Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome.
Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F).
This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.
In general, lateral gene transfer (LGT) can be defined as the transfer of DNA between organisms that does not result from sexual reproduction. It can allow organisms to acquire novel traits that are unique from those that are vertically inherited and occasionally results in higher fitness. However, since the transferred DNA has little to no effect on fitness in most cases, it is often lost from the population shortly after the transfer has occurred. Although LGT has been described in many different organisms over the last decades, relatively fewer cases have been found in animals compared to many other taxa. However, when identified in animals, the donors are often cytoplasmic inhabitants, such as mitochondria or intracellular bacteria. For example, nuclear mitochondrial transfers (numt) have been found in nearly every eukaryotic genome and typically represent 0.1% of the genome . In animals, most of the LGT events identified as originating from bacteria have occurred between Wolbachia bacteria and their various hosts.
Wolbachia endosymbionts are bacteria that colonize filarial nematodes and several different arthropods, including >20% of insect species [2–4]. While obligate mutualists in nematodes, Wolbachia are best known for the ability to manipulate host reproduction in arthropods in various ways which include induction of parthenogenesis, feminization, male killing, and cytoplasmic incompatibility . In general, Wolbachia bacteria are intracellular and maternally inherited through the egg cytoplasm [2, 3]. As some strains have also been shown to specifically target the germline stem cell niche , these endosymbionts are ideally located to bring about heritable LGT to eukaryotic genomes [6, 7], since they colonize the cells that will give rise to the germ line and therefore offspring.
Wolbachia-host LGT events are termed nuwts  for nuclear Wolbachia transfers (pronounced: noot) following the established nomenclature for numts and nupts, nuclear plastid transfers. The first nuwt described was found in the bean beetle Callosobruchus chinensis that contains ~30% of a Wolbachia genome on the X-chromosome, and where about half of the genes examined showed low level transcription . Nuwts have now been identified in the genomes of many diverse invertebrate taxa [6, 8–13] and several transfers of large segments of the Wolbachia genome have been described, e.g. in Monochanus alternatus (longicorn beetle)  and Glossinia morsitans morsitans (tsetse fly) . Furthermore, ~20% of the nematode and arthropod species where whole genome sequence data is available have evidence supporting the presence of nuwts , and 80% of the genomes of all known Wolbachia hosts have some evidence indicating the presence of nuwts . Interestingly, every filarial nematode genome examined thus far has been shown to contain nuwts [10, 11, 16, 17], including nematodes that currently lack a Wolbachia endosymbiont [16–18].
Although very little is known in general about the function of nuwts, several nuwts or nuwt-related sequences have been found that give rise to genes that are expressed and possibly functional in their new environment. For example, salivary gland genes have been transferred between mosquitoes and Wolbachia, possibly in both directions [19, 20] and experimental data for one of them showed specific localization of the expressed protein to the basal lamina of the salivary gland and indicated its importance for malaria sporozite invasion . Furthermore, expression of twenty genes of bacterial origin from the nuclear genome in mealybugs has been demonstrated . Although this does not directly infer functionality, the majority of these twenty genes has been lost in the obligate endosymbionts of the mealybug, and some of these nuclear encoded genes complement the genes retained in the endosymbionts leading to complete pathways. For example, riboflavin biosynthesis requires three insect genes that presumably arose via LGT from facultative endosymbionts belonging to the α-Proteobacteria and Bacteroidetes phyla, which include the genera Wolbachia, Cardinium, and Arsenophonus. A similar result of identifying LGT from extant facultative endosymbionts was observed through an exhaustive analysis of LGT in the pea aphid, Acyrthosiphon pisum. That analysis revealed twelve genes or gene fragments of bacterial origin , five of which are most closely related to bacterial genes from the order Rickettsiales , of which Wolbachia is a member. One of these genes, ldcA, is transcribed in the aphid bacteriocyte and is involved in bacterial cell wall metabolism . Interestingly, Buchnera aphidicola, an obligate mutualistic γ-proteobacterium associated with aphids, has all the components to synthesize the bacterial cell wall, except ldcA, and it has been hypothesized that expression of this nuclear-encoded gene in the bacteriocyte may allow aphids to control growth of its endosymbiont by manipulating biosynthesis of the endosymbiont's cell wall . Lastly, many filarial nematodes possess a ferrochelatase gene that was acquired by LGT from an α-Proteobacteria in the Rhizobiales . This ferrochelatase, which encodes the terminal step of heme biosynthesis, is functionally active in an enzyme assay and complements an E. coli hemH mutant. In Brugia malayi, the ferrochelatase is involved with filarial motility and viability, as assessed with a ferrochelatase inhibitor, and is essential, as assessed with RNAi silencing with abnormalities arising in nuclei of germ cells and embryos .
The nuwt in Drosophila ananassae, examined here, was initially estimated to consist of an entire ~1.4 Mbp Wolbachia genome integrated on the left arm of chromosome 2 in a D. ananassae line from Hawaii . Only low level transcription of 28 of the 1,206 nuwt genes tested was observed in the D. ananassae Hawaii line at the time , and was later confirmed by the low abundance of nuwt sequences in the transcriptome of Wolbachia-depleted adult flies . Although, the biological relevance of this low level transcription is not known, these results did not give strong indication for functionality of the nuwt. However, in fourteen lines from the Drosophila Stock Center, PCR screening performed in the same study revealed the presence of similar nuwts in three more lines of D. ananassae from Asia and the Pacific, warranting further investigation into the evolution of this large nuwt in D. ananassae.
Here, we describe the results of re-sequencing three of the four D. ananassae lines originally discovered to contain large nuwts and the results of further in situ hybridization to examine its chromosome position and structure. The analysis confirmed integration in all three lines and revealed that the integrated genomes are affected by extensive duplication and underreplication. We demonstrate that in the Hawaii line the nuwt is most likely located on the heterochromatic fourth chromosome, which is abnormally large in D. ananassae compared to its close relatives. Taken together, the results suggest that the nuwt in D. ananassae may not currently provide any function, but rather that it is tolerated in its new genome and contributes to the large size of a chromosome that is already riddled with non-coding DNA and repeats.
Three of the four D. ananassae lines (Hawaii, India, and Indonesia) that were previously found to have a nuwt  were re-sequenced. All three lines are infected with Wolbachia bacteria (Wb+) and contain the nuwt (nuwt+) . Because sequence data from contaminating Wolbachia bacteria would complicate the analysis of the nuwt, the flies were treated with tetracycline (tet) for three generations prior to DNA extraction in order to create Wb- nuwt+ lines. To further ensure the absence of Wolbachia DNA originating from the bacterial genome rather than the nuwt in the sequencing data, the ovaries of tet-treated flies were confirmed to be Wb- by fluorescence in situ hybridization (FISH) microscopy with Wolbachia-infected flies serving as a positive control. DNA for sequencing was then isolated from the remaining carcass. Separately, DNA was isolated from 3rd instar larvae from a cross between a tet-treated India male and a Florida female that is naturally Wb- and nuwt-. Female F1 offspring were subsequently backcrossed with males of the parental tet-treated India line, which resulted in a line without the Wolbachia endosymbiont, with Florida mitochondria, and a mix of Florida and India nuclear genomes slightly enriched for India.
Despite numerous attempts to cure the Malaysia line, which is the fourth line where a nuwt was previously detected , we were unable to generate the necessary numbers of Wolbachia-free insects from this line for sequencing. On three separate instances, the line was acquired from the stock center, successfully cured using tetracycline for two generations, and the absence of Wolbachia endosymbionts verified. However, following curing, the lines would not proliferate to the needed levels. It is not clear if this is due to the absence of Wolbachia, the absence of other members of the microbiome, or other factors. However, as a result, this line was not sequenced.
The DNA extracted from each line was used to construct an ~3-kbp Illumina mate pair library and an ~300-bp Illumina paired end library. The resulting reads were mapped against the ~231 Mbp D. ananassae caf1 assembly  and the ~1.4 Mbp wRi Wolbachia genome  using BWA . The wRi genome was chosen as the reference for further analyses because (a) previous analyses have shown that wRi is more than 99% identical to the Wolbachia sequences obtained from the D. ananassae genome sequencing project [27, 29, 30], (b) the mapped reads cover the whole wRi genome, including genes that are not shared between wRi and wMel , and (c) the Wolbachia sequences in the selected D. ananassae lines are estimated to differ by less than one SNP in 20,000 bp . Thus, the wRi genome should be useful as a reference for the Wolbachia reads in all of the sequenced D. ananassae genomes.
We considered the risk that mitochondrial sequences might erroneously map to the wRi genome. Across all three datasets, only nine reads of the more than 500,000 reads were identified that mapped to both locations (1 in Hawaii, 0 in India, and 8 in Indonesia). Given these very low numbers that would not influence the values presented nor the interpretation, we did not consider cross mapping an issue to address further. The only regions where we saw erroneous mapping of reads were in the rRNA genes (see below), which likely arose because fed adults with an active gut microbiome were used for preparing genomic DNA.
Approximately 5× coverage of the Drosophila nuclear genome was expected for single copy genes based on the sequencing output of the mate pair library of the Hawaii line. Although mapping of the reads from this library to the D. ananassae genome shows the expected 5× coverage, or 1 copy (Figure 1), when mapping the same dataset against the wRi genome, the coverage varies, suggesting 1–8 copies, with the majority of the genome having ≥10× coverage, or 2 copies (Figure 1). This suggests extensive duplications of the nuwt in this line with up to 12 copies per haploid genome in regions where the sequencing coverage is 60×.
Coverage of Hawaii sequencing data. The copy number for the 3 kbp mate pair library from the Hawaii genomic DNA was calculated over a 1 kbp window every 500 bp and plotted against the reference wRi genome (left) and the first 1.5 Mbp of the largest scaffold (gi|109914400|gb|CH902617.1|) of the Drosophila ananassae caf1 assembly. Approximately 5× coverage was expected to represent single copy Drosophila genes and single copy regions are apparent. However, most of the nuwt is present with at least two copies.
Previously, similar copy number differences were observed using Illumina sequence reads and were successfully validated for the nuwts in B. malayi. Therefore, a qPCR assay was developed using 24 different primer pairs that amplify 4–5 regions predicted to be at approximately 1, 2, 4, 8, and 12 copies in the nuwt of the Hawaii line. In this assay, relative abundance is estimated by measuring the difference between the cycle threshold (Ct) values, i.e. the number of PCR cycles required for the fluorescent signal to exceed the background level, for the amplicons in the nuwt relative to the average Ct value of 6 single copy D. ananassae genes to yield the ΔCt value (Ct(nuwt amplicon)-Ct(average D. ananassae genes). The ratio of the abundance between the nuwt amplicon region and the average single copy gene is then 2ΔCt and should equal the number of copies of the nuwt amplicon in the genome as determined by genome sequencing. As expected, 2ΔCt has a positive linear correlation with the copy number over the length of the amplicon (Figure 2A) for the Hawaii line, where up to 8 gene copies per haploid genome could be validated. This suggests there may be as many as 16 copies per nucleus of large portions of this nuwt.
Confirmation of coverage metrics using a qPCR based assay. The copy number was confirmed and found to be accurate up to 8 copies per haploid genome using a qPCR assay measuring the ratio of the LGT abundance to that of six single copy Drosophila genes as shown for the 3 kbp mate pair library for the cured Hawaii (Panel A), India (Panel B), and Indonesia (Panel C). A significant positive correlation (p < 0.001, linear regression) is found for the cured Hawaii and the India line The results in Panels A and C represent the accumulation of multiple qPCR experiments while those in Panel B result from a single experiment.
When performing the same analysis on the India line, a consistently lower coverage over the nuwt is observed when compared to single copy Drosophila genes (Figure 3), suggesting <1 copy per haploid genome. Even so, India has a highly similar pattern of coverage variation to the one observed in the Hawaii line, indicating the presence of similar duplications (Figure 4). This result was consistent in three independent sequencing runs of (a) third instar larvae from the cross between India and Florida (I × F) maintained by LK and sequenced at NCGR, Santa Fe, NM (Table 1); (b) third instar larvae from I × F maintained by LK and sequenced at IGS from DNA isolated on a separate occasion (Table 1, Figure 3); and (c) adult flies from a tet-treated India line obtained from the stock center at a separate time and maintained in the JCDH lab and sequenced at IGS (Table 1, Figure 3). This sequence coverage was validated with the qPCR assay described above, and a correlation similar to that for the Hawaii line was observed (Figure 2B).
Coverage of India sequencing data. The copy number for the 3 kbp mate pair library from the India (Panels A and B), and India x Florida (Panels C and D) genomic DNA was calculated over a 1 kbp window every 500 bp and plotted against the reference wRi genome (left) and the first 1.5 Mbp of the largest scaffold (gi|109914400|gb|CH902617.1|) of the Drosophila ananassae caf1 assembly. The India genome shows the same relative pattern of duplication but is less abundant than single copy nuclear genes, as is the genome from offspring of a backcross of India with Florida. The spikes in the coverage for the India × Florida line reflect the mapping of transposase sequences.
Comparison of the coverage patterns. The coverage of the Indonesia genome is shown relative to that of the Hawaii line by dividing the values for Hawaii line by those of the Indonesia line across the wRi genome (Panel A) and the Drosophila ananassae genome (Panel B). The coverage of the India line relative to the Hawaii line is illustrated in the same manner (Panels C and D). While the India line has the same pattern of duplication as Hawaii, that pattern is different in the Indonesia line.
The sequencing statistics for line and sequence sequence strategy are provided, including the combined read length, number of reads sequenced, number of non-duplicate reads mapping to the D. ananassae caf1 assembly, number of non-duplicate reads mapping to the wRi genome, and the ratio of the reads mapping to the wRi and D. ananassae genomes.
*This library had a significant number of duplicate mate pairs such that >84% of reads mapped, but >80% of mapped reads were duplicates.
In contrast to Hawaii and India, the Indonesia line has very even sequence coverage, indicating that there is no variation in copy number between different regions of the nuwt in this line (Figure 5). As expected, Indonesia does not show a positive linear correlation using the qPCR assay since all of the genes have the same copy number. Therefore, the plot only shows the random variation present in the data (Figure 2C) supporting the assessment of coverage using the sequence read mapping. In all three cases examined, paired end reads yield approximately the same results as mate pair reads (data not show).
Coverage of Indonesia sequencing data. The copy number for the 3 kbp mate pair library from the Indonesia genomic DNA was calculated over a 1 kbp window every 500 bp and plotted against the reference wRi genome (left) and the first 1.5 Mbp of the largest scaffold (gi|109914400|gb|CH902617.1|) of the Drosophila ananassae caf1 assembly. The Indonesia line has a nuwt with even coverage similar to the rest of the Drosophila genome. The spikes in the coverage for the Indonesia line reflect the mapping of conserved DNA sequences (e.g. those from rRNA or tRNA) from bacterial contaminants like those in the guts of adult flies.
To ensure that the duplications seen in the nuwt are not an indirect effect of removing the Wolbachia infection or due to the tetracycline treatment itself, the relative abundance of the nuwt in whole non-tetracycline treated adult Hawaii flies was investigated (Wb+, nuwt+). These flies show the same relative levels of duplication when comparing nuwt genes, although the qPCR results are shifted 4-fold due to the additional amplification of genes in the bacterial genome (Figure 6AB). This 4-fold shifting suggests that there are four bacterial genomes for every copy of the fly chromosomes. The conservation of the relative levels of duplication in the presence of Wolbachia endosymbionts indicates that the extensive duplication of the nuwt has not occurred during the tetracycline treatment of the flies.
Comparison of copy number of the nuwts in homozygous and heterozygous fly lines and cured v. uncured flies. Cured (Panel A) and uncured (Panel B) Hawaii flies show the same correlation of qPCR results and sequencing coverage, although the qPCR results are shifted by the amplification of genes in the bacterial genome as they are plotted against the sequencing coverage obtained from the cured insects. This indicates that the duplication of the nuwt does not occur during the tetracycline treatment of the flies to compensate for the loss of the infection. The results of the qPCR assay for coverage was compared for homozygous cured Hawaii (Panels A) and the offspring of a Hawaii/Mexico cross which is heterozygous for the nuwt (Panels C) for the 3 kbp mate pair library library. Should the Hawaii line be heterozygous, the cross should have resulted in 50% of the offspring being homozygous for absence of single copy regions of the nuwt. Instead, each of the 44 offspring (half with Hawaii mothers and Mexico fathers and half from the reciprocal cross) tested positive by PCR for single copy genes in the nuwt and the nuwt was at half of the abundance of the Hawaii line relative to internal single copy Drosophila nuclear gene controls in 4 of these as seen by a shifting of the y-intercept by ~1 ΔCt.
Given that coverage can be noisy and very few regions in the nuwt were inferred to be present in a single copy, we wanted to ensure that regions with low coverage were not due to heterozygosity of the nuwt. Tet-treated flies from the Hawaii line (Wb- nuwt+) were crossed with flies from the Mexico line that does not have a Wolbachia infection nor any nuwt (Wb-; nuwt-). If the nuwt is heterozygous, i.e. some genes are only present on one of the chromosomes in a pair, we expect that some of the F1 offspring from the cross with a nuwt- line will lack regions identified as single copy in our coverage analysis. However, the abundance of the nuwt, including single copy regions, in the F1 offspring was half that of the parental Hawaii line using the qPCR-based coverage assay (Figure 6A,C) indicating that the nuwt is homozygous. This is not unexpected given that the Hawaii line was selected for genome sequencing because it was the most inbred line available .
To determine whether the overrepresentation of the nuwt relative to nuclear genes could be due to something other than stable DNA duplications, we examined if there is any variation in the duplication level throughout the life cycle in the Hawaii line. Thus, the copy number of the nuwt was examined by qPCR in single individuals as embryos, third instar larvae, pupae, and adults (Figure 7). In the Hawaii line, the copy numbers were stable throughout the life cycle except in third instar larvae, where the nuwt had the same coverage pattern as the genome sequencing data, albeit with a 4-fold underrepresentation (Figure 7B) relative to single copy Drosophila genes. Interestingly, two Drosophila genes examined with the qPCR assay demonstrate the same behavior, dsx and an RNA polymerase III gene. In salivary glands of third instar larvae, DNA is found in the form of polytene chromosomes, which contain multiple linked chromosomes that are formed through massive replication without cell division, i.e. endoreplication . During endoreplication, not all parts of the chromosome are equally replicated; instead regions like the heterochromatin become underrepresented while euchromatin can become overrepresented. Since the dsx gene is found in heterochromatin in Drosophila melanogaster, an underrepresentation of these genes in the larval stage might suggest they are underreplicated and hence are present in heterochromatin. While the position of dsx with respect to heterochromatin/euchromatin is not known in D. ananassae, it is consistently 2- to 8-fold underrepresented in our qPCR studies. The similar levels of underrepresentation of dsx and the nuwt are consistent with both being located in heterochromatin in the Hawaii line.
Comparison of copy number of the nuwts through the life stages. The copy number from the genome sequencing of adult Hawaii flies have a significant positive correlation to the qPCR results obtained for embryos (Panel A), 3rd instar larvae (Panel B), pupae (Panel C), and adults (Panels D). While the slopes for embryos, pupae, and adults are approximately one, the slope for 3rd instar larvae is almost four suggesting that the nuwts are 4-fold underreplicated in 3rd instar larvae.
While qPCR on 3rd instar larvae suggests that the inserts in the Hawaii line are heterochromatic, this is inconsistent with the previous results demonstrating a single band in a euchromatic region when polytene chromosomes from the salivary glands of 3rd instar larvae were hybridized with a probe amplified from the nuwt for the gene homologous to WD_0484 . WD_0484 is a hypothetical protein in the genome of Wolbachia strain wMel from D. melanogaster with 1–2 copies per genome equivalent in the nuwt in the Hawaii line using the coverage values described above, consistent with a single band if euchromatic. If the nuwt is euchromatic, regions with 8–12 copies per genome equivalent should reveal multiple bands. Therefore, we probed polytene chromosomes from the salivary glands of 3rd instar larvae from the Hawaii lines with WRi_004290 amplified from genomic DNA of the nuwt+/Wb- Hawaii line. WRi_004290 encodes the DNA repair protein RadC and is estimated to be present in 8–12 copies. Instead of seeing multiple bands in the polytene chromosomes, only a single band was revealed (Figure 8A,B,C) that appeared to be in a different location than the band for WD_0484. However, as both of these probes were prepared directly from genomic DNA, low level contaminating amplification products could result in hybridization to the wrong locus. When repeating the experiment with a probe amplified from a sequence-verified clone containing WRi_004290, no bands were identified in the polytene chromosome FISH (Figure 8D) even though a single control band was seen when using a probe with a different fluorescent label amplified directly from a sequence-verified clone containing the D. ananassae actin gene in the same polytene chromosome preparation (Figure 8E,F). However, when the WRi_004290 probe generated from a clone was used on mitotic chromosomes isolated from larval brain ganglia, hybridization to the heterochromatic fourth chromosome (Figure 9) was observed. Considering that the various copies of the nuwt genes co-segregate , we believe that all, or most, of the LGT is on the heterochromatic fourth chromosome. This is consistent with the FISH results reported here with regards to polytene chromosomes, since the fourth chromosome cannot be visualized in the polytene for D. ananassae. Additionally, this result is more consistent with the genome sequence and mapping of scaffolds to the chromosomes in D. ananassae, relative to the previous results, because there is not a sufficiently large gap in the scaffold spanning the euchromatic portions of the second chromosome that could house the ~5 Mbp nuwt.
Fluorescence in situ hybridization of polytene chromosomes. Polytene chromosomes were hybridized with a Wolbachia-specific WRi_004290 probe amplified directly from genomic DNA from the Wb- Hawaii line and visualized at 40X magnification. A single band is observed and not the 8–10 bands expected (Panel A). Polytene chromosomes were again hybridized with the WRi_004290 probe amplified directly from genomic DNA from the Wb- Hawaii line and visualized at 20X (Panel B) and 40X (Panel C). A single band is observed and not the 8–10 bands expected. Polytene chromosomes were simultaneously hybridized with differentially-labelled probes for the Wolbachia-specific WRi_004290 (Panel D, pink) and a D. ananassae actin gene (Panels E &F, green) that were generated from sequence-verified plasmids and visualized at 40X. While hybridization by the plasmid-derived WRi_004290 probe is not detected, hybridization by the plasmid-derived actin probe is detected. This suggests that the previous detection with Wolbachia probes amplified directly from genomic DNA may be detecting hybridization of spurious amplification products and that the lateral gene transfer is heterochromatic.
Fluorescence in situ hybridization of mitotic chromosomes. Mitotic chromosomes were hybridized with a Wolbachia-specific WRi_004290 probe amplified from sequence-verified plasmids and visualized at 60X. While hybridization was not detected in polytene chromosomes with WRi_004290, it is detected in the mitotic chromosomes. The fluorescence appears to be localized to the centromere of the abnormally large fourth chromosomes of D. ananassae. The larger 2nd and 3rd chromosomes are visualized nearby as well as a likely X chromosome. Hybridization to the fourth chromosome is consistent with the LGT being largely heterochromatic.
The lower coverage of the nuwt in the India line relative to single copy nuclear genes indicates that it is underrepresented in the genomic DNA when compared to the majority of the D. ananassae genome. Unlike the putative underreplication in third instar larvae seen in the Hawaii line, underrepresentation of nuwt DNA in the India line is seen in adults. One possibility is that the population from the stock center was polymorphic for the nuwt and over time the population is losing the nuwt. Another possibility is that endoreplication is occurring or there is an episomal location for the nuwt that is underrepresented in individual flies.
A male from the untreated India line (Wb+, nuwt+) was crossed with a female from a line from Mexico that contains neither the nuwt nor any Wolbachia infection (Wb-, nuwt-), and an offspring was examined using the qPCR coverage assay. Such a cross should remove Wolbachia in a single generation without the use of antibiotics across multiple generations. The nuwt was undetectable by the F1 generation, suggesting it was lost in both the tetracycline treated and untreated populations.
Given that the India line was reared in the stock center for several years prior to us obtaining it, and that it was detected at levels like the Hawaii line at the time we obtained it, we are uncertain of what triggers this underrepresentation to occur. We re-acquired the line from the stock center and the nuwt in this line is now undetectable as well. Since the nuwt is no longer detected in the population, we could not examine this phenomenon further.
The even coverage of the nuwt in the Indonesia line (Figure 5), as seen from the mapping of reads to the wRi genome, was confirmed by the qPCR assay (Figure 2C). One concern with the even coverage observed in this line is that the sequences could stem from a residual Wolbachia infection, rather than from a nuclear insert. This is unlikely as the fly line was previously treated with tetracycline, and FISH on dissected ovaries using a probe for Wolbachia did not reveal the presence of any Wolbachia bacteria. However, to further confirm the presence of a nuwt in the Indonesia line, previously prepared DNA  from a F1 offspring of a cross between an Indonesia male and a Florida female (Wb-; nuwt-) was also examined. Such a cross should remove the maternally inherited Wolbachia endosymbionts. With a single insertion of the nuwt, these heterozygotes should contain half as much nuwt DNA as single copy nuclear genes. Instead, the ΔCt (average Ct for Drosophila – average Ct for nuwts) was found to be between 1.2 and 1.6 (average = 1.4; stdev = 0.18; n = 4) indicating that there is 21.4×, or 2.6×, more Wolbachia DNA than single copy nuclear genes. Given that these are F1 hybrids, this means the parental Indonesia lines have 5.2× more Wolbachia DNA than single copy nuclear genes, suggesting there were originally 5 or more copies of the nuwt covering the whole Wolbachia genome in the Indonesia line. This may suggest that when the genome sequencing of Indonesia was undertaken it was 5-fold under-represented relative to when the F1s were generated.
While the Indonesia line with a nuwt was present in the JCDH lab when sequenced, the line has since become underrepresented for the nuwt, similar to the India line. We and others have obtained further samples of the Indonesia line from the UCSD Drosophila Stock Center that all now seem to lack the nuwt, as shown by our qPCR assay and other PCR-based assays (data not shown). Therefore, further experiments to explore this were not possible.
There are multiple reasons for why not all parts of a chromosome are present in equal amounts in a cell. The most commonly known mechanism is that these different parts are not replicated an equal number of times, due to chromatin state and/or distance from origins of replication. The phenomenon of over- and underreplication is particularly common in cells that undergo endoreplication (i.e. DNA replication in the absence of mitotic division).
Apart from the endoreplication of polytene chromosomes found in salivary glands of third instar larvae, endoreplication has also been suggested to be evolutionarily conserved in ovarian follicle cells of Drosophilidae since all of the 38 species examined by Bosco et al. complete DNA replication with a ploidy consistent with 16 genome equivalents per cell . However, in the same cells the authors also find that some DNA is represented by only 4 genome equivalents, which is consistent with the presence of heterochromatic sequences that are not replicated to the general ploidy of the cell, but instead are underreplicated . While endoreplication and polytene chromosomes are best studied in third instar larva salivary glands and ovarian follicle cells in Drosophila species, nearly all differentiated cell types are polyploid with polytene chromosomes .
Given the bacterial ancestry of nuwts, it is likely that they lack Drosophila origins of replication. In general, if nuwts only contain a few genes, the distance from an origin of replication is inconsequential, and therefore underreplication might not occur. However, given the large size of the nuwt described here, underreplication could potentially occur merely due to a lack of Drosophila replication origins.
In Drosophila, a six-protein origin recognition complex (ORC) recognizes origins of replication, and binding of this complex is needed for replication to begin. A summary of predictions in D. melanogaster suggests that there are 7,329 non-overlapping ORC recognition sites with a maximum distance between ORC sites of 447 kbp in the genome that are potential origins of replication  (http://flypush.imgen.bcm.tmc.edu/pscreen/files/ORCsitenames.xls). Assuming that D. ananassae has a similar number of replication origins and that they are evenly spaced across the entire genome, one would expect to identify an origin every ~315 kbp assuming an ~231 Mbp genome, (size of the caf1 assembly [26, 33]), or every 293 kbp assuming an ~215 Mbp genome, (genome size from literature [33, 37, 40]).
Using either metric or even the maximum distance between D. melanogaster ORC sites, the distance between origins of replication is smaller than the size of the nuwts found in D. ananassae. It was previously shown that six physically distant nuwt loci co-segregate perfectly in F2 maternal inheritance crosses , indicating that the multiple copies of the nuwt are on the same chromosome and physically linked. Here we show that there are 2.3 Wolbachia reads for every 100 Drosophila reads in the Hawaii line (Table 1), meaning 2.3% of the D. ananassae Hawaii genome is from the nuwt. Assuming the D. ananassae genome is ~215 Mbp, this means that ~5 Mbp of the D. ananassae genome might consist of tandemly arrayed nuwts. Furthermore, we know of numerous integrations of retroelements  that make the chromosome region that is devoid of replication origins even larger, assuming origins of replication do not integrate in the nuwt along with retroelements. Therefore, the lack of Drosophila origins of replication may at least partially explain the underrepresentation observed in third instar larvae of the Hawaii line.
This would not explain the underrepresentation of the nuwt in the India and Indonesia lines. Although underreplication in somatic tissues, specifically the larval fat bodies and ovarian follicle cells, has been observed , we are not aware of any case with such high levels of underreplication as in the nuwts of the India and Indonesia lines. Therefore, while the underrepresentation of the nuwts in these two lines could result from underreplication in adult somatic tissues, we believe that they either are polymorphic in the population or that there is another, currently unknown, mechanism that explain these results. One possibility would be that nuwts are episomal or on dispensable B chromosomes in the India and Indonesia lines. However it is less clear why, before being lost, the nuwts were stable in these lines at the stock center for many years as well as in independently maintained collections.
Given the combination of extensive duplication containing polymorphic sites (data not shown) and the underrepresentation of the nuwt in the India and Indonesia lines yielding relatively low coverage, a robust analysis of SNPs is not feasible. However, from the coverage analysis of the nuwt in the re-sequenced lines we see a highly similar pattern of duplicated regions in the Hawaii and India lines suggesting that they share a recent common origin. Although an alternative explanation for this observation would be that the insertion of Wolbachia DNA in the nuclear genome is non-random, this explanation seems less likely given that the same pattern is not seen in the Indonesia line.
In the 14 lines that were originally screened in the study by Dunning Hotopp et al. only four were found to contain the nuwt. Of these four lines, two were from South East (SE) Asia, one from India and one from Hawaii, whereas all lines from Africa, the continental Americas, and Australia as well as one additional Hawaii line lacked a nuwt. Recently, an additional 23 D. ananassae lines were screened, of which eight tested positive for nuwts, including three from SE Asia, one from India, one from the South Pacific island Tonga, two from Australia and one from Ethiopia (although verification of lack of bacterial infection was not performed, leaving the possibility of a residual infection) . All additional nine samples from the Pacific in this screen tested negative, including one sample from Hawaii . Based on both screens, it is clear that the nuwt has wide geographic distribution, but that individual lines from the same geographic regions can differ in nuwt status. Given that we have experienced loss of detection of the nuwt in the Indonesia and India lines, it is possible that some lines may have nuwts that are undetectable or that some lines might have lost the nuwt during cultivation. It is however interesting to note that all five lines from SE Asia, which likely represents the ancestral species range of D. ananassae, contain a nuwt. Colonization of other parts of the world, including India and Hawaii, could be a consequence of human activity and hence these populations could represent independent migrations from the same ancestral population or human-enabled migrations between the two locations. Hence, integration of Wolbachia DNA into the nuclear genome of D. ananassae might have occurred just once and then spread to different locations via human activity. One possible scenario is thus that the most recent common ancestor of all three lines (Hawaii, India and Indonesia) was a line that had extensive duplication of the whole Wolbachia genome and that the Indonesia lineage maintained the complete copies of these integrated genomes while the lineage leading to Hawaii and India lost some parts of the nuwt.
The chromosomal location of the integration site of all three nuwts could inform our understanding of how nuwts form and perhaps also about their ancestry. However, due to the integration of numerous host retrotransposons inside the nuwt, we could not identify its exact location based on sequence data in any of the lines despite numerous attempts. Although FISH on mitotic chromosomes could be used to locate the nuwt in the Hawaii line to chromosome four, the inability to detect the nuwt in the two other lines currently eliminates the possibility of exploring this further.
In most Drosophila species, chromosome four (Muller element F) is referred to as the dot chromosome because of its small size. In D. melanogaster, chromosome four is the size of a typical bacterial genome at ~4.2 Mbp, containing 92 genes and ~3.0 Mbp of repeated sequences . Most Drosophila species sequenced have a similarly sized fourth chromosome with only 1.0-3.4 Mbp of contigs mapped to it [33, 42]. Interestingly, even though part of chromosome four can normally polytenize in most Drosophila species, the whole chromosome shows properties similar to heterochromatin.
In contrast, the fourth chromosome in D. ananassae is equivalent in size to the 37 Mbp X chromosome (Muller element A)  and has not been observed to polytenize. The increased size of the fourth chromosome of D. ananassae is thought to be the result of an expansion of repeats, with 32.5% of the sequence arising from retro-elements (27.1%) and other DNA-based repeats (5.4%) . However, the real repeat content is probably even higher, since gaps are likely to contain even more repeats. Based on our results, we propose that the ~5 Mbp of nuwt sequence described here, as well as the retro-elements known to be integrated into the nuwt, are located on the fourth chromosome of D. ananassae. The nuwt would thus represent ~20% of the genetic material in chromosome four and partially explain its larger size in D. ananassae. However, as there is no described size variation of chromosome four in D. ananassae, although there is variation in the presence of the nuwt, it might just indicate that this species is prone to accumulate genetic material on this chromosome and perhaps also in general, since D. ananassae has the highest repeat content of the sequenced Drosophila species .
In conclusion, we find that multiple Wolbachia genomes are transferred to the Drosophila ananassae nuclear genome. The Hawaii and India lines show uneven coverage suggesting that different parts of these nuwts are duplicated to different levels whereas the even coverage seen across the nuwt in the Indonesia line suggests nearly perfect duplicates. Furthermore, the large nuwt in the Hawaii line becomes underreplicated in third instar larvae suggesting that it is located in heterochromatin. However, the underrepresentation of the nuwt in the India and Indonesia lines is more severe than for heterchromatic regions and might reflect a gradual loss of the nuwt in the lab population or a yet unknown mechanism causing underrepresentation of DNA. Hybridization to mitotic chromosomes indicates that the nuwt is located on the fourth chromosome, which is enlarged in D. ananassae compared to other Drosophila species. Taken together with low levels of expression from genes in the nuwt [10, 25], it seems unlikely that the nuwt genes are functional. This might explain why the nuwt has not spread to more populations and other continents. However, it is still possible that the nuwts are functional under a yet unknown condition, which could explain their presence in multiple geographic locations. Taken together, each line is unique in its combination of attributes and present interesting aspects for further research on LGT between symbionts and their hosts.
The Wb + nuwt + Hawaii (14024–0371.13), India (14024–0371.31), Indonesia (14024–0371.34) lines and Wb- nuwt- Mexico (14024–0371.00) and Florida (14024–0371.12) lines were obtained from the Drosophila Species Stock Center (University of California, San Diego, San Diego, CA, USA). Populations were grown on Jazz-Mix Drosophila food (Applied Scientific, Waltham, MA, USA) in plastic bottles at room temperature and humidity, or in an insect growth chamber (Caron, Marietta, OH, USA) at 25°C and 68% humidity.
All Wb + nuwt + lines were treated with tetracycline to remove the Wolbachia endosymbiont and generate Wb- nuwt + versions. Populations were grown for at least two generations on Jazz-Mix media containing 0.025% (25 mg per 100 mL media) tetracycline (Teknova, Hollister, CA, USA). Absence of the Wolbachia endosymbiont in the lines was confirmed by FISH microscopy on ovaries using Wolbachia-infected insects as a positive control.
Flies were examined by FISH to verify presence or absence of Wolbachia endosymbionts. Freshly dissected ovaries were fixed in methanol for 1 h at 4°C and washed twice in 1× PBS. To confirm Wolbachia infection, two Alexa Fluor 488-labeled Wolbachia-specific oligonucleotides (W1: AACCCGGCCGAACCGACCC; W2: CTTCTGTGAGTACCGTCATTATC) were hybridized to the fixed ovaries at 37 °C overnight in hybridization buffer containing 50% formamide, 5× SSC, 200 g/L dextran sulfate, 0.5× Denhardt's solution, 0.1 M dithiothreitol (DTT), 250 mg/L salmon sperm DNA, 250 mg/L tRNA, 250 mg/L poly(A). Hybridized ovaries were washed in 1× SSC + 10 mM DTT at room temperature once followed by 1× SSC + 10 mM DTT at 55 °C twice, in 0.5× SSC + 10 mM DTT at 55°C twice, and in 0.5× SSC + 10 mM DTT at room temperature. Ovaries were placed on a microscope slide and mounted with glycerol, a drop of SlowFade Gold antifade reagent (Invitrogen, Grand Island, NY, USA), stained with DAPI, and visualized with a Nikon TE-2000 using inverted fluorescence microscopy (Nikon, Melville, NY, USA).
For the India x Florida cross (I × F) sequenced at IGS, DNA was isolated from ~100 larvae in 6 different extractions using the Gentra Puregene Tissue kit (Qiagen, Valencia, CA, USA) with an incubation with Proteinase K (Finnzymes, Vantaa, Finaland) for at least 4 h, but in several cases much longer. All samples were re-purified so that protein precipitation was done twice. For the DNA sequencing conducted by NCGR, DNA was extracted from 20 larvae with the Aqua pure tissue kit (Bio-Rad, Solna, Sweden) with a proteinase K incubation for 3.5 h at 55°C followed by an overnight incubation at room temperature. In addition, the protein precipitation was repeated for this sample. For all other samples, DNA was isolated using with a DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD, USA). Up to 50 mg insects were ground in liquid nitrogen with a disposable mortar and pestle and then processed according to the standard DNeasy protocol.
The library sequenced at NCGR was prepared using standard Illumina protocol for mate pair library preparation. For all other DNA samples, both a 300-bp paired-end and a ~3000-bp mate-pair library were constructed for sequencing on the Illumina platform using the NEBNext® DNA Sample Prep Master Mix Set 1 (New England Biolabs, Ipswich, MA, USA). First, DNA was fragmented with the Covaris E210. Then libraries were prepared using a modified version of manufacturer's protocol. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA). The PCR amplification step was performed with primers containing a 6 bp index sequence. Sequencing was performed using both an Illumina Genome Analyzer IIx and an Illumina HiSeq2000. Because the experiments were conducted during a period of rapid evolution of the Illumina sequencing technology and the read-length was maximized at each stage, the paired-end data here represents reads that range from 57 bp to 101 bp (Table 1). Conversely, in order to minimize the number of chimeric sequences generated by sequencing across the untagged junction in the mate-pair libraries, the reads for these libraries were limited to between 35–40 bp (Table 1). Base calling and quality scoring was performed using Illumina software followed by in-house quality assessment and control pipelines to truncate and eliminate poor-quality reads. Sequencing data is available in the SRA (Hawaii line: SRA052201; Indonesia: SRA052189; India: SRA052191; I × F: SRA052193).
Reads were mapped against the D. ananassae caf1 assembly  and the wRi Wolbachia genome  using BWA  version 0.5.9-r16 with the default parameters except that the maximum number of alignments reported in the XA tag was 1000. Reads from the mate pair libraries were reverse complemented prior to alignment using fastx_reverse_complement (http://hannonlab.cshl.edu/fastx_toolkit/). Duplicate sequences were removed and reads were position sorted using Picard version 1.48 (http://broadinstitute.github.io/picard/) with the default parameters. Genome sequencing coverage was calculated with MPILEUP in SAMTOOLS version 0.1.19-44428 cd  using the default parameters. Copy number was calculated by dividing the coverage by the mode of the coverage using a bin size of 1. Plots were generated using the R statistics and analysis package version 2.15.2.
Given the shared ancestry of mitochondria and Wolbachia, we wanted to ensure that reads were not mapping to both. Across all three datasets, only nine reads were identified that mapped to both locations (1 in Hawaii, 0 in India, and 8 in Indonesia). Given these very low numbers that would not influence the values presented or the interpretation, we did not consider cross mapping to be an issue to address further. The only regions where we saw erroneous mapping of reads were in the rRNA genes as discussed in the text, which likely arose because fed adults with an active gut microbiome were used for preparing genomic DNA.
Relative coverage of the Wolbachia insert in the Drosophila genome was examined with qPCR on genomic DNA. Primers (Additional file 1) were designed based on sequence coverage and loci were selected that were single copy in the wRi genome. Primers were designed using Primer3 and synthesized by Sigma-Aldrich (St. Louis, MO, USA). Dilutions of genomic DNA (0.2-1 μL stock gDNA per 20 μL reaction) were used as templates in a qPCR reaction containing 2× QuantiTect SYBR Green (Qiagen, Germantown, MD, USA), RNase-free water, and coverage primers using the standard protocol. The assays were conducted using an ABI 7900 instrument (Applied Biosystems, Foster City, CA, USA). The reactions were denatured at 95°C for 15 min followed by amplification with 45 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 30 s. Reactions were followed by a melt curve analysis that starts at 55°C, with a dissociation step at 95°C for 1 min plus 0.5°C/cycle for 80 cycles. Comparisons of normalization by (a) aggregating the results for the six single copy Drosophila genes or (b) using each individual single copy nuclear Drosophila gene showed little difference since the six single copy Drosophila genes routinely have <1 Ct variance. Linear regression analysis was completed using the standard R statistical package.
Genomic DNA was isolated from tetracycline-treated D. ananassae Hawaii flies using the Qiagen DNeasy Kit (Qiagen, Valencia, CA, USA). The PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA) was used to synthesize probes from 25 ng of genomic DNA with primers specific for a highly duplicated region of the nuwt (WRi_004290), as indicated by whole genome sequencing and qPCR. The labeled probe was purified using the Qiagen Qiaquick PCR Purification Kit and suspended in 150 μL hybridization buffer consisting of 2× saline-sodium citrate (SSC), 10% dextran sulfate, 50% formamide, and 0.8 mg/mL salmon sperm DNA.
Genomic DNA was isolated from tetracycline-treated D. ananassae Hawaii flies using the Qiagen DNeasy Kit (Qiagen, Valencia, CA, USA) and used as a template for PCR with NEB 2× Master Mix (New England Biolabs, Ipswich, MA), using primers specific for a highly duplicated region of the nuwt (WRi_004290), as indicated by whole genome sequencing and qPCR. The amplicon was cloned into a TOPO TA subcloning vector (Life Technologies, Grand Island, NY, USA). Plasmid DNA was isolated with the Qiagen Qiaprep Miniprep Kit. For the digoxigenin probes, 50 pg was used as a template in a reaction using the PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA) with primers specific for WRi_004290. The labeled probe was purified using the Qiagen Qiaquick PCR Purification Kit and suspended in 150 μL hybridization buffer consisting of 2× saline-sodium citrate (SSC), 10% dextran sulfate, 50% formamide, and 0.8 mg/mL salmon sperm DNA. For the biotinylated probes, 1 μg of purified plasmid was used as a template in the Roche biotin high prime labeling protocol, following the manufacturer's instructions, and suspended in 150 μL hybridization buffer as described above.
Salivary glands were dissected in 1× phosphate buffered saline (PBS) and incubated in 40 μL 45% acetic acid for 2 min followed by incubation in 10 μL 1:2:3 lactic acid:water:glacial acetic acid for 3 min on an 18 mm × 18 mm cover slip. The glands and cover slip were picked up with a SuperChip poly-L-lysine slide (Thermo Scientific, Waltham, MA, USA), flipped over, and the areas of the glands were gently tapped with forceps, followed by tapping concentric circles in an outward spiral. Excess fixative was blotted from an inverted slide applying gentle pressure. The face-up slide was gently streaked rapidly in a zig-zag manner in one plane, rotated 90°, and rapidly streaked again. Excess fixative was again blotted off, and with the cover slip facing down, the slide was squashed firmly with a stack of cleaning tissues. The spreads were checked with a microscope for refractivity. The slides were incubated at 4°C overnight, immersed in liquid nitrogen for 15 s, and the cover slip was flipped off using the edge of a razor blade as a lever. Slides were then incubated in 96% ethanol for 10 min, air dried, and checked for refractivity.
Brains were dissected from the 3rd instar of D. ananassae Hawaii larvae in 1% sodium citrate and incubated for 10 min at room temperature. The ganglia were then incubated in 3:1 methanol:glacial acetic acid for 30 s, 45% acetic acid for 30 s, and 60% acetic acid for 4 min on a poly-L-lysine slide under an 18 mm × 18 mm cover slip. The slide was then placed between tissue paper and the perimeter was tapped with an eraser. Excess fixative was again blotted off, and with the cover slip facing down, the slide was squashed firmly with a stack of cleaning tissues. The spreads were checked with a microscope for refractivity. Slides were immersed in liquid nitrogen for 15 s, and the cover slip was flipped off with a razor blade. Slides were then incubated in cold 70% ethanol for 5 min, cold 80% ethanol for 5 min, and cold 100% ethanol for 10 min, air dried, and checked for refractivity.
Chromosome preparations were incubated in 2× SSC for 1 h at 70°C and dehydrated in 70% ethanol for 5 min, 96% ethanol for 5 min, and air dried. The preparations were denatured in 0.07 NaOH for 10 min and washed in 2× SSC for 1 min, 1 min, and 5 min. The slides were then dehydrated as described above. The DIG- and/or biotin-labeled probes were incubated at 95°C for 5 min, snap cooled on ice, and warmed to 37°C. A volume of 10 μL probe was loaded onto the chromosome preparation under an 18 × 18 mm coverslip and incubated at 37°C for 16–20 h. . Following the overnight incubation, slides were washed three times in 2× SSC for 5 min at 37°C followed by one wash in 1× PBS for 10 min. Secondary, or indirect, labelling for single fluorophore and double fluorophore hybridizations were conducted as described separately below. Subsequently, preparations were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Roche Applied Sciences, Indianapolis, IN, USA) for 10 min and washed briefly in 1× PBS, mounted with SlowFade Gold Antifade Reagent (Life Technologies, Grand Island, NY, USA) under a cover slip, and imaged with a Nikon TE2000 fluorescence microscope (Nikon Instruments, Melville, NY, USA).
For detection of DIG-labeled probes, chromosomes were incubated with 1:250 mouse anti-DIG primary antibody (Roche Applied Science, Indianapolis, IN, USA) in 1% BSA and 2 mg/mL RNase A in 1× PBS for 1 h, followed by washes in 1× PBS for 5 min, 1× PBS + 0.1% Triton X-100 for 5 min, and 1× PBS for 5 min. Chromosomes were then incubated with 1:500 Alexa 488 goat anti-mouse secondary antibody (Life Technologies, Grand Island, NY, USA) in 1% BSA and 2 mg/mL RNase A in 1× PBS for 1 h, followed by washes in 1× PBS for 5 min, 1× PBS + 0.1% Triton X-100 for 5 min, and 1× PBS for 5 min.
For simultaneous detection of DIG- and biotin-labeled probes, 1:250 Alexa 555-streptavidin primary antibody (Life Technologies, Grand Island, NY, USA) was combined with the mouse anti-DIG antibody in 1% BSA and 2 mg/mL RNase A in 1× PBS for 1 h, followed by washes in 1× PBS for 5 min, 1× PBS + 0.1% Triton X-100 for 5 min, and 1× PBS for 5 min. Chromosomes were then incubated with 1:500 Alexa 488 donkey anti-mouse secondary antibody (Life Technologies, Grand Island, NY, USA) and 1:250 goat biotinylated anti-streptavidin secondary antibody (Vector Laboratories, Burlingame, CA, USA) in 1% BSA and 2 mg/mL RNase A in 1× PBS for 1 h, followed by washes in 1× PBS for 5 min, 1× PBS + 0.1% Triton X-100 for 5 min, and 1× PBS for 5 min. The signal was again amplified with 1:500 Alexa 488 donkey anti-mouse secondary antibody and 1:500 Alexa 555-streptavidin primary antibody in 1% BSA and 2 mg/mL RNase A in 1× PBS for 1 h, followed by washes in 1× PBS for 5 min, 1× PBS + 0.1% Triton X-100 for 5 min, and 1× PBS for 5 min.
The data sets supporting the results of this article are available in the Sequence Read Archive repository: SRP012548, SRP012550, SRP012551, and SRP012555.
This work was supported by a grants to JCDH from the NSF Microbial Sequencing Program (EF-0826732) and NIH New Innovator Award Program (1-DP2-OD007372) and grants to SGEA from the Swedish Research Council, the European Union, and the Knut and Alice Wallenberg Foundation, and a grant to LK from the Swedish Research Council Formas.
LK, LJT, SGEA, JCDH designed research; LK, NK, RB, MF, SHO performed laboratory-based research; LK, NK, KS, JCDH analyzed data; LK, NK, SGEA, JCDH wrote the paper. All authors read and approved the final manuscript.


text:
Q: ScriptManager.RegisterStartupScript e a Key Boa tarde,
Queria entender como funciona essa Key, tenho uma função no aspx que necessito no code-behind, mas quando a chamo com uma Key estática, colocando csname = "x"; Ela só é chamada uma vez, mas preciso que seja chamada quantas vezes for necessário, daí tentei colocar o nome como variável e ela nunca é chamada, mesmo sem o if (!Page.ClientScript.IsStartupScriptRegistered(Page.GetType(), csname))
Eis o código, este código coloca os itens em um mesmo row de uma gridview, depois ele verifica se colocará o ícone de Expand naquele row, para que o mesmo possa ser expandido quando clicarmos no ícone.
protected void gvProduct_RowDataBound(object sender, GridViewRowEventArgs e)
    {
        if (e.Row.RowType == DataControlRowType.DataRow)
        {
            HyperLink hlProcesso = (HyperLink)e.Row.FindControl("hlProcesso");
            GridView gvRepetidos = (GridView)e.Row.FindControl("gvRepetidos");
            DataTable dtNew = dtFormsFilhos.Clone();
            bool isRepetido = false;

            foreach (DataRow row in dtFormsFilhos.Rows)
            {
                if (row["ProcessoFilho"].ToString().Contains(hlProcesso.Text))
                {
                    dtNew.Rows.Add(row.ItemArray);
                    isRepetido = true;
                }
            }

            if (isRepetido)
            {
                //se coloco o csname como "x" ou "inclui", funciona 1x
                //mas se coloco como está, não funciona. Não entendo como essa key funciona!
                string csname = hlProcesso.Text.ToString();
                string cstext = "IncluiExpand('" + hlProcesso.Text + "')";
                if (!Page.ClientScript.IsStartupScriptRegistered(Page.GetType(), csname))
                    ScriptManager.RegisterStartupScript(this.Page, this.Page.GetType(), csname, cstext, true);
            }

            gvRepetidos.DataSource = dtNew;
            gvRepetidos.DataBind();
        }
    }

A: Finalmente consegui!
Ao invés de ScriptManager.RegisterStartupScript(this.Page, this.Page.GetType(), csname, cstext, true);, coloquei this.Controls.Add(new LiteralControl("<script type='text/javascript'>"+csText+";</script>"));
Nem precisa daquele if pra ver se o controle já existe, no meu caso.

redpajama_set_name: RedPajamaStackExchange


Pickup from Railway Station/ airport. Check in at the hotel. Afternoon depart for local sightseeing of Bangalore visiting the Cubon Park, Lalbaug, Bull Temple, Vidhan Soudha & High Court, Evening will be at your leisure. Overnight at the hotel.
After Breakfast leave for Mysore Approx. 110 Kms/ 3 Hrs. Hrs. Evening visit the Brindavan garden to witness the musical fountain show. Overnight at the hotel.
Morning depart for half-day excursion to Srirangapatanam and visit Palace of Tipu sultan. Later in the afternoon visit the local sights in Mysore visiting the Mysore Palace, Chamundi Hill & the church. Afternoon depart for Ooty. 135 Kms – 3 Hrs. On the way visit Mudumalai Sanctuary. On arrival check in at the hotel. Overnight at the hotel.
Morning you go for a half-day excursion of Coonor. Visit the local sights in Coonor & the Tea Gardens. Later in the afternoon visit Ooty Lake, Dodabetta Peak. Evning will be at your leisure. Overnight at the hotel.
Morning depart for Kodaikanal by surface approx. 350 kms/8 hrs. arrive check inn at the hotel. Dinner & Night Hotel.
Proceed for half Day sightseeing of Kodaikanal visiting Kurinji Temple, Coakers Walk, Silver Cascade Falls, Byrant Park, Pillar Rock & the Golf Course. Evening at Leisure or enjoy boating in the Kodai Lake(on own). Overnight at the hotel.


This is the compound that is thought to induce weight loss. There are no known interactions between garcinia cambogia amoxicillin but this may simply be due to the lack of available data. Garcinia Cambogia Complex is a natural supplement that comes from the tamarind fruit found primarily in southern Asia.
In fact it has been reported to reduce appetite , · Taking apple cider vinegar , garcinia cambogia, inhibit the production of fat in the v 06, the extract of a tropical fruit is claimed to aid weight loss. Although garcinia likely will not have any negative effects on amoxicillin your body' s ability to fight infection, it may be prudent to avoid supplementation with it until you have completed your course of amoxicillin your infection has cleared. The extract found in Garcinia Cambogia is called HydroxyCitric Acid ( HCA). This supplement may aid in weight loss through the following mechanisms: 1.
Some believe that garcinia cambogia can suppress appetite and block fat production. It could help keep blood sugar cholesterol levels in check too. Garcinia cambogia ( GC) is a small pumpkin- shaped fruit that grows in Southeast Asia India. In fact it has been reported to reduce appetite inhibit the production of fat in the body.
Jan 12 claims to have beneficial weight reduction , · One supplement, Garcinia cambogia lipid- lowering effects. Some believe that garcinia cambogia can suppress appetite and block fat.
Suppresses The Appetite 2. Taking apple cider vinegar the extract of a tropical fruit, garcinia cambogia is claimed to aid weight loss. Garcinia cambogia is a fruit that is native to India Southeast Asia is high in a compound known as Hydroxycitric acid.
It could help keep blood sugar Southeast Asia , · Garcinia cambogia is a fruit that is native to India , cholesterol levels in check is high in a compound known as Hydroxycitric acid. Pharmavits garcinia cambogia.
The key active ingredient found in the rind of garcinia cambogia is hydroxycitric acid ( HCA), which some research suggests can help certain people lose weight. Cambogia and HCA The active ingredient in G.
Cambogia is a competitive inhibitor of adenosine triphosphate citrate lyase, cholesterol, hydroxycitric acid ( HCA) triglycerides. Weight loss supplement fat burner appetite suppressant non gmo gluten free quality garcinia cambogia hca 95% 80% 50% pharmacists blend amazon safe. Inhibits the this Article.


I am passionate about interdisciplinary projects merging Product- and Interface design. Please scroll down for a selection of my work.
loui a new way to control light at your workspace.
this multidirectional kitchen brings us back to the fireplace.
servicedesign for easy ridesharing in rural areas.
explore cities and meet people - through an interactive window.
a modular plant pot, designed to rearrange and explore.
redesigning an often seen distributor for local internet and energy supplyment.


text:
Macau Grand Lisboa Palace (SJM) Patron Mobile App with CMS Development
A leading owner, operator and developer of casinos and integrated entertainment resorts in Macau
SJM Holdings Limited (HKEx: 880) is a leading owner, operator and developer of casinos and integrated entertainment resorts in Macau. Our principal subsidiary SJM Resorts, S.A. (formerly Sociedade de Jogos de Macau, S.A.) ("SJM") is one of the six concessionaires in Macau, authorised by the Government of the Macau Special Administrative Region to operate casinos and gaming areas. SJM is also the only casino gaming concessionaire with its roots in Macau.
SJM owns and operates the Grand Lisboa Palace Resort, the Grand Lisboa hotel and casino, as well as other casinos located in prime locations in Macau. The Company's operations cater to a wide spectrum of patrons, with casino gaming, leisure entertainment and hospitality services including fine and casual dining and luxury accommodation.
The Grand Lisboa Palace, SJM's integrated resort on Cotai, opened its doors to the public on 30 July 2021. In the initial phase opening, the resort offers luxury hotel rooms and suites, fine and casual dining, gaming, shopping, wellness and spa facilities and events spaces. THE KARL LAGERFELD hotel tower was added to the resort's diversified offerings on 3 December 2021. Additional features to be inaugurated include the hotel tower Palazzo Versace Macau, as well as more facilities for meetings and conferences, shopping and dining.
#UI/UX & Web Design #Mobile App #Custom CMS

redpajama_set_name: RedPajamaCommonCrawl


text:
static NSString* const KHybridNSURLProtocolHKey = @"KHybridNSURLProtocol";
@interface HybridNSURLProtocol ()<NSURLSessionDelegate>
@property (nonnull,strong) NSURLSessionDataTask *task;
@end

@implementation HybridNSURLProtocol

+ (BOOL)canInitWithRequest:(NSURLRequest *)request
{
    NSString *scheme = [[request URL] scheme];
    if ( ([scheme caseInsensitiveCompare:@"http"]  == NSOrderedSame ||
          [scheme caseInsensitiveCompare:@"https"] == NSOrderedSame ))
    {
        //看看是否已经处理过了,防止无限循环
        if ([NSURLProtocol propertyForKey:KHybridNSURLProtocolHKey inRequest:request]
            ){
            return NO;
        }

        return YES;
    }
    return NO;
}

//+ (BOOL)replaceAdHostsWebString:(NSArray *)adhosts response:(NSString **)response{
//    BOOL isContain = NO;
//
//    for (NSString *adhost in adhosts) {
//        isContain = isContain | [[self class]  replaceWebString:adhost response:response];
//    }
//    return  isContain;
//}
//
//
//+ (BOOL)replaceWebString:(NSString *)adhost response:(NSString **)response{
//    BOOL isContain = NO;
//    if ([*response containsString:adhost]) {
//        *response = [*response stringByReplacingOccurrencesOfString:adhost withString:@""];
//        isContain = YES;
//    }
//    return  isContain;
//}

+ (NSURLRequest *)canonicalRequestForRequest:(NSURLRequest *)request
{
    NSMutableURLRequest *mutableReqeust = [request mutableCopy];
    [mutableReqeust setValue:@"Mozilla/5.0 (iPhone; CPU iPhone OS 11_0 like Mac OS X) AppleWebKit/604.1.38 (KHTML, like Gecko) Version/11.0 Mobile/15A372 Safari/604.1" forHTTPHeaderField:@"User-Agent"];

    NSString *requestUrl = request.URL.absoluteString;

    //request截取重定向
    if ([self reloadURL:requestUrl])
    {
        NSURL* url1 = [NSURL URLWithString:[self reloadURL:requestUrl]];
        mutableReqeust = [NSMutableURLRequest requestWithURL:url1];
        NSLog(@"\n重定向URL:%@", requestUrl);
    }else
    //拦截广告
    if ([self stringContainsAdTypeType:requestUrl]) {
        NSLog(@"\n拦截广告URL:%@", requestUrl);
        mutableReqeust = nil;
    }else
    //拦截视频
    if([self stringContainsMediaTypeType:requestUrl])
    {
        //NSArray *urlArray = [requestUrl componentsSeparatedByString:self.sepType];
        NSArray *urlArray = [requestUrl componentsSeparatedByString:@"=http"];
        NSString *url = urlArray.lastObject;
        if (urlArray.count>1) {
            url = [NSString stringWithFormat:@"http%@",url];
        }

        NSLog(@"++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++");
        NSLog(@"拦截视频URL:%@--请求URL--:%@", url,requestUrl);
        NSLog(@"++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++");
        dispatch_async(dispatch_get_main_queue(), ^{
                    [[NSNotificationCenter defaultCenter] postNotificationName:@"SPVipVideoCurrentDidChange" object:nil userInfo:@{@"url":url}];
        });

        mutableReqeust = nil;
    }else
     {
        NSLog(@"\n请求URL:%@", requestUrl);
    }

    return mutableReqeust;
}

+ (BOOL)requestIsCacheEquivalent:(NSURLRequest *)a toRequest:(NSURLRequest *)b
{
    return [super requestIsCacheEquivalent:a toRequest:b];
}

- (void)startLoading
{
    NSMutableURLRequest *mutableReqeust = [[self request] mutableCopy];
    //给我们处理过的请求设置一个标识符, 防止无限循环,
    [NSURLProtocol setProperty:@YES forKey:KHybridNSURLProtocolHKey inRequest:mutableReqeust];

    //这里最好加上缓存判断,加载本地离线文件, 这个直接简单的例子。
//    if ([mutableReqeust.URL.absoluteString isEqualToString:sourIconUrl])
//    {
//            NSData* data = UIImagePNGRepresentation([UIImage imageNamed:@"medlinker"]);
//            NSURLResponse* response = [[NSURLResponse alloc] initWithURL:self.request.URL MIMEType:@"image/png" expectedContentLength:data.length textEncodingName:nil];
//            [self.client URLProtocol:self didReceiveResponse:response cacheStoragePolicy:NSURLCacheStorageAllowed];
//            [self.client URLProtocol:self didLoadData:data];
//            [self.client URLProtocolDidFinishLoading:self];
//    }
//    else
    {
        NSURLSession *session = [NSURLSession sessionWithConfiguration:[NSURLSessionConfiguration defaultSessionConfiguration] delegate:self delegateQueue:nil];
        self.task = [session dataTaskWithRequest:self.request];

        //NSLog(@"请求 request %@", self.request.URL.absoluteString);

        [self.task resume];
    }
}
- (void)stopLoading
{
    if (self.task != nil)
    {
        [self.task  cancel];
    }
}

- (void)URLSession:(NSURLSession *)session dataTask:(NSURLSessionDataTask *)dataTask didReceiveResponse:(NSURLResponse *)response completionHandler:(void (^)(NSURLSessionResponseDisposition))completionHandler {
    [[self client] URLProtocol:self didReceiveResponse:response cacheStoragePolicy:NSURLCacheStorageAllowed];

    completionHandler(NSURLSessionResponseAllow);
}

- (void)URLSession:(NSURLSession *)session dataTask:(NSURLSessionDataTask *)dataTask didReceiveData:(NSData *)data {
    [[self client] URLProtocol:self didLoadData:data];
}

//- (void)URLSession:(NSURLSession *)session task:(NSURLSessionTask *)task didCompleteWithError:(nullable NSError *)error {
//    [self.client URLProtocolDidFinishLoading:self];
//}

- (void)URLSession:(NSURLSession *)session task:(NSURLSessionTask *)task didCompleteWithError:(NSError *)error {
    if (error) {
        [self.client URLProtocol:self didFailWithError:error];
    } else {
        [self.client URLProtocolDidFinishLoading:self];
    }
}

//- (void)URLSession:(NSURLSession *)session dataTask:(NSURLSessionDataTask *)dataTask didReceiveResponse:(NSURLResponse *)response completionHandler:(void (^)(NSURLSessionResponseDisposition))completionHandler {
//    [self.client URLProtocol:self didReceiveResponse:response cacheStoragePolicy:NSURLCacheStorageNotAllowed];
//
//    completionHandler(NSURLSessionResponseAllow);
//}

//- (void)URLSession:(NSURLSession *)session dataTask:(NSURLSessionDataTask *)dataTask didReceiveData:(NSData *)data {
//    [self.client URLProtocol:self didLoadData:data];
//}

+ (BOOL)stringContainsMediaTypeType:(NSString *)str {
    __block BOOL isContains = NO;
    NSArray *filterTypes = [self mediaType];
    [filterTypes enumerateObjectsUsingBlock:^(NSString * obj, NSUInteger idx, BOOL *  stop) {
        if([str containsString:obj]){
            isContains = YES;
            *stop = YES;
        }
    }];
    return isContains;
}
+ (BOOL)stringContainsAdTypeType:(NSString *)str {
    __block BOOL isContains = NO;
    NSArray *filterTypes = [self adType];
    [filterTypes enumerateObjectsUsingBlock:^(NSString * obj, NSUInteger idx, BOOL *  stop) {
        if([str containsString:obj]){
            isContains = YES;
            *stop = YES;
        }
    }];
    return isContains;
}

+ (NSString *)reloadURL:(NSString *)orginURL{
    __block NSString *url;
    [[self reloadType] enumerateKeysAndObjectsUsingBlock:^(NSString *key, NSString *obj, BOOL *  stop) {
        if([orginURL containsString:key]){
            url = obj;
            *stop = YES;
        }
    }];
    return url;
}

+ (NSArray <NSString *>*)mediaType{
    NSArray *types = [[NSUserDefaults standardUserDefaults] arrayForKey:@"mediaType"];
    return types? types : @[@".m3u8"];
}

+ (NSArray <NSString *>*)adType{
    NSArray *types = [[NSUserDefaults standardUserDefaults] arrayForKey:@"adType"];
    return types? types : @[];
}

+ (NSDictionary*)reloadType{
    NSDictionary *types = [[NSUserDefaults standardUserDefaults] dictionaryForKey:@"reload"];
    return types? types : @{};
}

//+ (NSString *)sepType{
//    NSString *type = [[NSUserDefaults standardUserDefaults] stringForKey:@"sepType"];
//    return type? type : @"url=";
//}

@end

redpajama_set_name: RedPajamaGithub


text:
https://podcast.playmas.today/dmc/brunch%20setting.mp3
Brunch Settings | Personal Pull Up Party 03.18.2022
The Blue Marlin Bistro 5495 Old National HWY, Atlanta, Ga is now open for business. Saturday, May 18 management is kicking off the celebration with a Caribbean & Soul Food Brunch. Food, Drinks and Music provided by #BTeamDJs AfricaAllah from 11am -6pm.
Why Can't We Be Friends                                             00:03:04
The Payback                                                         00:03:10
Shape of You                                                        00:01:44
Real Love                                                           00:01:55
Casanova (CLEAN) (SE) (MM Edit)                                     00:01:42
I Love Your Smile (Driza Bone Single Remix)                         00:01:45
Sending My Love                                                     00:01:58
Right Here (Human Nature Remix) (CLEAN) (MM Edit)                   00:02:21
Return of the Mack                                                  00:02:56
Every Little Step (With Rap)                                        00:02:12
My Lovin' (You're Never Gonna Get It)                               00:01:57
Tom's Diner (CLEAN) (ACA In) ((MM Edit)                             00:02:20
I'm So Into You                                                     00:02:40
She's Playing Hard To Get                                           00:02:55
Back To Life                                                        00:02:19
Nasty                                                               00:02:06
Get It Up (CLEAN) (MM Edit)                                         00:02:16
Do Me!                                                              00:01:40
Groove Me                                                           00:01:51
Remember the Time                                                   00:02:53
Touch (CLEAN) (MM Edit)                                             00:02:44
I'll Be Good To You (CLEAN) (SE) (MM Edit)                          00:01:27
If I Had No Loot                                                    00:02:28
Round and Round (Soul Mix Edit)                                     00:03:31
Run It!                                                             00:02:04
Beautiful Music                                                     00:02:22
Track Star                                                          00:02:17
Love Will Never Do (MM Edit)                                        00:02:22
By Yourself (feat. Jhené Aiko & Mustard)                            00:02:04
Ain't 2 Proud 2 Beg                                                 00:02:57
Never (Feat. Eve)                                                   00:03:07
All Night Long (All Night)                                          00:02:46
Essence                                                             00:03:42
Burna_Boy_-_Rock_Your_Body_(mp3.pm)                                 00:03:38
Hotel (feat. Ty Dolla $ign & Burna Boy)                             00:02:56
Peru                                                                00:01:42
StarBoy - Soco ft. Wizkid, Ceeza Milli, Spotless, Terri             00:02:07
Soco [CLEAN]                                                        00:02:18
Slow Down                                                           00:01:42
Banyana                                                             00:02:00
Ke Star (Remix)                                                     00:03:33
Yaba Buluku (Remix)                                                 00:04:09
Hooka [DJ ECool Extend]                                             00:02:41
Uptown Funk (feat. Bruno Mars)                                      00:01:43
Get Lucky (feat. Pharrell Williams & Nile Rodgers)                  00:02:03
Want to Want Me                                                     00:02:20
Electricity (feat. Diplo & Mark Ronson)                             00:01:56
Call Me Maybe                                                       00:01:24
TiK ToK                                                             00:01:20
Rumour Has It                                                       00:01:29
Treasure                                                            00:01:54
Blurred Lines                                                       00:03:42
Fireball (feat. John Ryan)                                          00:02:07
One Kiss                                                            00:01:57
This Is What You Came For (feat. Rihanna)                           00:01:39
Don't Start Now                                                     00:01:53
Time of Our Lives                                                   00:02:14
What Do You Mean?                                                   00:02:22
Only Girl (In The World)                                            00:01:39
Super Bass                                                          00:01:19
Run the World (Girls)                                               00:01:13
Don't Stop the Party (feat. TJR)                                    00:01:48
Moves Like Jagger (Studio Recording From The Voice Performance)     00:03:55
We Found Love (Album Version)                                       00:03:25
Stage Gawd                                                          00:05:11
Dear Promoter
Why Can't We Be Friends 00:03:04 The Payback 00:03:10 Shape of You 00:01:44 Real Love 00:01:55 Casanova (CLEAN) (SE) (MM Edit) 00:01:42 I Love Your Smile (Driza Bone Single Remix) 00:01:45 Sending My Love 00:01:58 Right Here (Human Nature Remix) (CLEAN) (MM Edit) 00:02:21 Return of the Mack 00:02:56 Every Little Step (With Rap) 00:02:12 My Lovin' (You're Never Gonna Get It) 00:01:57 Tom's Diner (CLEAN) (ACA In) ((MM Edit) 00:02:20 I'm So Into You 00:02:40 She's Playing Hard To Get 00:02:55 Back To Life 00:02:19 Nasty 00:02:06 Get It Up (CLEAN) (MM Edit) 00:02:16 Do Me! 00:01:40 Groove Me 00:01:51 Remember the Time 00:02:53 Touch (CLEAN) (MM Edit) 00:02:44 I'll Be Good To You (CLEAN) (SE) (MM Edit) 00:01:27 If I Had No Loot 00:02:28 Round and Round (Soul Mix Edit) 00:03:31 Run It! 00:02:04 Beautiful Music 00:02:22 Track Star 00:02:17 Love Will Never Do (MM Edit) 00:02:22 By Yourself (feat. Jhené Aiko & Mustard) 00:02:04 Ain't 2 Proud 2 Beg 00:02:57 Never (Feat. Eve) 00:03:07 All Night Long (All Night) 00:02:46 Essence 00:03:42 Burna_Boy_-_Rock_Your_Body_(mp3.pm) 00:03:38 Hotel (feat. Ty Dolla $ign & Burna Boy) 00:02:56 Peru 00:01:42 StarBoy – Soco ft. Wizkid, Ceeza Milli, Spotless, Terri 00:02:07 Soco [CLEAN] 00:02:18 Slow Down 00:01:42 Banyana 00:02:00 Ke Star (Remix) 00:03:33 Yaba Buluku (Remix) 00:04:09 Hooka [DJ ECool Extend] 00:02:41 Uptown Funk (feat. Bruno Mars) 00:01:43 Get Lucky (feat. Pharrell Williams & Nile Rodgers) 00:02:03 Want to Want Me 00:02:20 Electricity (feat. Diplo & Mark Ronson) 00:01:56 Call Me Maybe 00:01:24 TiK ToK 00:01:20 Rumour Has It 00:01:29 Treasure 00:01:54 Blurred Lines 00:03:42 Fireball (feat. John Ryan) 00:02:07 One Kiss 00:01:57 This Is What You Came For (feat. Rihanna) 00:01:39 Don't Start Now 00:01:53 Time of Our Lives 00:02:14 What Do You Mean? 00:02:22 Only Girl (In The World) 00:01:39 Super Bass 00:01:19 Run the World (Girls) 00:01:13 Don't Stop the Party (feat. TJR) 00:01:48 Moves Like Jagger (Studio Recording From The Voice Performance) 00:03:55 We Found Love (Album Version) 00:03:25 Stage Gawd 00:05:11 Dear Promoter
Monster Music Spotlight vol1_01
Better | Pull Up Mix
B Team DJs Dance RnB
The Truth about the Business of Carnival #diDocumentaries: Building an Empire
Dexta Daps Kranium | 01.13.23 Personal Pull Up Party
Last One Out | Get Familiar Friday 12.09.22


text:
Oswego Paralympian Wins Gold In London
Filed Under:2012, Gold Medal, london
Gold medallist Joseph Berenyi of the United States poses on the podium during the medal ceremony for the Men's Individual Cycling C3 Pursuit final on day 2 of the London 2012 Paralympic Games at Velodrome on August 31, 2012 in London, England. (Photo by Bryn Lennon/Getty Images)
(CBS) –A U.S. Paralympian from the Chicago region is bringing home the gold from London.
Joseph Berenyi, an Aurora native now living in Oswego, this week earned a gold medal in the 3-km, individual pursuit cycling race. He finished less than 1 second ahead of the silver medalist.
LISTEN: WBBM Newsradio's Mike Krauser Reports
Earlier in the day, he set a new world record in the qualifying round.
Berenyi, 43, lost his right arm and left kneecap in a construction accident 18 years ago.
"It's a dream come true, a big dream just to make the team," he said.


text:
package org.openfact.models.ubl.common;

import java.math.BigDecimal;
import java.util.List;

public interface BillingReferenceLineModel {

    String getID();

    void setID(String value);

    BigDecimal getAmount();

    void setAmount(BigDecimal value);

    List<AllowanceChargeModel> getAllowanceCharge();

    void setAllowanceCharge(List<AllowanceChargeModel> allowanceCharge);

    String getId();

}


From the Poor, Pitiful, Underpaid Teachers File. . .
A bit of investigative reporting from Marcha Richards at the Evergreen Freedom Foundation: Peninsula public school teachers Brenda and David Aston were featured on the front page of the August 17, 2005, edition of The Peninsula Gateway, picketing school district officials for higher pay. They held signs reading "Our family qualifies for W.I.C. and subsidized health care" and "Ask me about Job #2." . . . According to data provided by the Office of the Superintendent of Public Instruction, Brenda and David Aston each earned base teaching salaries of $45,169 this year (2004-05). In addition, both had supplemental contracts with the district, from which they earned $3,599 and $4,546 respectively. On top of that, Brenda's benefits package (health insurance, etc.) was worth $11,232 and David's was worth $11,319. Thanks again to Joanne. Who notes: Together they earned just under $100,000 a year, not including income from the husband's second job as an evidence custodian for a local police department. The family does not qualify for WIC — the cut-off for a couple with four children is less than half their pay — or subsidized health care.


text:
ICE's Attempts to Normalize Immigration Raids Must be Challenged at Every Turn
ICE's Attempts to Normalize Immigration…
Contact: Armando Carmona, 323-250-3018; armando@ndlon.org;
Los Angeles, CA – On Friday, Immigration and Customs Enforcement (ICE) issued a press statement acknowledging that they detained more than 160 individuals during immigrations on Thursday February 9th 2017. This statement comes as ICE agents repeatedly attempted to counter claims made by immigration advocates and lawyers regarding the arrests of "over 100 immigrants" through home raids in several cities in southern California.
In response, David Abud, Regional Organizer from the National Day Laborer Organizing Network issued the following statement:
"We recognize these raids as an effort by ICE and the new administration to sow chaos and intimidate our communities. They are attempting to normalize these attacks towards our community and we must be ready, at a moments notice, to mobilize and resist. In the face of these efforts, we will remain vigilant, focused, and organized. We are now more than ever emboldened to fight every single deportation and all further attacks on our communities in whatever form they may appear."
"Ironically, this most recent attempt to terrorize Los Angeles residents will likely compel local elected officials to take more aggressive actions themselves. When Mr Trump was first elected, we heard many statements offering moral support for communities the has threatened. Now it is time for leaders to action and to demonstrate the courage that immigrants are showing in response to Mr Trump's bullying."
"In Los Angeles, we will continue to push for Universal Representation and to make it a Sanctuary City that provides safety for all of its residents. Throughout California, a consensus is emerging in support of SB-54 (The "Values Act," Senator de Leon) which would ensure public safety for all Calfornia residents."
Category: Press ReleasesBy Armando Carmona February 10, 2017
Author: Armando Carmona
PreviousPrevious post:NDLON Responds to Confirmation of Jeff Sessions as US Attorney GeneralNextNext post:Day Laborers and Domestic Workers celebrate Celebrate 25th Anniversary of SF Day Labor Program and Women's Collective


text:
Spencer gets preview of Park Plaza
By Rebecca Rider
Published 12:00 am Wednesday, June 13, 2018
SPENCER — The Spencer Board of Aldermen got its first look at a revamped Park Plaza on Tuesday night.
Keith Wales of Studio Wales Architecture presented designs and architect's renderings of what the finished municipal complex would look like.
"We're very excited to present our vision tonight," Wales said.
Much of Wales' presentation was spent going over designs for the property's future park — which will occupy space near South Salisbury Avenue. The green space would be, in many ways, the heart of the project.
"The town wanted to bring back the park," Wales explained. "There once was a park, and then it became parking — and we want to take it back that way."
The park will preserve an old tree in the area, lined with alders and filled with other trees to provide lots of shade and to prevent passers-by from seeing straight through the park to another parking lot. Wales referred to the area as "an oasis."
"We don't want it to become a barren wasteland of grass," he said. "We want to make sure there's plenty of shade for everyone."
The design for walking paths in the park was inspired by subway systems and the national layout of the Norfolk-Southern Railway system, with Spencer at its heart.
"Spencer becomes kind of the center gathering place for this system," he said.
The park will also feature a pavilion inspired by the design of train engines.
When it comes to the municipal complex itself, everything is designed with the comfort, safety and ease of Spencer residents in mind — from a wall separating the booking and interview rooms in the Police Department to placing two closely related municipal departments in the same office space to planting holly trees outside the police chief's window to deter snooping.
When the space is finished, town offices will occupy only about 8,000 square feet of the 20,000 square feet purchased. The rest will be available for lease. But leased spaces, Wales explained, were set up in such a way that the town could easily repurpose them with some quick upfitting.
"This will allow you to expand without any major renovation," Wales said.
Town board members said they were impressed with the designs.
"You've done a fine job," Mayor Jim Gobbel said. "Gosh, it's just very nice."
If all goes according to plan, Wales estimated that the town should be able to put out construction bids by early July and begin construction by fall. The existing shell should help speed the project along, as well.
"Hopefully, it'll be a very fast process for you all," he said.
• Aldermen voted to close streets and waive security deposits and rental fees for vendors during this year's October Tour.
• The board agreed to partner with Life Church in funding transportation for North Rowan Elementary School's summer camp. The board voted to pay about $1,200 toward transportation costs.
• Police Chief Mike James cautioned residents to lock their car doors and to not leave valuable items in cars after a rash of vehicle break-ins in the area.
Contact reporter Rebecca Rider at 704-797-4264.
Forsyth County man challenges Rowan County arrest as 1st Amendment rights violation
SALISBURY — A Forsyth County man who has challenged other law enforcement agencies in the past regarding his right to... read more


Modernizing Clarissa: the Affordances and Limitation of Letters and Blogs. Kendra Sheehan.
As a blog, Clarissa loses many traditional literary qualities associated with a print novel. For instance, the dramatic scenes Lovelace writes in Letter 214 move from being written like a play in print to resembling an Instant Messaging conversation in an electronic blog. That is to say, the letters that appear so formal in print become casual and modern in an electronic medium such as the blog.
Further, the idea of editions, as found in printed texts, is lost as Clarissa becomes a blog. Clarissa's author, Samuel Richardson, made changes through various editions that were based in part on his engagement and correspondence with his audience. As a blog, Clarissa would have the potential to be similarly changed and edited, but its original format may be lost with revisions unless each draft was saved and uploaded separately or cached in an online database. Only in later editions can a print author make changes or revisions based on audience response. Richardson was a printer and publisher who had the unique rights to change and print his work at will. Today it is much harder for an author to make numerous editions of a work without consent of a publisher or even his literary agent.
In a blog post, one can easily edit and make changes, whereas with a hand-written letter (of the sort contained in Clarissa) one must start over or leave a visible marker of mistakes, such as scratched out words or the use of white-out. Also, a letter cannot be changed, "deleted," or called back once it has been sent to its recipient. A blog post, however, can be easily posted and deleted before anyone reads it, or can even be set to "private" so that it's available to only a select few. While blogs can be "written" in different fonts, the fonts are not necessarily unique to the blog and are quite standard to many word processors. Letters, on the other hand, can display handwriting unique to the individual. Blogs can provide links and images to things that a blogger likes, but letters can have drawings by the same hand that wrote the letter. Then again, a blog can upload images of letters complete with the blogger's handwriting. The voice present in the blog can tell the readers when something is rushed or written in an emotional moment, and so on. Hand-written letters can also show these emotions and hurriedness at first glance based on the handwriting alone.
Writing functions similarly and differently in blogs and letters. For instance, blogs can be updated nearly anywhere at any time via one's cellular phone, laptop, or tablet, whereas letters can be written anywhere but not immediately updated or posted because the letter needs to be mailed to a recipient rather than uploaded through the internet. Finally, both blogs and letters can be written using the present tense, but are often about the past or recently past events.
In terms of an audience, letters are typically meant for one person whereas blogs are often meant to be read by an audience of innumerable people. A blog's audience can be affected by the community in which the blog is posted or linked to. Despite the audience number, blogs and letters can still be intimate because the reader is being provided with thoughts as well as sharing in the private moments of the writer's life. The audience for letters is often one person and can be tailored to just that specific reader. A similar thing happens in blogs when they are specifically tailored to a member of the online community to which they belong. Most importantly, both letters and blogs provide a way for the writer to connect to at least one person in the world at large.
As a blog, Clarissa would connect to more than just college students reading 18th century literature. A blog offers an opportunity to modernize Clarissa's story for a modern and technologically literate audience, with little to no change to the story's plot. The intimacy of the letters in Clarissa allows others to sympathize with her plight and even create communities based around it. As a blog, Clarissa goes beyond the scope of the 18th century audience that Richardson engaged with and introduces the novel on a global scale for a new generation of readers.
This entry was posted in LBI on December 3, 2013 by awesomekendora.


text:
HOME / DOCUMENTS / THE BIOLOGY OF CULTURE (FALL 2016) /
The Best of ReVista Photo Contest
The Best of ReVista is a prize for the best photograph published in ReVista in the previous academic year sponsored by DRCLAS publications and ARTS@DRCLAS.
This year's winning photographs chosen by a jury of professional photography experts are Walter Iraheta and David Huamani B. Honorable mentions in the professional category go to Mauro Arias and Leslie Searles.
WINNER, PROFESSIONAL PHOTOGRAPHER CATEGORY
WALTERIO IRAHETA for "Shoes" Iraheta studied Applied Arts in El Salvador, Mexico and Chicago. He lives and works in El Salvador as a photographer, curator and professor.
WINNER, EMERGING PHOTOGRAPHER CATEGORY
DAVID HUAMANI B./OJOS PROPIOS for "Marcona" Huamani B. is a member of the photo collective Ojos Propios in Lima, Peru.
MAURO ARIAS of El Salvador for "Boy in the Coffee Fields"
LESLIE SEARLES of Peru for "Newborn"
THIS YEAR'S JUDGES
Rodrigo Abd is an Argentine staff photographer for the Associated Press. He won the 2013 Pulitzer Prize for breaking news photography. He is a 2016 Maria Moors Cabot Award winner for a distinguished body of work that has contributed to inter-American understanding.
Marcelo Brodsky is an Argentine photographer and conceptual artist whose best-known work deals with the legacy of the dictatorship. A recipient of the Jean Mayer Award from the Tufts Institute for Global Leadership, Brodsky created the installation "Human Tides" at Tufts to draw attention to contemporary global migrations and refugees.
Andrea Bruce, a 2016 Nieman Fellow at Harvard, is a U.S. docu- mentary photographer who brings attention to people living in the aftermath of war. She was named Photographer of the Year four times by the White House News Photographers Association and received a 2011 Alicia Patterson Foundation Fellowship.
Andrea Josch, a Chilean photogra- pher and academic, is the editor- in-chief of the South American photography magazine Sueño de la Razón. The academic director of the Photographic Research and Creation Master's program at the Finis Terrae University in Santiago de Chile, she is also a researcher at the same institution.
João Kulcsar is a Brazilian photog- rapher, curator and academic who works on issues of citizenship and visual literacy. He was a 2002-03 Fulbright Scholar at Harvard's Graduate School of Education and collaborates frequently with Project Zero. He teaches at Senac University in São Paulo, Brazil, and is the editor of the site www. alfabetizacoavisual.com.br.
The Biology of Culture (Fall 2016)
Editor's Letter: Questions and Answers (sort of)
First Take: The Biology of Culture
From Chocolate to Potatoes
Cacao Biology: Chocolate a Superfood
Agrobiodiversity as Jazz Improvisation: Foodscape Change and Continuity
Fungi and Forests: Shaping Landscapes, Cuisine and Commerce
Healing, Sacredness and Beyond
Living with Landscapes: The Primacy of Nature
The Coolness of Cleansing: Sacred Waters, Medicinal Plants and Ritual Baths of Haiti and Peru
Indigo in Guatemala: Textile Dye and the Biology of Culture
The Biology of Consciousness: From William James to Richard Schultes
Expecting the Unexpected: Looking for Lady Beetles and their Parasites
Democratic Transitions: Conversations with World Leaders
A Ditadura Acabada
Latin America in International Politics: Challenging US Hegemony
Volunteering for a Cause: Gender, Faith, and Charity in Mexico from the Reform to the Revolution
La danza hostil: Poderes sub-nacionales y Estado central en Bolivia y Perú (1952-2012)
Spinning Mambo into Salsa: Caribbean Dance in Global
Commerce
Beyond Geopolitics: New Histories of Latin America at the League of Nations
Teaching and Learning for the Twenty-First Century. Educational Goals, Policies and Curricula from Six Nations
Añil o Indigo en Guatemala