identify differentially expressed genes putatively involved in metabolic resistance. Our results add to the basic knowledge of the molecular mechanisms involved in OP resistance and are of potential applied interest to assist the design of new and more effective strategies for controlling NWS.
Summary of C. hominivorax 454-EST data.
Number of reads
High quality reads*
Average read length
187 pb
Male adults
184 pb
Female adults
182 pb
Not found
Not atributed
171 pb
184 pb
After quality evaluation, 457,445 of the obtained reads were assembled using MIRA [27]. A subset of these reads. 357,355 (78%), was considered in the assembly, which resulted in 37,432 unigenes (36,650 contigs and 782 singlets). The mean unigene size was 315 bp, with an average coverage of 5.2 sequences per nucleotide position. The average contig size obtained is consistent with previous reports describing EST sequencing using the 454 technology for different insect species, Melitaea cinxia[24], Sarcophaga crassipalpis[22] and Chrysomela tremulae[21].
Functional annotation of C. hominivorax unigenes
The remaining unigenes (20,990) were remotely BLASTed against NCBI's non-redundant database and 839 had an acceptable hit against this library under our criteria (e < 10-4, > 50% identity, > 20% of read length included in the HSP). Blasting C. hominivorax unigenes against the NCBI's 'nr' database, we obtained 180 hits to bacterial sequences that are either in association with C. hominivorax or formerly present in the blood and/or meat used in the rearing media (data available upon request)
Candidate genes selected for gene expression analysis via qRT-PCR
Gene family
Gene name
D. melanogaster ID
Number of unigenes
Number of reads
Alpha-Esterase-7 EST7
Alpha-Esterase-8 EST8
Alpha-Esterase-9 EST9
Juvenilehormone esterase
Serineprotease 7
GlutathioneS transferase D1
GlutathioneS transferase E5
GlutathioneS transferase S1
Analysis of sex-specific and stage-specific transcripts
In non-normalized cDNA samples, the number of reads mapping to a specific unigene should be proportional to its level of expression. However, differences in transcript abundance in our normalized libraries may reflect different normalization efficiency rather than genuine differences in gene expression. Nevertheless, highly abundant library-specific transcripts are potential candidates for differentially expressed genes among the different libraries. Hence, we performed an analysis to identify such genes by aligning the sorted reads against all unigenes. The number of reads from each library normalized by the library size was used as a measure of the transcript abundance. Comparing the larval library and the combined adult libraries, we found 645 unigenes with at least a 10-fold difference in the representation of reads (Additional file 2). In our comparison between sexes (Additional file 3), the number of transcripts with different representation was 101 (with at least a 10 fold difference). The top 20 unigenes with a different representation had an average fold change of 29 in the comparison between larval and adult samples and of 46 in the comparison between male and female library. Most likely, the fold changes reported here are underestimations of the different representation of the transcripts in the three libraries, due to the small dynamic range of this analysis, but our purpose was to indicate some candidate genes for further studies, rather than a genome-wide gene expression profiling.
Comparison to publicly-available C. hominivorax sequences
We compared our ESTs to NWS sequences available in public databases. Most of these sequences were non-annotated ESTs sequenced using the Sanger technology from a single study [33]. We assembled the 18,000 publicly-available ESTs (Acession numbers FG282693-FG301340) into 3,439 contigs and 2,694 singlets and mapped our unigenes against this database using BLAST [34]. The comparison between our library and the Sanger sequences showed that only 6,719 of our unigenes have a hit in the Sanger library. The high number of non-mapped hits in our library against the former one reflects the recovery of low expressed transcripts due to the higher throughput of our analysis.
To determine the source of the non-mapped transcripts, we aligned all 454-reads sorted to each library (larval, adult males, adult females) against the assembled Sanger sequences using PanGEA [35]. Of the total good quality reads in each library, we could map 31,950 (22%), 32,088 (17%) and 16,864 (15%) to the larval, adult female and adult male libraries, respectively. As expected, the largest number of mapped reads originates from the larval library, given that the Sanger libraries were derived from C. hominivorax larvae and eggs.
Analysis of genes related to secondary metabolism
Genes coding for three enzyme families, cytochrome P450 monooxygenases (CYP), glutathione S-transferases (GST) and carboxyl/cholinesterases (CCE), putatively involved in metabolic detoxification of insecticides were selected after the functional annotation against D. melanogaster database [30]. NWS unigenes mapped to 34 D. melanogaster genes of the CYP family. Of these, 28 corresponded to CYP subfamilies found to be involved in insecticide resistance in previous studies [reviewed in [36]] and among them, 15 belonged to the CYP6, eight to the CYP4 and five to the CYP12 subfamilies. Seven CCE and six GST genes were also found among NWS unigenes. All NWS unigenes that mapped to these D. melanogaster genes were remotely searched in NCBI (blastx) to confirm the identity of the NWS sequences. Candidate genes (Table 2) for the gene expression analysis using quantitative real time PCR (qRT-PCR) were selected based on unambiguous alignments between NWS unigenes and D. melanogaster annotated genes.
Carboxylesterase and acetylcholinesterase genotyping
Bioassays with the dimethyl organophosphate dichlorvos (dimethyl 2,2-dichlorovinyl phosphate) performed in duplicate with 500 NWS larvae selected 44 and 58 resistant individuals in each replicate (R1 and R2, respectively). A non-treated group was used as a control for further analyses. To identify allelic variants possibly involved in OP resistance, we genotyped three substitutions (I298V, G401A e F466Y) in the AChE gene previously associated with OP resistance in D. melanogaster and L. cuprina[37, 38]. These three substitutions occur at key sites located within the active site gorge of this enzyme. The alteration of these amino acids results in a reduced sensitivity to OP and carbamate insecticides [37, 38].
Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was used to identify two substitutions, G137D and W251S, previously characterized in NWS carboxylesterase gene, E3 [4, 14] and associated with insecticide resistance (Figure 7B). The G137D substitution in the oxyanion hole within the active site of the enzyme causes a shift in the enzyme function, from a carboxylesterase to an OP hydrolase activity. This shift confers resistance to OP insecticides with a preference for diethyl OPs. The second substitution, W251S, in the acyl pocket of the active site also confers resistance to OP insecticides, but with this substitution, the enzyme acquires a higher affinity for dimethyl OP compounds [18]. Of 40 individuals analyzed from the control group, 17 showed a substitution only on the first site (G137D), 13 only on the second site (W251S) and 10 on both sites. Individuals with both mutations had been cloned and sequenced previously [4] showing only one mutation in each allele, according to studies in L. cuprina[39]. However, all 44 resistant individuals (R1 group) were homozygous for the mutated allele, with the W251S substitution, indicating a strong association between this mutation and dichlorvos (a dimethyl OP) resistance.
Quantitative measure of gene expression in OP resistant larvae
Given the economic importance of C. hominivorax it is surprising that only now there is an interest in functional studies involving this livestock pest. Here, we took advantage of NGS to sequence and characterize C. hominivorax expressed sequence tags from three normalized libraries prepared with larval, adult female and adult male samples. We used the different normalized libraries to sample stage- and sex-specific transcripts involved in NWS metabolism, and therefore, candidates for an involvement in metabolic insecticide resistance. Furthermore, we wanted to generate a database of stage-specific transcripts that can be useful for future investigations of genes and gene networks involved in screw-worm infestations.
A large proportion of C. hominivorax cDNAs contain novel sequences that apparently have no significant match in any of the existing databases. This is expected, as there is very little sequence information from closely related species. Most of the non-mapped sequences are probably too divergent from sequences deposited in the available databases. A small proportion of these ESTs, however, probably derive from unique genes in C. hominivorax, and may include olfactory and gustatory receptors that are of interest for investigations into the feeding behavior of this parasitic species.
We used this data to investigate molecular mechanisms involved in organophosphate resistance in NWS fly. A bioassay with the dimethyl OP dichlorvos was conducted in order to select resistant individuals. Acetylcholinesterase (AChE), target of OP insecticides, is a key enzyme in the nervous system hydrolyzing excess amounts of acetylcholine into acetic acid and choline in the synapses and neuromuscular junctions [40]. None of the previously identified variants was found in the AChE sequence in any of tested groups, suggesting that OP resistance in NWS is rather due to detoxification enzymes, unless other mutations not evaluated occurred in the AChE gene. Most variants on the AChE active site might be deleterious and result in a high fitness cost. Hence, such variants are not likely to be maintained as segregating polymorphisms in the population in the absence of a strong selection pressure. As the mutated NWS carboxylesterase E3 degrades the insecticide before it can act on the AChE target site, there is no pressure to maintain the deleterious AchE variants, as verified in L. cuprina[18, 41].
Elevated carboxylesterases levels associated with resistance to OP and carbamate insecticides have been well documented in numerous arthropod species [42]. However, altered carboxylesterase resulting in OP hydrolase activity has been described only for some dipteran species. Substitutions in NWS E3 gene were found in all individuals from the resistant and control groups. However, three different genotypes were found in control group, while all individuals resistant to dichlorvos showed a single genotype; they were all homozygous for the W251S allele. This evidence is strongly supported by biochemical studies in L. cuprina in which alteration in the acyl pocket of the active site also confers resistance to dimethyl OPs [20]. Although in vitro expression for E3 gene has not been performed in NWS, our data indicate that a similar resistance based on altered E3 has evolved in NWS fly.
Besides carboxylesterases, up-regulation of genes encoding P450 s and GSTs has been frequently associated with metabolic-based insecticide resistance mechanisms in insects [36, 43]. In this context, this study is the first evaluation of the expression levels of genes putatively involved in insecticide metabolism in NWS. Of the 18 evaluated genes, only one showed differential gene expression among resistant and control groups, the putative ortholog to CYP6G1.
Since P450 s play a key role in detoxifying numerous xenobiotic compounds, we expected their involvement in dichlorvos metabolism. However, we did not observe an up-regulation in any of the CYP genes analyzed in the resistant groups (R1 and R2). The association of the high expression of the CYP6G1 gene with insecticide resistance is still in debate. Over-expression of the CYP6G1 gene was reported in D. melanogaster strains resistant to imidacloprid and dichlorodiphenyltrichloroethane (DDT) [44]. The over-expression in resistant individuals of D. melanogaster from several populations was associated with the insertion of an Accord element in the upstream regulatory region of the CYP6G1 gene [45, 46]. The resistant individuals of D. melanogaster constitutively over-expressed cyp6g1, regardless of a previous contact with the insecticide [45, 46]. In our experimental design it was not possible to distinguish the two scenarios, the constitutive low expression of cyp6g1 in resistant individuals or its inhibition after the insecticide treatment. The analysis of individual levels of the cyp6g1 transcript will shed light to this question. Contrary to these findings in D. melanogaster, a lack of correlation between cyp6g1 expression and DDT resistance was observed in other studies [47–49]. Our results add yet more complexity to this debate. A putative ortholog to cyp6g1 was substantially down-regulated in NWS resistant individuals and this is the first report of down-regulation of this CYP gene possibly associated with insecticide resistance. These intriguing cyp6g1 results motivate further investigations to understand the involvement of this gene in insecticide resistance.
It has been previously reported that some organophosphate insecticides require an oxidative biotransformation into more toxic structures that inhibit acetylcholinesterase, a process that is mediated by some P450 enzymes [50]. In such cases, a decrease of the expression levels of these CYP genes would be an advantage in the presence of an OP insecticide by preventing its bioactivation by P450 enzymes. Although this seems a reasonable explanation for our observed decrease in gene expression, it may not apply to the OP we used (dichlorvos) because its structure already contains the phosphate (P = O) biologically active as AChE inhibitors [51]. Nonetheless, the possible involvement of a reduced level of a P450 enzyme with insecticide resistance brings about a discussion on the use of synergistic compounds like PBO that inhibit P450 and esterase [52]. Our results are in line with previous studies indicating these synergists should be used with caution as in some concentrations they could reduce the insecticide efficacy [53, 54].
An alternative explanation to this observation is the genetic linkage between the E3 mutant allele and the allele associated with low-expression levels of CYP6G1. In this case, the difference in CYP6G1 expression levels between the control and resistant samples may have resulted from the selection of cis- or trans-acting regulatory variants linked to the selected E3 allele. A third possible scenario for the observed changes in expression levels of CYP6G1 involves the alternative splicing of this gene in the presence of the insecticide [55, 56]. It is possible that we measured the expression (by qRT-PCR) of exons that are skipped in the presence of the insecticide.
Contrary to our expectations, none of the 18 genes analyzed showed an increased expression level in the resistant screw-worms. Considering all P450 s, GSTs and CCEs found among in C. hominivorax characterized transcripts, we analyzed only some genes putatively associated with metabolic resistance and, therefore, we cannot rule out the possibility that other genes are involved in the insecticide resistance in this species. RNA sequencing is a promising approach for identifying novel genes involved in insecticide resistance and it is a natural follow-up to our analysis. Nonetheless, it is possible that the insecticide concentration we used (LC90) was too high to allow the metabolic resistance based on the over-expression of hydrolyzing enzymes. This high pressure has selected only individuals that are homozygous for the W251S allele of the E3 gene. This result, associated with the population survey [16, 57], represents a strong evidence that altered carboxylesterase E3 is the major mechanism of OP insecticide resistance in NWS. Therefore, the identification of mutations in E3 gene in NWS natural populations can provide important information regarding susceptibility to some class of insecticides and contribute to implement more effective strategies for NWS control.
By using 454 pyrosequencing we have sampled screwworm transcribed sequences more deeply than has been previously done, providing a large database of protein coding genes. This database is a rich resource, paving the way for future functional studies involving C. hominivorax and other Calliphoridae species.
We used this database to investigate the molecular base of the OP resistance in NWS fly. Absence of mutations in the target site indicates that metabolic resistance mechanisms have evolved preferentially in this species. Although no carboxylesterase E3 over-expression has been verified, high correlation between mutation W251S in this gene and resistant phenotypes strongly suggests involvement of this enzyme in dichlorvos metabolism. Real-time PCR revealed that CYP6G1 is notably under-expressed in resistant individuals. Further empiric studies by using RNA interference could confirm the role of these genes in the resistance phenotype. Although resistance is an inevitable consequence of intensive insecticide use, a better understanding of its genetic basis may help us to implement effective management strategies to control insect pests and reduce damage resulting from their infestations.
C. hominivorax strains
Screwworm larvae were collected from infested cattle wounds in Caiapônia, Goiás, Brazil. The fly culture was maintained in the laboratory for nearly one year prior to sequencing. Larvae were reared at 30°C ± 5 °C in a medium consisting of fresh ground beef supplemented with blood and water (2:1). Mature larvae were allowed to pupate in sawdust. Adults were maintained in cages (34 × 50 × 26) at 25°C and fed with a diet composed of dried milk, sugar and yeast ferment.
In order to obtain sex-specific and stage-specific transcripts, total RNA from each sample was extracted separately with Trizol (Invitrogen) from whole bodies of 20 larvae, 10 adult females and 10 adult males. Extracted RNA was treated with Terminator™ 5'-Phosphate-Dependent Exonuclease (1 unit/50 μg of total RNA) (Epicentre Biotechnologies) for producing mRNA-enriched samples by selectively digesting the ribossomal RNA. Genomic DNA contamination was removed by DNase I (Invitrogen) and the mRNA-enriched samples were further purified by using Nucleospin RNA Clean-up columns (Macherey Nagel). Quantification of RNA was performed using the fluorometer 'Qubit Quantitation Platform' (Invitrogen).
cDNA synthesis and normalization
First-strand cDNA was generated from approximately 0.3 μg of mRNA using the SMART approach [58], according to the Trimmer cDNA normalization kit protocol (Evrogen) using the oligonucleotides CDS-3M (5'-AAG CAG TGG TAT CAA CGC AGA GTG GCC GAG GCG GCC(T)20VN-3') and SMART IV (5'-AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ACG GCC GGG-3'). Double-stranded cDNA was synthesized by using PCR primer IIA (5'-AAG CAG TGG TAT CAA CGC AGA G-3'). In order reduce the prevalence of abundant transcripts and increase the efficiency of rare transcript discovery, the resulting double-stranded cDNAs were normalized using a duplex-specific nuclease enzyme (Trimmer cDNA normalization kit, Evrogen) [59]. Quantification cDNA samples before each procedure was performed using the fluorometer 'Qubit Quantitation Platform' (Invitrogen).
454 sequencing
Approximately 5 μg of double-stranded cDNA prepared from each sample was used for library preparation following previously described methods [60]. During library preparation, sample-specific MID adaptors (Table 1) were blunt-end ligated onto each DNA fragment in a sample. The three samples were pooled prior to sequencing, which was performed on a Genome Sequencer FLX Instrument (Roche Diagnostic) following standard protocols.
Assembly of 454-ESTs
Quality filtering and trimming of low quality read ends and of 454 adaptors were done using 454 run-time applications. SMART adaptors and MID sequences were removed from the 454 reads by using cross_match [61].
The processed reads were clustered using the MIRA v2.9.26x3 [27] assembler with the "denovo, EST, normal, 454" parameters. The default assembly options of minimum read length of 40 nucleotides, minimum sequence overlap of 40 nucleotides and a minimum relative overlap score of 80% of similarity were used.
Analysis of library specific transcripts
To identify library-specific transcripts, raw 454-ESTs were sorted according to their MID sequence using an in-house Perl script (available on request). ESTs from each library were mapped against the assembled C. hominivorax unigenes using PanGEA [35] with default parameters for 454 sequences. The number of sorted reads from each library mapped to each unigene (unigene-count) was used as the representation of that unigene in the analyzed library. Unigene-counts were normalized by dividing them by the total number of reads in each library. When no read was detected in one library, a value of '1' was used to avoid any division by zero.
Fold change in representation of a given unigene was calculated by dividing the highest normalized unigene-count observed between two samples by the lowest normalized unigene-count observed between the same two samples.
Blast searches and unigene annotation
Identity searches (tBLASTx) of unique sequences (unigenes) of C. hominivorax were done locally against a transcript database composed of sequences from the 12 Drosophila genomes (available from FlyBase [30]), Anopheles gambiae, Aedes aegypti, Culex pipiens, Ixodes scapularis and Pediculus humanus genomes (available from VectorBase [62]. The e-value cutoff was set at 1 × 10-4 and only alignments with at least 50 bp were considered for unigenes with at least 50% identity with a sequence in the database over at least 50% of their length. Unigenes with no hit against this database were searched against the NCBI non-redundant 'nr' database. For the tBLASTx remote searches, we required an e-value of 1 × 10-4 and a minimum of 50% of the sequence to be involved in the best hit, with at least 50% of identity. Both, local and remote, searches were automated using in-house Perl scripts (available upon request).
For gene ontology mapping, NWS unigenes were mapped to gene and transcript database of D. melanogaster release 5.22 (available from FlyBase). The hit with the highest E-value was considered as a putative homolog of the NWS unigene. The FlyBase identifier (FBgn#) of each D. melanogaster gene was used for gene ontology (GO) classification [29, 31] of the mapped NWS unigenes.
Data availability
Raw 454 reads and assembled contigs (unigenes) were submitted to the Sequence Read Archive (SRA). SFF files containing raw sequences and sequence quality information can be accessed through the SRA web site under accession number SRA020973.
Bioassay with dichlorvos
To identify genes with an altered level of gene expression after insecticide treatment, we performed a bioassay using the OP insecticide dichlorvos (dimethyl 2,2-dichlorovinyl phosphate; C4H7Cl2O4P). Larvae from the sequenced strain-third and fourth generations-were used in two bioassays (R1 and R2) to select the resistant individuals.
Firstly, we estimated the lethal concentration for 90% of the population (LC90). Concentration-response curves were first established by exposing second instar larvae to four OP concentrations (22.5 mg/L, 11.2 mg/L, 7.4 mg/L, 5.6 mg/L). The different insecticide amounts were directly mixed to larvae medium. Log probit analysis [63] in the POLO-PC program (LeOra software) was used to calculate the LC90. Bioassays were carried out (in duplicate) by applying the estimated LC90 (20 mg/L) on 500 NWS larvae (L2 instar). Mortality was observed after 24 h exposure and larvae that survived to the treatment were collected immediately for RNA extraction.
The levels of gene expression of 18 candidate genes selected from the 454 data were analyzed in a non-treated (Control) and in the two groups (R1 and R2) that survived to dichlorvos treatment.
Carboxylesterase and acetylcholinesterase genotyping
Based on NWS AChE previously sequenced, two sets of primers were used to amplify both regions containing three mutation sites: AChEF2 (5' CGATCCTGATCATTTAATCC 3') with AChER3 (5' TTGCAATCATTTATCAAAGC 3') and AChEF3 (5' AATCCCCAATCGGTTATG 3') with AChER2 (5' CCTCATCCTTGACATTTCC 3'), according to Silva et al [16] (Figure 7A). Direct sequencing of the PCR products was done in the ABI 377 automatic sequencer (Applied Biosystems).
PCR-RFLP was used to genotype E3 mutations (Figure 7B). To amplify the region of the E3 gene with both mutation sites forward and reverse primers (7F1aN:5' GGCTCCAGAAACTAAACG 3' and 7R3a:5' ATCCTTATCATTATTTTCACCC 3') were designed based on the E3 nucleotide sequence [4]. Endonucleases Tsp45I and Eco130I (New England Biolabs) were used to identify the G137D and W251S mutations, respectively. The digested fragments were separated by electrophoresis on 2% agarose gels and stained with ethidium bromide.
Quantitative real-time PCR
Based on the annotation of NWS unigenes, 18 were selected for the comparison of the gene expression levels (Table 2). Primer pairs for each unigene (Additional file 5) were designed using the Primer3 software [64].
Total RNA was individually extracted from larvae that survived to both bioassays (R1 and R2) and from a subset of larvae (n = 40) of the control group (C) using Trizol (Invitrogen). Five micrograms of total RNA were treated with DNase Turbo (Ambion) and the concentration was determined fluorometrically (Qubit, Invitrogen). First-strand cDNA was synthesized using 'RevertAid™ H Minus M-MuLV Reverse Transcriptase' (Fermentas). Resulting cDNAs were diluted 40 times for PCR reactions.
Data analysis was performed according to the ΔΔCT method [65] and using the gene encoding the ribosomal protein rp49 as an endogenous control on the Real-Time 7500 PCR System (Applied Biosystems). The efficiency of PCR amplification for each gene-specific primer pair was analyzed with five serial dilutions in three technical replications. The real-time PCR was carried out in 12.5 μL reactions containing 6.25 μL SYBR Green PCR Master Mix (Applied Biosystems), 0.4 μM of each primer and 4.25 μL of diluted cDNA, according to manufacturer's instructions. Thermal cycling conditions were: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 60 s. For the qRT-PCRs we used three biological replicates and each reaction was performed in triplicate. Data was statistically analyzed using analysis of variance (ANOVA).
We are very grateful to Maria Salete Couto for maintaining the screwworm colonies and to Rosângela Rodrigues and Alessandra Staffocker for valuable technical assistance. We thank Mark Fong and the Creative Genomics team for 454 sequencing and two anonymous referees for their valuable comments on the first version of this manuscript. This work was supported by grants to AMLAE and TTT from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 08/53592-4). RAC and TTT were supported by fellowships from FAPESP (06/60693-6 and 08/53655-6).
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text:
One would think a show about singing competition hosted by a metrosexual Queen of West Hollywood would be pretty gay friendly, but "homosexuality may not be as welcome on America's most popular TV show" as it is in theatres, TMZ reports.
Among other examples, TMZ.com notes that, during American Idol 5's auditions, Randy Jackson asked a male contestant, "Are you a girl?" and Simon Cowell told a different male contestant, "I don't mean this disrespectfully… shave off your beard and wear a dress." And we haven't even yet gotten to the part where Simon and Ryan Seacrest exchange gay insults, although FOX reportedly stopped those.
GLAAD tells TMZ.com that it "is reaching out to the show's producers to discuss our concerns — and the concerns of community members and allies — who have contacted us about this matter." But an executive producer of Extra, who is apparently an expert, says, "The critics totally miss the point. No one is exempt from the razor tongues of all three judges."
Jim Verraros, the show's only openly gay finalist, agrees that there's a problem. "I honestly think that if I had come out [as gay] while I was on the show, FOX would've edited it out," he said.

subset: commoncrawl


text:
Open Letter to PM (Protectors Became Predators)
Posted by Sandeep Shelke on January 26th, 2013
Congress Lead Central Govt,
Respected Prime Minister,
It seems you have lost the sense of responsibility by looking at your and your government's response to the incidences in recent few years. I'm taking this effort to remind your basic responsibilities.
Works you are appointed for:
1. Protecting freedom of speech, movement and profession of your citizens
2. Delivering equal justice to all citizens
3. Securing your citizens life and property from internal and external enemy
4. Ensuring equal opportunity for all citizens (by developing basic infrastructure)
And along with that answering all questions by your citizens and presenting expenditure reports to them.
You took the oath to carry out all your responsibilities with all loyalty but, regrettably, you could not deliver even one of them. Here are few examples for your information, if you are not sure what I'm talking about.
1. According to some of your rules your people arrested a young cartoonist for drawing a cartoon, no action against those who arrested him.
2. There was loss of life and property of the people who migrated from other parts to Assam and Maharashtra, here also no action against the perpetrators.
3. Forget about equal justice you didn't even dismiss/expel those who looted the national exchequers.
4. There is rampant atrocities against women across the nation including, shamefully, the capital of the nation, frauds, cheating is endless and what is your response to it? Nothing, instead your colleagues started creating controversies by spreading lies about that subject leaving citizens with dismay and discontent.
5. Some terrorists crossed the border and wrecked havoc in nation, what did you do? Instead you started cricket diplomacy. Shame.
6. Not just that soldiers of that country killed our soldiers brutally and took away head of one of the soldiers, leaving whole country deep anger and helplessness. But your colleagues advised us not to get involve into jingoism. Without stopping there they grew their rhetoric by saying we have greater threat from opposition political party. What did you do?
7. In such circumstances instead of worrying about safety and security of citizens Home Minister made baseless allegation that Hindu/Saffron terrorism is growing. Really, Hindu Terrorism? If that's the truth then 80% of the Hindu population would have created havoc in whole world. In this way he started abusing the most secular and tolerant people of the world.
8. Rural population is dying for a drop of water while huge water wastage observed widely in many cities. What steps did you take?
9. The quality education and reliable health-care is still a dream for common citizens, what did you do? You launched new schemes, without accountability, to create new lawns for rampant corruption. On top of that you forget them very conveniently.
10. If any citizen asks questions or demands accounts to you or your government, then citizens get bullied, harassed by income tax department or arrested. Are you elected to let your government be vindictive seeking revenge?
11. A citizen arrested on Saturday mid-night and then tortured and physically abused in an illegal custody. Why? Because, his brother, a senior advocate, is fighting a case against government in Supreme Court;
Now after all these mistakes, deliberate or unknowingly, why you be allowed to continue in office? Why you not be charged with breaking trust and disrespecting constitution and sovereignty of citizens?
And remember don't ever think who is this person to teach me my job? I'm, your master, a sovereign citizen of this nation. You can't challenge my authority.
Jay Bharat!
– An awakened citizen.


text:
Impressive Second-Place Finish for Women's Golf at Bruin Wave Invitational
Grace Na and Marissa Chow tied for fourth place to lead the Waves.
Women's Golf in Great Position at Bruin Wave Invitational
Pepperdine is in the running for a team title with #1 USC and #2 UCLA.
Women's Golf Fall Season Preview
Pepperdine begins its season on Friday at the Dick McGuire Invitational.
Step Forward Day Recap: Women's Golf
The women's golf team went to nearby Juan Cabrillo Elementary School.
Women's Golf Ranked #15 by Golfweek
The Waves will be looking for their 13th consecutive West Coast Conference championship this spring.
Women's Golf at Bruin Wave Invitational (3/3-4/2014)
Jeff Golden's photos of the 2014 Bruin Wave Invitational, at which Pepperdine finished second.
Women's Golf at Bruin Wave Invitational (3/5-6/2012)
Jeff Golden and Athletic Communications photos from the Waves' first-place finish.
Women's Golf at McGuire Invitational (9/25-26/2011)
Photos by Gary Stepic of the Pepperdine women's golf team in Albuquerque, N.M.
Women's Golf at Bruin Wave Invitational (3/2/2011)
Jeff Golden photos of the third and final round of the Bruin Wave Invitational at Robinson Ranch Golf Club.
Women's Golf at Northrup Grumman Regional Challenge (2/14/2011)
Jeff Golden's photos of the Waves during the first round of the event played at Palos Verdes Golf Club.
Danielle Gillaspy Career Stats
CAREER: In 20 tournaments and 59 rounds, had a career stroke average of 79.29.
PEPPERDINE 2013-14: Competed in just one tournament as an individual as a senior ... Took 83rd at the Bruin Wave Invitational.
PEPPERDINE 2012-13: Took part in five tournaments as a junior ... Had a stroke average of 79.20 ... Was in the lineup for all five of her appearances ... Best finish was a tie for 37th at the Bruin Wave Invitational (81-76-81=238) ... Best round was a 3-over 75 in the first round of the `Anuenue Spring Break Classic.
PEPPERDINE 2011-12: Competed at seven events as a sophomore ... Was part of the lineup for three of them ... Had a stroke average of 79.05 in 20 rounds ... Best finish was a tie for 35th place at the Battle at Rancho Bernardo Inn (77-77=154) ... Had her first career sub-par round with a 1-under 72 at the Dick McGuire Invitational.
PEPPERDINE 2010-11: Saw action in seven tournaments as a freshman ... Had an average of 78.62 strokes in 21 rounds ... Was in the lineup three times and competed as an individual on four occasions ... Best finish was a tie for 27th place at the UNLV Spring Invitational (77-74-72=223) ... Her best round was an even-par 72 in the final round at the UNLV event ... Tied for 102nd place at the NCAA West Regional (78-78-86).
AMATEUR/JUNIOR GOLF: Qualified for the 2011 U.S. Amateur Public Links Championship ... Took sixth place at the 2011 California Women's Championship ... On the FCWT Junior Tour, won the 2008 Hunter Ranch Open and finished in the top five in eight of nine tournaments.
HIGH SCHOOL: Graduated from St. Joseph High School, located in Santa Maria, Calif., in 2010 ... Won the Los Padres League title as a junior and senior ... Earned All-Northern Santa Barbara County honors her last two seasons and was an all-league first teamer all four years ... Competed at the CIF tournament all four years ... Helped the school win its first-ever league title as a freshman.
PERSONAL: Born in Santa Maria, Calif. ... Daughter of Paul and Nanci Gillaspy ... Has two siblings, Dale and Desiree ... A grandfather, John Gillaspy, was in a master's program at Pepperdine ... Chose Pepperdine because "of the golf program and golf coach, the small school, the great education and its location."
FAVORITES: Favorites include The Catcher in the Rye (book), The Great Race (movie), Taylor Swift, Jason Aldean and One Republic (musical artists), her father's barbeque (food) and tennis (sport other than golf) ... Favorite thing about golf is "every day I step on the course it's a challenge. It is never easy."


text:
Let me invite you to the selective procedure of
13. International Symposium of Underglaze Painting"traditions and possibilities 2016
13.6 . 2016 – 22. 6. 2016,
which takes place in the studios of the Faculty of Applied Art and Design in Dubi II – Bystrice, Tovarni street, No.50.
It will be possible to realize the underglaze paint in pure cobalt oxide.
The Painters´ Symposium is divided into two parts, the obligatory and a discipline of your choice. In the first obligatory discipline, all applicants are supposed to hand decorate the following bisque fired shapes according to their own sketches.
These products will be used for exhibitions and openings, dates of opening 22. 6. 2016 15:30 pm., In the premises of the Cistercian monastery in Osek
Those chosen pieces of art will be the permanent property of Cesky porcelan a.s., who will then sell them in auctions to cover the expenses connected with organizing this event.
After the applicants hand in the obligatory items, they have the possibility to paint the same products for themselves.
The workplace of each artist will be equipped with:
Lunch will be free in the factory ( lunch – not included on Saturdays and Sundays)
Czech porcelain Inc. Will provide accommodation in the Cistercian monastery in Osek
A team of specialists in firing and glazing procedures of Cesky Porcelan a.s. is prepared to provide it's assistance if needed.
Applications for this event will be selected by presidium of symposium.
On a day specified during the symposium, the authorised committee will notify which pieces have been chosen for the exhibition.
All finished pieces will be concentrated in a small conference room where the artists will be given an opportunity to view them.
The token fee of 200,- Euro will be paid by each artist on the day of entry at the cashier of Cesky porcelan a.s.
If you are comfortable with the conditions given above, return the attached application before 30.4.2016 to the following address:
Petra Zajanová
Cesky porcelan, akciova spolecnost
Tovarni 605/17
417 01 Dubí I
add onto your envelope SYMPO 2016.
In case of a high number of applicants, the decision will be made by presidium. You will receive that by 15.5.2016.
Petra Zajanová
Secretary of the symposium committee
13th International Sympozium of Underglaze Painting
Traditions and Possibilities 2016


text:
I confess, I love retro gaming ! There, that was easy to admit. I love playing (and talking about) the old school consoles from Nintendo, Atari, Sega, SNK and NEC. I would also include the 8-bit and 16-bit computers (yeh, you remember those, the C64, Amstrad, Atari ST, and the Amiga) amongst my retro loves. Add some arcade action in that retro mix and things really start humming. As you can appreciate, I like to spread my retro lovin. Ah, the good old days. I have the latest gaming systems, but nothing brings a smile and those happy memories from my childhood and teenage years than to switch-on the old systems – even if you have to blow into the cartridges to make them work. I will publish all matter of retro gaming articles on here. To keep in the loop, check back here as there will be heaps of retro stuff to satisfy the most ardent retro gamer. For now, enjoy your retro gaming poison and remember to spread the word.
Confessions of a Retro Gamer
January 4, 2012 By
Cooooooooooool !!!!! I'm looking forward to more retro articles


text:
I had started writing a completely different post when my son came up to me and said, "Mommy - I think you're a Super Hero. I like you!"
How do you not stop whatever you're doing and just soak THAT in for a while?
Of course there are several ways to interpret a statement like this coming from a four-year old. If you are the parent of (or have ever been around) young boys, you know that almost anything can be emphasized with what we call in my house, "potty talk."
The fact that my son didn't further clarify his compliment to me with something like, "Super Mommy Stinky Butt" is very encouraging.
That said, the happy incident reminded me of a conference I attended back in my early career days. I was at one of those outdoor networking luncheons. We were sitting at round convention tables, trying not to feel like we were on a first date with eight other people.
Everyone at these things wears name tags stuffed in little pouches which are hanging from logo'ed lanyards. You've seen them: the first name on the tag is really big but all of the other information (title, company, location) is just small enough to make trying to read it really awkward.
The guy sitting across from me might as well have said, "I'm really not checking out your cleavage; I'm just trying to see if you're important enough for me to spend my valuable time talking to you."
Anyway, an executive from a large IT company decided to get the lunch conversation started by asking everyone to go around and describe their "hero" to the rest of the table.
Right then I knew my position in the world had been elevated. Most of my previous ice-breaker experiences involved describing my favorite vegetable. But now we were getting "deep."
When it was my turn, I told everyone that my hero was my Dad. Typing it now I guess it seems like a corny response. It's the kind of thing a pageant contestant would say if she had no intellectual or historical frame of reference to come up with a "real" hero (as the rest of my table did.)
But at the time, people seemed genuinely surprised. The IT executive said, "Wow - I'd say your dad is a pretty successful guy....for his daughter to say that. I sure hope my kid says that someday."
So when my son called me a "hero" last night -- despite the fact that he's four and goodness only knows what he really meant -- it made me think of that luncheon and my dear ol' Dad.
As I tried to fall asleep, I kept thinking of the various lessons I learned from the man who is still my "hero."
Here are the first ten things that came to mind. I'm keeping them concise on purpose so I don't spoil the chance to provide embellishment in future blog posts.
1. You can't burn a candle from both ends... at least not for very long.
2. If you're forcing something too hard, you're doing it wrong.
3. Sometimes pain doesn't actually teach you anything but it sure will show you what you're up against.
4. Never wrestle with a pig. You both get dirty but the pig likes it.
5. If you want to get more done in the day, wake up earlier.
6. If you really want something, it might be worth going to 15 different places to look for it.
7. Good education is about learning how to learn.
8. You don't have to like everyone and not everyone has to like you.
9. People in love think they're invisible.
10. When you're thinking about someone, let them know -- it will make their day.
THANKS DADDY! I love you and am thinking about you!
Happy Father's Day.


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STARKVILLE, Miss. — With his 26-yard touchdown run with 13:16 remaining in the first half Saturday night, Florida senior QB Tim Tebow joined former Georgia All-American RB Herschel Walker atop the SEC list for career rushing touchdowns.
It was also the longest touchdown run of Tebow's career.
Tebow and Walker each have 49 career rushing touchdowns. Walker achieved the feat in just three seasons — 1980-82.
Florida coach Urban Meyer called it an amazing accomplishment for his quarterback.
"I was a Herschel Walker fan, like nuts about him when I grew up," Meyer said. "He was (in college) right at the point I was in high school. I was actually a huge Georgia Bulldogs fan because of Herschel Walker. I got to meet him at the Heisman ceremony and sat and talked to him.
"Everything I know about him, he's first class. He represents Georgia, he represents the Southeastern Conference. My era was Herschel Walker and I was in awe of, (and) here's Tim Tebow in the same, I get to see him every day, and you're matching him in a rushing record that's the same as Herschel Walker. That's pretty powerful."
Tebow gets a chance to break the record next week in Jacksonville … against Georgia.
RED ZONE WOES: Florida's struggles inside the red zone continued. Coming in, the Gators scored touchdowns inside the red zone just 50 percent of the time this season (15-of-30). Through the first half Saturday, the Gators were inside the red zone four times with the following results: two field goals, one missed field goal and an interception that was returned 100 yards for a score by Mississippi State DB Jonathan Banks.
STREAK ENDS AT LAST: The Gators finished 3-0 against the SEC West, ending a decade-long streak that had plagued them. Florida had lost to at least one West team each of the past 10 years. The breakdown: 1999 — Alabama, 40-39 in OT (and again 34-7 in the SEC title game); 2000 — Mississippi State, 47-35; 2001 — Auburn, 23-20; 2002 — Ole Miss, 17-14, and LSU — 36-7; 2003 — Ole Miss, 20-17; 2004 — LSU, 24-21, and Mississippi State, 38-31; 2005 — Alabama, 31-3, and LSU, 21-17; 2006 — Auburn, 27-17; 2007 — Auburn, 20-17, and LSU, 28-24; 2008 — Ole Miss, 31-30.
FEELING THE HEAT: The constant attention and off-the-chain expectations might be taking a toll on the Gators, Meyer said this week. The fifth-year Gator coach said he has noticed that everyone associated with the program, from football office secretaries to coaches and players, are all showing signs of the pressure associated with this season.
"I think there's tremendous pressure on this team to perform at a very high level …," Meyer said. "My job is to make sure that I tell them constantly you don't have to be perfect, you just have to be the better team on the field that day."


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At some point during planning, I began to notice a theme to the inspiration images that stuck with me. They were airy, ethereal, soft, and romantic—all qualities that I hoped my bridal style would convey on my wedding day. I noticed one simple detail that I think makes a big impact: ribbons!
Photo by Erich McVey
I love this look—the soft pink color reminds me of the ribbons I used to lace up my pointe shoes back in my (middle school) ballerina years. You can use any type of ribbon, but silk ribbon was best for the look I wanted—it is super lightweight, so it blows romantically in the breeze, and it is just so delicate and pretty in person! I purchased six yards of blush pink silk ribbon (1.25 inches wide) from this Etsy seller.


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This property has been removed by the agent. It may be let or temporarily removed from the market
Property Description
Letting information:
|Not Specified
|Added on Rightmove:
|22 November 2011 (2132 days ago)
Key features
- Commercial
Full description
Modern offices available located in Manor Royal Industrial Estate, Crawley. Originally constructed in the early 1990 s, the whole terrace underwent a substantial refurbishment in 2008. The offices provide open-plan accommodation which has been refurbished to a good standard. ACCOMODATION First Floor Office 1,520sq.ft / 141.2sq.m AMENITIES Allocated Parking, Gas Central Heating (Not Tested), kitchenette, Partitioned Offices, Suspended ceilings, Double Glazing, TERM New FRI lease on flexible terms to be agreed RENT/PRICE From £5 per sq.ft RATES We have been verbally informed that the rateable values for the first floor - £19,000 (2010) VAT VAT may be chargeable on the terms quoted LEGAL COSTS Each party is to be responsible for their own legal fees VIEWING Viewing by appointment.
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Nicolas Cage details home invasion by naked man eating Fudgesicle
Nicolas Cage is selling his new movie Trespass at the Toronto International Film Festival, and he's picked a very interesting method to push the story of a man and his family held for ransom: He told reporters a story about a home invasion at his own house way back when. And it wasn't even about repo men coming to get his keys for not paying his taxes.
"It was two in the morning. I was living in Orange County at the time and was asleep with my wife. My two-year old at the time was in another room. I opened my eyes and there was a naked man wearing my leather jacket eating a Fudgesicle in front of my bed," Cage told Reuters. "I know it sounds funny ... but it was horrifying." That would explain his move to Nassau, Bahamas, then.
Cage said he talked the man into leaving the house and called cops, but didn't press charges because the man obviously was a couple Fudgesicles short of a snack pack. Maybe that's where Joel Schumacher got the idea for this new flick.
[Photo: Getty Images]


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A play centre could soon be opened, if planning permission is granted.
An application has been submitted to open the centre in the industrial estate in Hitchin Road, Shefford.
The building has stood empty for several years.
The application states: "The centre would offer a ball park and an activity climbing area, a sensory room for babies and toddlers, the disabled and the elderly together with a games room for electronic gaming for eight to 12-year-olds.
"The facility would also provide a mini gym area and one or two party rooms for private bookings and a cafe type food and light beverage facility."
The centre would be open from 9am to 9pm on weekdays and it would create up to 12 jobs.
There would only be two parking spaces and these would be used by staff; the application states that most people would walk. There would be 25 cycle spaces and there is general public parking for the industrial estate.
The report concludes: "The proposed facility will not only provide a positive addition to the town and local residential community but also a facility that forms an essential recreational space with great potential value."


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Pomegranate Extract in Treating Patients With Rising Prostate-Specific Antigen Levels After Surgery or Radiation Therapy for Localized Prostate Cancer
This study has been completed.
Roll International Corporation
National Cancer Institute (NCI)
Information provided by:
Roll International Corporation
ClinicalTrials.gov Identifier:
First received: June 13, 2006
Last updated: August 8, 2011
Last verified: August 2011
- Full Text View
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- No Study Results Posted
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- How to Read a Study Record
No Study Results Posted on ClinicalTrials.gov for this Study
|Study Status:
|This study has been completed.
|Study Completion Date:
|December 2010
|Primary Completion Date:
|July 2010 (Final data collection date for primary outcome measure)


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kenjennings I signed up with Repifi 2 months ago and its actually not bad. They don't just remove bad reviews but they create a system using my existing customer base to bring in additional positive reviews. Like I said I've only been with them for 2 months, but I can say it appears they do what the say they can do.
kittensmashr I've tried that with yelp, but they are a complete scam. They now offer a "membership" that I can pay for that will remove a bad review upon my request.


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Polling eBay Shareholders
On Wednesday, Bernstein Research analyst Carlos Kirjner published a poll of 178 eBay investors that indicated investors were undecided on whether or not eBay should split its Marketplace and PayPal businesses.
Of those that responded, 51% said a split was not wise, while 43% supported a split. Nearly 30% of shareholders responded that Carl Icahn should get a board seat, while 55% said eBay's board composition should change. Sixty percent of respondents said unlocking PayPal's value was the best reason for a split.
"[We] are in the minority and are not convinced that there is indeed a large value creation opportunity simply from separating the businesses. We think there are multiple cross-subsidies between Marketplaces and PayPal and a cursory valuation of PayPal as a standalone entity often will fail to account for the potential elimination of these cross-subsidies," Kirjner said.
"In other words, the relationship between the pieces would be identical to today's and all we would get (again, in theory) is a valuation bump," the analyst concluded.
Place Bid, Buy It Now
At about the same time Swan took the stage at the Morgan Stanley conference, Reid Hoffman published an extremely supportive letter of eBay's current strategy and accused Icahn of focusing on short-term trading profits over long-term growth. "It's classic market exploitation," Hoffman wrote.
By Thursday morning, Icahn responded.
"We have not yet begun to fight," the activist wrote on a website he recently created to champion shareholder rights. In late February, Icahn filed a proxy solicitation to split PayPal from eBay and nominate two new directors to eBay's board at the company's annual shareholder meeting later in 2014.
Sometimes, amid the cacophony of an activist campaign and a corporation digging in its heels, the narrative moves to tangential subject matter.
In the eBay case, Icahn has identified two board members, venture capitalist Mark Andreessen and Intuit (INTU) founder Scott Cook and their perceived conflicts of interest as the flagpole for his campaign.
Icahn has painted both Andreessen and Cook as the Manchurian Candidates of eBay's board, who've used their seats to invest in competitors to PayPal. Icahn also accused Andreessen of self-dealing in eBay's sale of Skype to private equity firm Silver Lake Partners, a deal which his VC firm Andreessen Horowitz had a 3% stake that paid off handsomely when the business was sold 18-months later to Microsoft for $8.5 billion.
EBay founder and Chairman Pierre M. Omidyar rejected those assertions earlier in March and Andreessen took to his blog to reject Icahn's claims.
On Thursday, Andreessen categorically denied Icahn's allegations surrounding the Skype deal, which it is worth noting, eventually made money for eBay shareholders.
"Mr. Icahn is making up a fake conspiracy theory out of thin air," Andreessen wrote, citing a Wall Street Journal article that said Microsoft passed on Skype when eBay put it up for sale. Seventy percent of the business was sold to Silver Lake for $2.75 billion in 2009. In 2011, Silver Lake flipped Skype to Microsoft for $8.5 billion.
EBay shares were gaining over 1% in Thursday trading at $59.48. Shares have gained over 11% in the past twelve months.
-- Written by Antoine Gara in New York


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The suspects were accused of culpable homicide.
An Ilorin Magistrate's Court on Tuesday remanded the traditional ruler of Tewu Village in Baruteen Local Government Area of Kwara, Sinapatiagi Sule, for alleged inhuman treatment.
The monarch was arraigned on a four-count charge with his seven chiefs for criminal conspiracy, culpable homicide, cursing grievous hurt, and trial by ordeal.
The chiefs are: Musa Lafia, Gunu Adamu, Muhammed Akpo, Usman Sabi, Sule Taku, Gunu Ibrahim and Amuda Tabe.
The prosecutor, Adekunle Iwaleye, told the court that the monarch invited the seven complainants to his palace for allegedly belonging to witches and wizards.
Mr. Iwaleye said the accused beat up the complainants with sticks, tortured them by putting fire into their mouths as well as forcing them to drink concoction made of urine.
He said the complainants were grievously hurt, fell sick due to the inhuman treatment and hospitalised.
The prosecutor said the offences contravened Sections 97, 229, 246 and 214 of the Penal Code.
The accused persons pleaded not guilty to the charges.
The Magistrate, Joel Michael, ordered that the accused be remanded in Mondala Prison Yard, Ilorin.
He adjourned the case to November 26 for mention.


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Is the Munis Pool dead in the water?
Supporters of Hamilton's crumbling Municipal Pool are treading water, although not in the lightly chlorinated, warm embrace they've been without for nearly two years.
As the barred and worn-out Victoria St complex awaits its fate, the city rushes past, and shiny new buildings thrust skywards nearby.
It is the city's forgotten oasis, a sanctuary from Hamilton's past, whose advocates have been fighting a rearguard action against its demolition even as they plan for its future.
But whether it even has a future as a pool remains unknown, and 19 months and counting after its doors closed to the public, tangible progress is hard to find.
The complex has suffered from years of maintenance neglect by the council and closed last year after a series of mechanical and structural failings.
Municipal's supporters also last year unveiled a $6.8 million plan that may yet save the much-loved but often overlooked central-city pool complex.
Their efforts to fundraise the millions of dollars to rebuild it have been knee-capped by the lack of committed council funding.
Pools lobby group Sink or Swim and city architect Mark Wassung's concept plan would see the century-old pools renovated and rebuilt in three major stages, the first estimated to cost $4.1m.
The city council last December passed up the chance to commit to a rescue package for the pools, instead deferring a decision until February 2014 and asking supporters to come up with a funding plan.
In a telling sign of their determination, they did. Stage one includes demolition of the existing large pool, considered beyond repair, and the development of new changing rooms, office space, green space and relocation of the existing plant, with a projected cost of $4.1m plus GST.
Stage two would involve the construction of a new 25-metre pool suitable for junior swimming education, construction of a cafe or restaurant and a grandstand upgrade, costing a further $1.4m.
Stage three includes boutique office space next to the pools and the Celebrating Age Centre, at an estimated cost of $1.3m.
But Sink or Swim spokesperson Katherine Luketina says potential external funders for the pools have balked at contributing without a council commitment.
"It's a council facility and, without council funding, external funders will not fund it to any extent," she said earlier this year.
However, Hamilton City Council, faced with a clamour from schools and the city's northeast over water space, too, has also been determined - to wait for an over-arching regional sports facilities plan due shortly, to give politicians the "big picture".
The Waikato Mayoral Forum last year asked Sport Waikato to lead the development of the Waikato Regional Sports Facilities Plan.
Eleven councils are completing a stocktake of their recreational facilities and analysing how well they are used. This data capture is due to be compiled by the end of the month and is overseen by a project control group and a technical working group.
The plan is to be presented to the city council in April and will complete the first stage in the planning of Waikato sport and recreation facilities,city council staff say.
Included will be a prioritised list of the region's current and future recreational facility needs.
It's almost certain to undermine the case for retaining some facilities and bolster the standing of others.
Sport Waikato head Matthew Cooper says the project control group is collecting and analysing data. "We'll then begin shaping a plan based on 'informed need' that will provide direction around existing and future sporting facilities across the Waikato region," he said.
The final report with recommendations will guide decisions in terms of upgrading existing facilities, developing new facilities, encouraging shared facilities and "decommissioning or disinvesting unused facilities". The last will have municipal pool supporters' attention, come April.
In the meantime, Luketina says Sink or Swim has been working behind the scenes as the new council bedded in and the review of sporting facilities was carried out. They are regularly lobbying councillors and Sport Waikato.
The group is aware of pushes by several city secondary schools to partner with the council to provide swimming space for the schools and public.
But Luketina says the city's partner pools programme, also under review, "can never fully address the needs of the swimming public because it only gives access to swimming clubs, which only cater to youth swimmers, and provides very limited opportunity for community access."
"It does not address the needs of the individual adult lane swimmers, of which Hamilton has many, or the needs of schools for somewhere to have their swimming sports, or the needs of the inner-city workers and dwellers."
Their aim is simple: raise funds in partnership with the city council to get the pools refurbished "so the people of Hamilton can enjoy their city asset".
Outsiders may wonder why the council is considering levelling a central city pool at the same time it is ramping up incentives and rule changes to persuade more people to live in the central business district.
Certainly, that apparent contradiction is not lost on several councillors who have expressed support for the pools complex in the past.
City council lawyers have already advised that public consultation would be required on any plans for the future of the municipal pools.
Waikato Times


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Seven area high school football teams remain alive in the state playoffs, and each of those seven programs knows it will be put to the test this weekend, as the second round gets underway in Classes AA-AAAAAA, while the first round kicks off for Class A.
Three of the postseason showdowns will be broadcast live on Friday by the Jacobs Media family of radio stations, including Westminster at Jefferson, Morgan County at North Hall and Gainesville at Kell.
In Class AA, Jefferson is looking to make history this week, as the Dragons aim to progress into the quarterfinal round for the first time since 1977.
Jefferson (11-1) will play host to Westminster (5-6) in a game that will play out on 550 AM, and should the Dragons prove victorious it would prove their deepest playoff charge since losing to Lincoln County in the 1977 Class B title game.
The Wildcats may own a losing record, but Westminster cruised through the first round, 34-0 over Pepperell, and all six of the Wildcats' defeats came to playoff teams, including four to Class AAA playoff programs -- three of which are also still alive in the state championship hunt.
Jefferson had little trouble with first round foe Kendrick, winning 42-8, but will face a stout Westminster defense that allowed just 182 yards and returned two interceptions for scores in its first round win.
North Hall too will expect a battle from Region 8-AAA No. 2 seed Morgan County when the Bulldogs travel to The Brickyard on Friday in a game broadcast live on 102.9 FM.
The Trojans (9-2) outfought Woodward Academy in the first half before cruising down the stretch in a 38-7 first round win last week, while Morgan County nipped Gordon Central 24-21 in Madison.
The Bulldogs (9-2) enter the second round averaging 33.9 points per game on offense behind a rushing attack that features a number of dangerous ball carriers, including Marquis Benton, Michael Gray, Melvin Davis and Andrew Couch -- North Hall, meanwhile, is yielding just 102.8 yards on the ground. Morgan's defense is allowing 17.7 points per contest on defense and will face a Trojans offense that is scoring 42.1 points per game while averaging just under 350 yards rushing.
The Trojans are gunning for their first state quarterfinal berth since reaching the Class AAA semifinals in 2007. Morgan County has not reached the third round of the postseason since 1990.
Gainesville is coming off a Class AAA semifinal berth and, on Friday night, will gun for the program's first-ever Class AAAAA quarterfinal spot in a face-off at Kell in a game that will be broadcast live on 1240 AM.
The Red Elephants (8-3) bounced back from a two-game losing skid in emphatic fashion last week, hammering Rome 41-14 at City Park -- marking the fifth straight season Gainesville has advanced past the first round. The Red Elephants will look to make it five straight quarterfinal trips this week, but that won't be easy against the Longhorns (10-1).
Region 7-AAAAA champs Kell battled past Southwest DeKalb 19-14 last week, using a last-minute, 32-yard touchdown pass from quarterback Matthew McGuigan to Julian Burris to secure the victory.
The Longhorns enter the contest averaging 31.2 points per game on offense -- which includes standout running backs Jamari Moore and Khalil Bell. Kell also features a number of FBS recruits, including defensive backs Brendan Langley and Quincy Mauger (both Georgia commits).
Gainesville's spread attack has put up points all season (43.4 ppg), but the key could be the Red Elephants' defense, which clamped down on Rome's wing-T attack for its lowest scoring total allowed since a 49-6 defeat of Salem on Oct. 12.
Kell is looking for its second ever quarterfinal berth after reaching the third round in 2010 courtesy a 21-13 defeat of Flowery Branch.
The trio of contests represent just part of a big night for area squads, as Buford also looks to keep rolling in the postseason.
The third-ranked Wolves (8-3) stormed past Cedar Grove 41-27 last week and will travel to take on North Oconee (10-1) this Friday.
The Titans have won back-to-back region titles (8-AA in 2011, 8-AAA in 2012) and will be battling Buford for their first ever state quarterfinal berth. The Wolves have not fallen short of the third round since 1999.
Both teams lean on stingy defenses, with Buford allowing just 8 points per game (last week's 27 points was the first time the Wolves gave up more than 19 in a game in 2012) and the Titans yielding 13.7, including three shutouts.
In Class AAAAAA, Mill Creek will play host to Brookwood (7-4) in a rematch of a 31-21 Broncos win from Sept. 7. The Hawks (8-3) defeated Alpharetta 41-39 in the first round, putting up 562 yards of offense. Meanwhile, in a top 10 showdown, No. 3 Grayson will play host to No. 5 North Gwinnett. The Bulldogs (10-1) hammered Johns Creek 49-19 in the first round, while Grayson (10-1) smacked Wheeler 49-9.
Meanwhile, in Class A, the new public/private postseason will get underway this week, and Commerce will travel almost 300 miles to face Seminole County. The Tigers (5-5) will try to win a playoff game for the first time since 2009. The Indians (8-2) reached the Class A quarterfinals last season.
FIRST-ROUND PLAYOFF RESULTS FOR AREA TEAMS, Nov. 16
Blessed Trinity 35, White County 14
Buford 41, Cedar Grove 27
Carrollton 40, Stephens County 7
Cartersville 56, Jackson County 21
Gainesville 41, Rome 14
Heard County 56, Rabun County 29
Jefferson 42, Kendrick 8
LaGrange 59, Chestatee 46
Mill Creek 41, Alpharetta 39
North Gwinnett 49, Johns Creek 19
North Hall 38, Woodward Academy 7
North Paulding 63, Flowery Branch 43
St. Pius X 72, Dawson County 28
Class AAAAAA
North Gwinnett at Grayson
Brookwood at Mill Creek
Class AAAAA
Gainesville at Kell -- Radio: 1240 AM
Class AAA
Morgan County at North Hall -- Radio: 102.9 FM
Buford at North Oconee
Class AA
Westminster at Jefferson -- Radio: 550 AM
Class A Public (First Round)
Commerce at Seminole County


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Take your pick: Tarpon can be targeted off of any bridge. The Gandy, Howard Frankland and Sunshine Skyway would be my top choices. While waiting for a tarpon to strike, I will drop smaller baits for Spanish mackerel. Snook are on the beaches in the swash channel. I like to cast my bait just short of the sand, then pull it back into the swash. Redfish have been hanging around oyster bars on the extreme high tides in the afternoon. A small pinfish under a small float is my bait of choice. You can pitch right next to the mangroves and monitor the bait. Trout provide action in deeper grass flats. Blue skies make it easy to find deeper grass; just look for the dark areas in 3 to 5 feet of water and work the edges with a quarter-ounce grub tail jig. Cobia have been roaming the flats on top of monster stingrays and spotted eagle rays looking for easy meals. When you see a huge ray, toss a pinfish around the ray; cobia can come out of nowhere and strike. Blacktip sharks have been roaming the sandbars outside of the gulf passes. Anchor on the edge of the channel and hang a chum block over the side. Use a large free-lined pinfish rigged with heavy wire.
Rob Gorta charters out of St. Petersburg and can be reached at captainrobgorta.com or (727) 647-7606.


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University of Minnesota, Morris
February 21, 2007
Members Present: Andy Lopez, Maddy Maxeiner, Sarah Mattson, Sara Haugen,
LeAnn Dean, Dan Moore, Lowell Rasmussen, Sharon Van Eps,
Bryan Herrmann, Arne Kildgaard
Guests: Bob Kvavik, Lincoln Kallsen, Orlyn Miller,
Carol Marxen, Jacquie Johnson, Sandy Olson-Loy, Dave Swenson
Bob Kvavik reported that they had spent the fall visiting with the various Deans on the Twin Cities campus as well as the coordinate campuses listening to what people want/need regarding capital planning. The University has never had a long term development plan and we need a better space management plan in place. Andy noted that in the last 15 years, UMM has been very blessed with buildings here and we hope we can continue. Bob stated that the six year capital request is very friendly to Morris. Andy asked if our priorities are clear.
Minutes prepared by Carrie Grussing