Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels.The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation,  The~30 kilobase positive-strand coronavirus genome and subgenomic (sg) mRNAs of shorter lengths are 5’ capped and 3’ polyadenylated as are most eukaryotic mRNAs . They alHere, we report studies using a previously-described technique of head-to-tail ligation of intracellular coronaviral positive-strand RNAs and negative-strand RNAs which demonstrate that both the poly(A) on the positive-strand RNAs and poly(U) on the negative-strand RNAs can reach a length of ~65 nt, and that both, surprisingly, vary in length during infection suggesting there is a regulatory mechanism –9. ExperThe ability to ligate positive-strand viral RNAs head-to-tail and determine poly(A) tail length soon after viral infection has enabled us to measure poly(A) tail length differences as a function of time post-infection  6,10]. ,10. 6,106 PFU/ml was used to infect freshly-confluent HRT-18 cells with an MOI of ~10 viruses per cell and total cellular RNA was extracted at the time points indicated in To determine poly(A) tail length on total intracellular viral RNA as a function of time post-infection, the characterized inoculum containing an infectious titer of ~10in vitro-ligated components from mock-infected cell RNA mixed with in vitro synthesized positive-strand subgenomic mRNA 7 transcripts and in vitro synthesized transcripts representing full-length BCoV genomic RNA  tail and poly(U) tract are made are unknown. Considering that a difference in the kinetics of appearance between them might yield a clue as to how they are synthesized, the extent and timing of antigenomic poly(U) synthesis were measured and compared with the appearance of poly(A). For this, the same primers were used as for poly(A) measurement but in reverse order . A majoromic RNA . Therefoin vitro-ligated mixture of mock-infected cell RNA mixed with positive-strand subgenomic mRNA 7 transcript and full-length transcripts representing full-length BCoV genomic RNA (To determine the poly(A) and poly(U) sequence lengths on subgenomic mRNA, advantage was taken of the greater abundance and shorter length of subgenomic mRNA 7 from its leader to the poly(A) tail   was was11] wlication ,12–16. Tlication . The polin vitro and studied in vivo. For this, equal amounts (3µg) of transcript were transfected into BCoV-infected HRT cells and accumulation of the DI RNA-encoded protein was measured by Western blot analysis  tail and translation has not been previously described. On eukaryotic mRNAs, a widely-documented behavior of the 3’-terminal poly(A) tail is that an increase in its length positively correlates with an increased translation efficiency ,19. Suchanalysis , row 1 aanalysis , row 2. analysis . To relaanalysis , row 3 aanalysis , row 3, analysis , row 4, analysis , row 5.One interpretation of these results is that the protein is synthesized more efficiently from constructs with longer poly(A) tails, and although the RNA template from the non-replicating DI RNA degraded with time, the accumulating His-tagged protein persisted in the cell because of a longer half-life. Another interpretation is that the His-tag coding sequence could have recombined with the helper virus genome and become expressed from helper virus sgmRNAs containing this sequence. Recombination of this nature has been demonstrated when experimentally-applied selective pressures are used . To seekIn this study we report that the coronaviral 3’-terminal poly(A) tail length in total viral RNA, sgmRNA7, and DI RNA is relatively short ~26-45 nt) in infected cells at 0-2 hpi, increases to peak length (~65 nt) at ~6-10 hpi, and gradually decreases in size (~30-45 nt) after ~10 h of infection. An analysis of poly(A) sequences in 16 isolated bacterial colonies at 39 hpi showed tail lengths to range from 39 to 45 nt, suggesting the mean sizes measured by group sequencing is a good reflection of lengths in the major population at the times measured. This finding appears to represent a regulation of polyadenylation in a coronavirus that mimics aspects of cytoplasmic regulation of poly(A) tail length in certain mRNAs during the maturation of oocytes. It suggests also that there may be a regulated control of poly(A)-influenced translation. This behavior, to our knowledge, has not been previously documented in coronaviruses nor in other poly(A) tail-containing positive-strand RNA viruses, although poly(A) addition following transfection of non-polyadenylated MHV MIDI-C defective interfering (DI) RNAs into infected cells has been reported . We also nt in inThe coronaviral poly(U) length at the 5’ end of negative-strand RNA in BCoV as reported here differs from two previous reports . (i) In AAUAAA signal or its variations in the 3’ UTR for cytoplasmic polyadenylation , was transfected into BCoV-infected cells with the use of Lipofectin (Invitrogen) as previously described [To identify the poly(A) tail length on DI RNA-M in packaged virus, supernatant fluids from BCoV-infected and DI RNA-M transfected HRT-18 cells at 48 hpt were collected and extracted with TRIzol (Invitrogen). To prepare RNA for identifying poly(A) and poly(U) lengths on DI RNA-M in infected cells, HRT-18 cells at approximately 4 × 10escribed . SupernaA head-to-tail ligation method previously used to identify terminal features on influenza virus  and coroMluI-linearized plasmid DNA constructs Δ50HisBM-25A, Δ50HisBM-45A and Δ50HisBM-65A were transcribed in vitro with the mMessage mMachine T7 transcription kit (Ambion) in a total reaction volume of 50 µl supplemented with 7.5 µl of 30 mM GTP. In vitro transcription was done at 37°C for 60 min, treated with 5 µl Turbo DNAse (Ambion) at 37°C for 30 min, and chromatographed through a Biospin 6 column (Bio-Rad) before use in transfection [5 cells/dish) were first infected with BCoV at a multiplicity of infection of 5 PFU per cell and then transfected at 2 hpi with 3 µg of transcript RNA using Lipofectin (Invitrogen) [sfection . For traitrogen) . Proteinitrogen) .5 cells/dish) which were then infected with BCoV at a multiplicity of infection of 5 PFU per cell. For replication assay, supernatant fluids were harvested at 48 hpt and 500 µl was used to infect freshly confluent HRT-18 cells in a 35-mm dish (VP1). Total cellular RNA was extracted with TRIzol (Invitrogen) at 48 hpi of VP1 and 10 µg of extracted RNA was used for Northern assay. For internal controls, 3 µg of DI RNA transcript was transfected into BCoV-infected HRT cells and total cellular RNA was extracted with TRIzol at 4, 8, and 21 hpt of virus passage 0 (VP0) as indicated in 32P-labeled oligonucleotides. For detecting DI RNA specifically, the reporter-detecting oligonucleotide probe named TGEV(+), 5’ CATGGCACCATCCTTGGCAACCCAGA3’, was used. For detecting viral N subgenomic mRNA, viral M subgenomic mRNA, and the viral RNA portion of DI RNA the oligonucleotide named BCoVN(+), 5’ CCAGAACGATTTCCAAAGGACGCTCT3’, was used. For detecting the His-tag-encoding sequence, the oligonucleotide His(+), 5’ GTGGTGGTGGTGGTGGTG3’, was used. For detecting 18S rRNA, the oligonucleotide 18S rRNA(+), 5’ GCCTGCTGCCTTCCTTGGATCTGGTAGCC3’, was used. The probed blot was exposed to Kodak XAR-5 film at -80°C. For quantitating DI RNA, N subgenomic mRNA and 18S rRNA, probed blots were read with a Packard InstantImager Autoradiography System.The Northern assay for detecting reporter-containing DI RNAs was performed as described previously ,17. Brie


Phaedon cochleariae, for example, secrete deterrents from specialized defensive glands on their back. The secretion process involves ATP-binding cassette transporters. Therefore, sugar as one of the major energy sources to fuel the ATP synthesis for the cellular metabolism and transport processes, has to be present in the defensive glands. However, the role of sugar transporters for the production of defensive secretions was not addressed until now.Phytophagous insects have emerged successfully on the planet also because of the development of diverse and often astonishing defensive strategies against their enemies. The larvae of the mustard leaf beetle P. cochleariae, a transcript catalogue was created by Illumina sequencing of cDNA libraries. A total of 68,667 transcripts were identified and 68 proteins were annotated as either members of the solute carrier 2 (SLC2) family or trehalose transporters. Phylogenetic analyses revealed an extension of the mammalian GLUT6/8 class in insects as well as one group of transporters exhibiting distinctive conserved motifs only present in the insect order Coleoptera. RNA-seq data of samples derived from the defensive glands revealed six transcripts encoding sugar transporters with more than 3,000 counts. Two of them are exclusively expressed in the glandular tissue. Reduction in secretions production was accomplished by silencing two of four selected transporters. RNA-seq experiments of transporter-silenced larvae showed the down-regulation of the silenced transporter but concurrently the up-regulation of other SLC2 transporters suggesting an adaptive system to maintain sugar homeostasis in the defensive glands.To identify sugar transporters in We provide the first comprehensive phylogenetic study of the SLC2 family in a phytophagous beetle species. RNAi and RNA-seq experiments underline the importance of SLC2 transporters in defensive glands to achieve a chemical defense for successful competitive interaction in natural ecosystems. Glucose is one of the major energy sources and an important substrate for both protein and lipid synthesis. Its catabolism fuels cellular respiration for ATP production. For glucose absorption in the mammalian small intestine two pathways are known: the passive, paracellular absorption which bats rely on for more than 70% of their total sugar uptake Phaedon cochleariae which causes yield losses on cruciferous crops in Europe Although beetles (Coleoptera) represent one of the most diversified lineages on earth with about 350,000 species described and total numbers probably an order of magnitude higher, SLC2 sugar transporters of beetles have not been in the focus of researchers so far. In particular, Chrysomelidae (leaf beetles) constitute, together with the Cerambycidae (longhorn beetles) and the Curculionoidea (weevils), the largest beetle radiation, namely “Phytophaga”, which represent about 40% of all known species P. cochleariae is known to produce defensive compounds in such glands, herein further referred to as defensive glands, in the larval as well as in the adult stage in vivo relevance for deterrent production.When it comes to producing defensive compounds to repel their omnipresent enemies, insect in general are very innovative de novo-assembled transcriptome created from the mRNA of P. cochleariae, we have performed comprehensive and statistically supported phylogenetic analyses of the identified sequences together with their orthologs selected from other insects and other Metazoa including the known human glucose transporter (GLUT) isoforms. Our data revealed an enormous expansion of the GLUT6/8 sister group in insects and a clade of sequences unique for beetles. Subsequent next generation sequencing-based expression studies revealed putative SLC2 transcripts highly expressed in the defensive glands of juvenile P. cochleariae. Single silencing of selected SLC2-candidates by RNA interference (RNAi) resulted only in two cases in a reduced production of defensive secretions. However, in two other cases silencing did not affect deterrent production suggesting an adaptive backup system which stabilizes the sugar level in the defensive glands. To prove the observed homeostasis, we subsequently sequenced the mRNA of silenced larvae to study the differential expression of putative SLC2 transporters not only on phenotypic but also on a transcriptional level.Here we focus on a first catalogue of putative members of the SLC2 family as well as trehalose transporters for a phytophagous leaf beetle species. By means of a P. cochleariae (F.) was lab-reared on Brassica rapa chinensis in a light/dark cycle of 16 h light and 8 h darkness, 14°C±1°C in light and 12°C±1°C in darkness.P. cochleariae larvae as described by Bodemann et al. P. cochleariae were used for paired-end sequencing. Therefore, the fragmentation step during library preparation of the pooled total RNAs was shortened to four minutes  to obtain longer fragments. This library was sequenced using a GAIIx  in 150 bp paired-end mode in one sample per lane. All reads were extracted in FastQ format using CASAVA v1.7 (HiSeq) and v1.8 (GAIIx)  for further analysis.Total RNA was isolated from tissue samples from gfp dsRNA-injected larvae and three samples of larvae injected with dsRNA of three highly abundant sugar transporters) – two biological replicates each, have been prepared as mentioned above and sequenced on a HiSeq2500  in 50 bp single-read mode (multiplexed in one lane). All reads were extracted in FastQ format using bcl2fastq v.1.8.3 . The raw sequence data are listed in P. cochleariae can be accessed at NCBI homepage (BioProject ID: PRJNA210148).Subsequently to the RNAi experiments (described below), additional sequencing was carried out. Four samples of glandular tissue (one sample of de novo assembly software de novo assembled transcripts were reassembled using the TGI Clustering tools (TGICL), a software program to cluster large EST datasets The paired-end reads were assembled using Trinity, a RNA-seq de novo transcripts were translated into their possible protein sequences  by applying the transeq script which is part of the EMBOSS package (http://imed.med.ucm.es/EMBOSS/). Thereafter, the script pfam-scan.pl (downloaded from the ftp://ftp.sanger.ac.uk/pub/databases/Pfam/Tools/site) was used with showing overlapping hits within clan member families in addition to default parameters to search the protein sequences against the Pfam-A database which consists of high quality protein families based on profile HMMs and clans. A clan is a collection of Pfam-A entries which are related by similarity of sequence, structure or profile-HMM The via BLASTp with an E-value smaller than 1e-3 against the Swiss-Prot protein database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/). Swiss-Prot is a high quality and manually annotated and reviewed, non-redundant protein sequence database The SLC2 sequences and trehalose transporters were identified by searching the Pfam results for Sugar_tr hits. Sugar (and other) transporters are part of the major facilitator superfamily Phylogenetic trees were calculated for identified SLC2 encoding sequences (see above). Amino acid sequences in multi-FASTA format were aligned using Probalign version 1.4 For every calculation of the phylogenetic tree only conserved parts were taken into account. Thus, the N-terminus, the loop between TM6 and TM7 as well as the C-terminus were excluded from calculating the phylogenetic tree.P. cochleariae to divide these into groups with respect to their sequence similarities. Second, sequences originating from other insects  that have been functionally characterized were added to these sequences . For this, orthologous sequences derived from other beetles namely Dendroctonus ponderosaeTribolium castaneum were adjoined.First of all, the trees were calculated only for the chosen sequences derived from nces see  [15], [1P. cochleariae’s glands were prepared and sequenced as described above. All short reads of three replicates of glandular tissue were mapped onto the reassembled transcripts using Bowtie, an ultrafast short read aligner Pcrpl6 and Pcrps3) by first calculating the normalization factor Three replicates of the cDNA of 12–18 primer . Two technical replicates were performed from three biological replicates each. Technical replicates with a Cq difference of >1 were repeated. To normalize the PCRs for the amount of cDNA template added to the reactions, Pcrpl6 and Pcrps3 were chosen for P. cochleariae as reference genes. Primers were designed using primer3PLUS: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi  were cloned into T7-promotor site lacking TOPO-plasmids pIBV5/HIS . Plasmids were sequenced prior to further processing. For double stranded RNA (dsRNA) production, sequences of these targets were analyzed in silico to avoid sequenced related off-target effects according to gfp sequence was amplified from pcDNA3.1/CT-GFP-TOPO  accordingly. The amplicons were subject to in vitro transcription assays according to instructions from the Ambion MEGAscript RNAi kit . The resulting dsRNA was eluted from silica membranes after nuclease digestion three times with 50 µl of injection buffer . The concentration of dsRNA was calculated with A260 = 1 = 45 mg/ml and adjusted to 2 µg/µl. The quality of dsRNA was checked by TBE-agarose-electrophoresis.The open reading frames encoding putative transporters of four highly abundant transcripts  driven by a three-axis micromanipulator. The larvae were injected dorso-central between the pro- and mesothorax.According to Larval secretions were collected in glass capillaries . Secretions were weighed in the sealed capillaries on an ultra-microbalance  three times; the weight of the capillaries was subtracted and the final weight was averaged. Sealed capillaries containing samples were stored at −80°C until needed.P. cochleariae were diluted in 1∶200 (w/v) ethylacetate, supplemented with 100 µg/ml methylbenzoate as internal standard. Of each diluted secretion, 1 µl was subjected to GC/EIMS analysis  equipped with Phenomenex  ZB–5–W/Guardian–column, 25 m. Substances were separated using helium as a carrier (1.5 ml/min). The column temperatures were set as followed: 50°C (2 min), 10°C min−1 to 80°C, 5°C mi−1 to 200°C, 30°C min−1 to 300°C (1 min). Inlet temperature was 220°C and transfer line was 280°C. Chrysomelidial was identified according to According to P. cochleariae larvae have been mapped onto the studied sugar transporters of P. cochleariae’s transcriptome using bowtie The short reads (sequenced in 50 bp single-mode) from the glandular tissue (4 samples) of the RNAi-silenced P. cochleariae with special emphasis on the defensive glands, we performed a comprehensive analysis of transcriptomic data. For this purpose, cDNA libraries prepared from different tissues of juvenile P. cochleariae have been sequenced by using the Illumina technique. In addition to the sequencing of cDNA derived from a tissue pool in 150 bp paired-end mode, three biological replicates of the RNA of P. cochleariae’s defensive glands were extracted, processed and each sequenced in 50 bp single-read modes. The raw sequence data  are listed in de novo assembly of those reads by using the Trinity software In order to predict sugar transporters in the larvae of PcSUTxx  PcSUTxx . AccordiThe phylogenetic relationships of our selected 68 transporter sequences were analyzed by applying Probalign . The GLUT8 of Solenopsis invicta also belongs to this group. Therefore, we suggest ), for cladistic analyses by MrBayes and RAxML. Generally, et al. PcSUT31 clusters together with mammalian HMIT. The class II  and the GLUT10/12 clade contain only vertebrate sequences. According to Wilson-O’Brien et al.To study phylogeny of sugar transporters in a larger context, we selected the  of life , includiDendroctonus ponderosae, Tribolium castaneum, and P. cochleariae belong to this purple branch. Whereas, looking at P. cochleariae show homologies to Diptera, Apocrita and other insect orders. According to our cladistic analyses, the purple branch of the phylogenetic trees seems to be the most interesting. All changed and additionally conserved motifs together with the motif QH lead to the hypothesis that the sequences belonging to the purple branch may provide a new class of sugar transporters in insects, especially in the order Coleoptera, not before described. However, their biological function remains to be elucidated.The bootstrap values of the tree of life support the notion that the purple branch may be restricted to the insect order Coleoptera. For deeper analysis of this branch, we have calculated trees with the sequences of this purple branch and its homologs/orthologs in insects. The MrBayes tree as well as the RAxML tree  shows thThe Illumina short reads derived from the glandular tissue sequenced using HiSeq2000 in 50 bp single-read mode (two or three samples multiplexed in one lane) resulted in 34,918,295 reads with length of 50 bp as well as 36,598,828 and 29,866,004 short reads, respectively. Those short reads have been mapped onto our transcriptome. 82.62% to 84.90% of all short reads could be mapped, whereas 14.28% to 16.31% of the reads failed to align .PcSUT2 and PcSUT6, clustering into the blue branch, are exclusively expressed in defensive glands  and Pcsut5  to get deeper insights into their biological function for the development of P. cochleariae larvae and the production of defensive secretions in vivo. Early second instars were injected with either dsRNA identical to a unique part of one of these transporters sequences or with dsRNA of gfp. As confirmed by quantitative real-time PCR, transcription of all targets was successfully silenced in the defensive glands 10 days after injection  and Pcsut2 (p = 0.001) and also in the amount of secretions produced by targeting Pcsut1 (p = 0.007) and Pcsut2 (p = 0.03)  was carried out, besides for njection . By moninjection . To scre = 0.03) . KnockinPcsut2 or Pcsut6 (as examples for SLC2 members exclusively expressed in glands) or Pcsut1 (as example highly expressed in defensive glands and gut) and analyzed differential expression of SLC2 transporters in comparison to gfp-treated larvae.In contrast to the silencing effect of the ABC transporter in the defensive glands which caused a total loss of defensive secretions Pcsut1, Pcsut2 and Pcsut6-silencing and dsgfp-injection, glandular tissues were dissected and two biological replicates for each treatment were sequenced. The normalized counts of all sugar transporters of all samples are listed in 2 fold-changes of the silenced transporters (dsgfp-injected samples as control) and adjusted p-values were determined using the DESeq package . In all samples , we observed varying transcript levels of SCL2 transporters owing to the individual biological variance and diversity despite similar developmental stage or living conditions.Ten days after kage see . In FiguPcsut1 knocking-down led to a significant decrease of its own transcript level  suggesting counter-regulation to the silencing effect to ensure sugar homeostasis in the defensive glands. This up-regulation, however, did not fully compensate the silencing effect of Pcsut1 indicated by the decrease in the production of defensive secretions (as shown before).Pcsut2 showed, on the one hand, a significant down-regulation of Pcsut2 itself (padj = 2.49E-06), but also a decrease of Pcsut6, Pcsut26, Pcsut30, Pcsut32 and Pcsut34  (The samples prepared after dsRNA-injection of egulated . Additio= 0.092) . But alsPcsut6 silenced via dsRNA-injection, Pcsut6 was drastically reduced (padj = 2.56E-12). Furthermore, Pcsut13, Pcsut29, Pcsut30 and Pcsut32 were also down-regulated  compared to the dsgfp-injected samples. Especially Pcsut25 was significantly higher expressed (padj = 0.008) (In the samples with = 0.008) , Table 4Sugars play an important role in all species’ metabolism. Transporters of the SLC2 family are key elements involved in the adaptive response to sugar demand that have important physiological implications to cell survival and growth. They are expressed in a tissue-specific manner with different affinity, specificity and capacity for substrate transport P. cochleariae in which we annotated 68 sequences as SLC2 transporters. The phylogenetic analyses of putative sugar transporters were performed with MrBayes and RAxML. MrBayes, on the one hand, is a program for Bayesian inference and model choice across a large space of phylogenetic and evolutionary models. On the other hand, RAxML is a maximum likelihood phylogeny estimation. In our case, we were able to show that both programs result in a division of the predicted transporters into 4 main groups. However, there are some differences in the results of the calculations with the two methods. For example, PcSUT8 belongs to the green branch of the phylogenetic tree showing P. cochleariae’s sequences as well as the functionally characterized sequences of the other insects calculated by MrBayes; RAxML sorted it into the blue branch  and NlST6 from the brown planthopper N. lugens which function as glucose and glucose/fructose transporters, respectively, in the gut P. vanderplanki and its orthologs from A. gambiae, B. mori, A. mellifera and D. melanogasterN. lugensThe mammalian GLUT proteins are well-studied, and phylogenetic studies have been carried out. In addition to the five distinct classes of mammalian GLUTs A. pisum, whose genome contains a conspicuous number of genes encoding predicted sugar transporters The large number of SLC2 transporters may result from gene duplications as has been suggested for et al. Interestingly, the mammalian GLUT6/8 form a sister group of the expanded insect clade. In accordance with the literature P. cochleariae’s sugar transporters, TMHMM as well as Memsat2 were used. For 40 of 68 sequences, Memsat2 was able to predict all 12 TM domains. TMHMM predicted those 12 TM domains for just 33 sequences. But, for three sequences Memsat2 failed to predict at least 10 TM helices and proposed 1 (twice) and 2 (once) TM helices. For those three sequences, TMHMM predicted 10 (once) and 12 (twice) TM helices. This leads us to the conclusion that neither of these prediction methods is perfect, but applying both gives us the required information. Furthermore, Cuthbertson et al.Membrane proteins, such as sugar transporters, seem to have a restricted range of folds than their water-soluble counterparts, making them more amenable to structural predictions Pcsut1 and Pcsut2, phenotypic analyses revealed a reduction of defensive exudates in the larvae. Silencing of Pcsut5 and Pcsut6 did not result in a changed phenotype. By combining the RNAi experiments  with subsequent mRNA isolation and RNA sequencing, we could show the down-regulation as well as up-regulation of sugar transporters in the defensive glands. Silenced and induced sequences belong to separate branches in the phylogenetic trees which also suggest that insect transporters belonging to different clades can have comparable substrate preference. Anyhow, a direct correlation of counter-acting transporters was difficult to identify. All predicted off-targets according to Bodemann et al.T. castaneumAfter silencing Homeostasis may not only be achieved by the induced expression of transporters but, for example, also by the induced trafficking of transport proteins within a cell. This trafficking is known, for example, from the mammalian GLUT4. The protein is hormonally induced to translocate from intra-cellular membranes to the plasma membrane for the absorption of excessive glucose from the blood Figure S1Bar plot showing the molecular functions assigned to the PcSUTs. First, BLASTx was performed to annotate the studied sugar transporters of P. cochleariae. Thereafter, BLAST2GO was applied to those BLAST hits. The assigned molecular functions are displayed.(EPS)Click here for additional data file.Figure S2Phylogenetic tree of the 68 chosen sugar transporters derived from P. cochleariae. The phylogenetic tree was calculated with RAxML showing the main 4 groups. The purple subbranch is separated from the other branches with a bootstrap value of 100%.(EPS)Click here for additional data file.Figure S3Multiple sequence alignment of the 68 chosen sugar transporters derived from P. cochleariae. The multiple sequence alignment was calculated using Probalign. The purple-branch-specific amino acids are framed.(TIF)Click here for additional data file.Figure S4Phylogenetic tree of PcSUTs derived from P. cochleariae and functionally characterized sugar transporters of other insects listed in  calculated with MrBayes.(EPS)Click here for additional data file.Figure S5Phylogenetic tree showing the selected organisms from the tree of life.(EPS)Click here for additional data file.Figure S6The phylogenetic tree of the nine sequences derived from P. cochleariae belonging to the purple branch  as well as from Tribolium castaneum (Tc), was calculated using MrBayes. Indicated in purple, it can be seen that the beetles’ sequences build up a separate branch.(EPS)Click here for additional data file.Figure S7Phylogenetic tree of the 68 chosen sugar transporters derived from P. cochleariae. The six most abundant glandular sugar transporters are circled and not branch-specific, but distributed all over the tree.(EPS)Click here for additional data file.Figure S8Relative mRNA levels of putative SLC2 transporters in the defensive glands of juvenile P. cochleariae determined by carrying out RNA-seq (A) and quantitative real-time PCR (B) experiments. The corresponding fold-changes of the RNA-seq samples are listed in (TIF)Click here for additional data file.Figure S9RNAi effects on the development of the larvae from P. cochleariae. A, The development of larval weight was documented and measured in a 24 or 48 h±3 h period. B, In neither the relative growth rate nor in the weight of freshly emerged pupae significant differences could be observed between dsgfp- and dsPcsut1-, dsPcsut2-, dsPcsut5-, dsPcsut6-injected larvae, n = 30.(TIF)Click here for additional data file.Figure S10MvA-Plot showing normalized mean values versus2fold-changes. log The fold-changes (log-transformed) were computed for the comparison of dsgfp-injected and dsPcsut1-injected samples. The transcript levels are significant at 10% FDR ). The differentially expressed transporters, the ones colored red, are listed in Pcsut1-injected and dsgfp-injected samples. Pcsut1’s transcript level was significantly reduced by RNAi (red dot in the lower right quadrant). (B) MvA-Plot of the comparison of dsPcsut2-injected and dsgfp-injected samples. Pcsut2’s transcript level was significantly reduced by RNAi (red dot in the lower right area). Additionally, the expression of Pcsut25 was significantly induced (red dot in the upper right part). (C) MvA-Plot of the comparison of dsPcsut6-injected and dsgfp-injected samples. Pcsut6’s transcript level was significantly reduced by RNAi (red dot in the lower right area). Additionally, the expression of Pcsut25 was significantly induced (red dot in the upper right part).(TIF)Click here for additional data file.Table S1Primer sets used in quantitative real-time PCR and RNAi experiments.(XLSX)Click here for additional data file.Table S2BLAST2GO results for all identified sugar transporters. Table shows sequence description, sequence length, minimal e-value of BLAST search, mean similarity to all 5 hits for each query sequence respectively, the number of assigned gene ontology (GO) categories as well as assigned GO categories.(XLSX)Click here for additional data file.Table S3Prediction of 12 transmembrane regions using TMHMM as well as Memsat2. Furthermore, the counts of each replicate as well as sequence length and coding region’s length are listed.(XLSX)Click here for additional data file.Table S4The short reads of glandular tissues were mapped onto the transcriptome using bowtie. The short reads of the three replicates of RNA derived from the glandular tissue were mapped onto the transcriptome by applying bowtie. The percentages of aligned reads and of reads that did not align or were suppressed (due to option m) are listed. Furthermore, the numbers of reads that have been mapped are shown.(XLSX)Click here for additional data file.Table S5Pcsut counts normalized to the effective library size, relatively to Pcrps3 and Pcrpl6 according to Livak and Schmittgen . Samples were derived from glandular tissue from juvenile P. cochleariae.(XLSX)Click here for additional data file.Table S6Counts normalized to the effective library size of all sugar transporters after dsRNA-injection. dsRNA targeting Pcsut1, Pcsut2 and Pcsut6 as well as dsRNA targeting gfp were injected. Samples were derived from glandular tissue from juvenile P. cochleariae.(XLSX)Click here for additional data file.


We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by  Prokaryotic RNomics has drawn unprecedented attention from molecular biologists and microbiologists in recent years. In studying RNomics, one would focus not only on the protein-coding mRNA, but also the small RNAs. A major obstacle in such studies is the relatively small amount of mRNA and small RNAs (sRNA) in comparison to the ribosomal RNA (rRNA), which constitutes over 90% of total prokaryotic RNA. In order to preserve and measure the subtle change in RNome, particularly the low abundant RNA, enrichment of mRNA or rRNA depletion is inevitable.in vitro transcription of the probes [Various methods have been developed for eukaryotic system such as mRNA enrichment based on their polyadenylation status. However, this method is not ideal for prokaryotic mRNA purification as the poly(A) tails are associated with only 2-60% of the mRNA molecules . Apart fe probes .Mycobacterium smegmatis, a laboratory model for Mycobacterium tuberculosis was used [In depleting rRNA, an unintended loss of mRNA is observed because of the off-target effect between the probes and the mRNA. Probe-based subtractive rRNAs capture approaches are proven to be better than exonuclease digestion in preserving RNA abundance and integrity . The nonwas used . The prohttps://sourceforge.net/projects/ospstools. The program is available in version 3.0 and version 3.0 LIR which have different system requirement. The version 3.0 LIR has an extra function which allows prediction of sites for self-annealing or forming secondary structures such as hair-pins. The program is written in JAVA with Biojava (19) and is divided into two modules - Pre-BLAST and Post-BLAST  were identified and extracted. The 23S  and 16S  ribosomal RNAs were identified, extracted and virtually fragmented to a window size of 100 bp (customable size) with leap size of 5 nucleotides (customable which must be >=1). The fragmented 16S and 23S rRNAs were then assigned as query and were subject to BLAST (18) against all the CDS. The results were then processed by the Post-BLAST module with an e-value less than 1 with both 90% of identity recovery and hit length. Final matches with smallest number of hits were then chosen for probe design.Probe selection was done by Organism-Specific Probe Selection (OSPS) which can be found with the user guide at st-BLAST . Pre-BLAM. smegmatis of strain MC2 155 were grown in mycobacterium Middlebrook 7H9 medium (BBL) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC) (BBL) in a conical flask. The cultures were grown at 37°C with shaking of 100 rpm under subdue light. The culture was harvested at stationary phase when the optical density 600 nm (OD600) reached 2.0. Cells were collected from 50 ml culture. The cell pellets were lysed in 500 µl of 20 mg/ml lysozyme (Sigma-Aldrich) at 37°C for 30 min and then further lysed in 10 ml TRIzol reagent (Invitrogen) at room temperature for 10 min. The total RNA was then precipitated by isopropanol as instructed in TRIzol protocol. Since total RNA isolated for M. smegmatis usually contains some impurities, the isolated RNA was subject to a second-round purification using acidic phenol-chloroform . The purified RNA was reconstituted with 50 µl nuclease-free water. The RNA quantity and quality was analyzed on a NanoDrop ND-1000 spectrophotometer  and TBE agarose gel respectively.First strand cDNA was synthesized using QuantiTect Reverse Transcription kit (QIAGEN) following the protocol provided by the manufacturer. One microgram of purified RNA in 12 µl of RNase-free water was mixed with 2 µl 7× gDNA Wipeout buffer. After incubation at 42°C for 2 min, 4 µl Quantiscript RT buffer (5×), 1 µl primer mix and 1 µl Quantiscript reverse transcriptase was added to the reaction and incubated at 42 °C for 30 min followed by 2 min incubation at 95 °C. OSPS-selected 16S or 23S sequences were amplified from the first strand cDNA by PCR using probe-specific primers incorporated with restriction sites. PCR reaction was performed with Phusion high-fidelity DNA polymerase (Finnzyme) with the following cycling profile: DNA denaturation at 94 °C for 3 min and 35 cycles of amplification at 94 °C for 30s, 60 °C for 30s, 72 °C for 1 min, plus a final extension step at 72 °C for 7 min. PCR products were then cloned into a modified pUC19 renamed as pT1 which carries a minimal SP6 promoter at the 3’ end of the multi-cloning site.BamHI (New England Biolabs) locating at the 5’ end of the cloned rRNA sequence. In vitro RNA transcription was done using MEGAscript® SP6 kit (Ambion). The reaction mixture containing 1 µg of linearized plasmid DNA, 2 µl of each of 10 mM ATP, CTP, GTP, 0.5 µl of 10 mM UTP, 1.5 µl 10 mM Bio-11-UTP (Ambion) and 2 µl RNA polymerase was incubated at 37°C for 12 hours.Probe-incorporated plasmid DNA was linearized with The transcription product was mixed with Gel Loading Buffer II (Ambion) and separated on an 8% urea-acrylamide gel at constant current of 25 mA for 2 hours. The gel containing probe was visualized under the UV light and exercised. The exercised gel was transferred into 400 µl of RNA elution buffer  followed by incubation at 42°C overnight. The probes were then purified with acidic phenol-chloroform (Ambion). Briefly, 400 µl of acidic phenol-chloroform (Ambion) was added to the gel-containing elution buffer. After vigorous sharking for 15 seconds and centrifugation at 12000 g for 15 min at 4 °C, the supernatants were collected and mixed with 40 µl of 5M ammonium acetate, 8 µl of GlycoBlue (Ambion) and 1.2 ml 96% ethanol. Probes were incubated at -20 °C for 2 hours and harvested by centrifugation at 12000 g for 15 min at 4°C. The probes were then washed with cold 70% ethanol and then dissolved in nuclease-free water.2, 200 mM NaCl) containing 1.5 mM EDTA. The mixture was denatured at 70 °C for 10 min, then cooled down to 25 °C for probe-RNA hybridization. Meanwhile, Dynabeads M-280 Streptavidin (Invitrogen) was washed with 100 volumes of 200 µl RSB-300  containing 0.01% NP40 by centrifugation at 9000 g for 30 seconds. Then the samples were mixed with the streptavidin beads and incubated at 4 °C on a rotator for 15 min. After centrifugation at 9000 g for 30 seconds, the supernatant was transferred to a collection tube containing 40 µl of 5M ammonium acetate, 8 µl of GlycoBlue (Ambion) and 1.2 ml absolute ethanol. Another 200 µl RSB-200 supplemented with 1.5 mM EDTA was added to the RNA-probe-beads containing tube and incubated at 4°C for 5 min. The supernatant was transferred to the same collection tube after centrifugation at 9000 g for 30 seconds. The enriched RNA was precipitated by incubation at -20°C for 2 hours and harvested by centrifugation at 12000 g for 15 min at 4 °C. The purified RNA was dissolved in 20 µl of RNase free water. For comparison, the total RNA sample was also treated with the MICROBExpress™ Bacterial mRNA Enrichment Kit (Ambion) following the manufacturer’s instructions.1 µg total RNA was mixed with 0.8 µg probe for 23S rRNA and 0.6 µg probe for 16S rRNA in 200 µl RSB-200 buffer  and visualized under UV light.Half of the enriched RNA sample was analyzed on 1.5% denaturing agarose gel  propanesulfonic acid/ MOPS electrophoresis buffer) with untreated T method (2-ΔΔCT) was used to calculate the relative expression level of the target 16S rRNA, 23S rRNA and other mRNA.Half of enriched RNA was used for reverse transcription reaction using QuantiTect Reverse Transcription kit (QIAGEN) as described previously. Real-time PCR was performed by using 2× Power SYBR Green PCR Master Mix (Applied Biosystems) in ABI 7500 Fast Real-time PCR system (Applied Biosystems). The PCR cycling program initiated from denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Melting curves were also determined by an auto-dissociation program. The house-keeping gene MSMEG_5072 was measured for normalization. Comparative CMycobacterium smegmatis were selected by OSPS for 16S and 23S rRNAs respectively . In Express™ Bacterial mRNA Enrichment Kit (Ambion) or using our probes were shown in lane 2 and lane 3 respectively. Both the commercial kit and our designed oligos efficiently removed the 23S and 16S rRNA to a level that was invisible on gel.The workflow of the program Organism-Specific Probe Selection (OSPS) and the experimental procedures of rRNA depletion were illustrated in ectively , and werExpress™ Bacterial mRNA Enrichment Kit (Ambion) and our OSPS probes reduced the amount of 16S and 23S rRNAs to a level lower than 0.25 fold relative to the original amount of rRNAs. In order to evaluate the effect on the rRNA depletion probes on the integrity of mRNA, the transcription level of several selected genes was determined by real-time PCR in parallel. Fourteen genes ranging from high abundance to low abundance in M. smegmatis were studied and compared to the level in untreated RNA sample. Compared to our OSPS probes, using MICROBExpressTM caused an overall greater decrease in transcript amounts for genes of different abundances (ExpressTM caused a decrease in transcript level by 50-75% (hybC) and MSMEG_2525 (amino acid permease); moderately abundant genes such as MSMEG_1742 (oxidoreductase), MSMEG_1777 (UsfY), and MSMEG_0965 (porin); and lowly abundant genes MSEMG_2389 (hup) and MSMEG_2079 . In comparison, OSPS retained 100% transcript level for MSMEG_2263, MSMEG_1777, MSMEG_0965, and MSMEG_2079. OSPS also performed better in retaining the transcript levels of MSMEG_2525 and MSMEG_2389 (glnA).As shown in undances . For insy 50-75%  for highMEG_2389 . HoweverMycobacterium smegmatis. This method consists of three stages. Firstly, Organism-Specific Probe Selection (OSPS) program was used to screen for probes that are specific to mycobacterial rRNAs and have no significant similarity to other mycobacterial transcripts. Secondly, probes were designed and produced using an in-house plasmid system in combination with easily available commercial kits and reagents. Thirdly, the generated probes were applied to deplete the target RNA from total RNA.Ribosomal RNA contamination has been a problem in studies that focus on either coding or non-coding RNAs. In account of the sequence complexity of bacteria, probes that are designed based on the conserved sequences of bacteria rRNAs may also bind to and hence deplete some coding RNAs as illustrated in this study. Here we propose the idea of using organism-specific probes instead of using universal probes that target conserved rRNA sequences in bacteria. We demonstrated our organism-specific rRNA depletion method on ExpressTM kit which is a bacteria-specific subtractive hybridization rRNA removal kit. Our results demonstrate that computationally-selected probes can efficiently deplete 23S and 16S rRNAs from total RNA in M. smegmatis to a level comparable to the commercial kit. In spite of no similarity between ours probes and mRNA in M. smegmatis, off-target mRNA depletion was still observed. This can be due to the nonspecific hybridization of the DNA probes to the mRNA as some bases on the mismatch DNA probe can effectively forms Watson-Crick pairings in the nonspecific duplexes [We compared our in-house organism-specific method with the commercially available MICROBduplexes . Yet whein vitro transcription, the RNA probe will be linked with an affinity tags which can be immobilized onto streptavidin beads. Target RNA species such as rRNAs will then be bound onto the streptavidin and therefore be removed.Our findings also suggest a potential system for bacteria-specific rRNA depletion using streptavidin beads. In theory, this program could be applied to any organisms. However, we anticipate a various efficiency of rRNA depletion depending on the genome complexity of particular organisms. This method can also be further advanced by modifying the program and the experimental procedures to suit different purposes. For example, the program may be modified to remove RNA species other than 23S and 16S rRNAs by selecting the specific sequence on the RNA of interest. In this study, biotinylated-UTP was incorporated into the RNA probes which are then able to bind to streptavidin beads. In consideration of the cost of biotinylated UTP, an alternate cost-effective probe “labeling” methods can be applied by using RNA affinity tags. For instance, streptavidin aptamers could be cloned in front of or at the end of the probe sequence . By in vIn summary, the combination of OSPS and our in-house pT1 system should provide a tool for depleting ribosomal RNA in an organism-specific manner. We anticipate this strategy to be widely utilized in preparing RNA samples extracted from diverse organisms for application in Next-generation sequencing.https://sourceforge.net/projects/ospstools.The computer program and manual described in this paper is available at Figure S1Designing probes using Organism-Specific Probe Selection (OSPS) program.(DOCX)Click here for additional data file.


Here we use theory and experiment to evaluate Fe2+ in the absence of free oxygen as a replacement for Mg2+ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg2+ in RNA folding and function can indeed be served by Fe2+. The results of quantum mechanical calculations show that the geometry of coordination of Fe2+ by RNA phosphates is similar to that of Mg2+. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4–P6 domain RNA is conserved between complexes with Fe2+ or Mg2+. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg2+, and the hammerhead ribozyme are enhanced in the presence of Fe2+ compared to Mg2+. All chemical footprinting and ribozyme assays in the presence of Fe2+ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe2+. The combined biochemical and paleogeological data are consistent with a role for Fe2+ in an RNA World. RNA and Fe2+ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg2+ alone.Mg Folded RNAs are stabilized in part by inorganic cations that accumulate in and around the RNA envelope. ‘Diffuse’ cations remain hydrated and make primary contributions to global stability by mitigating electrostatic repulsion of the negatively charged backbone. Chelated ions are less frequent, but in some instances are essential for achieving specific local conformation of the RNA. A special importance of Mg2+ can substitute for Mg2+ in RNA folding and catalysis. Mg2+ possesses important electronic and geometric properties that are key to its relationships with RNA. It is redox inactive, and does not cleave RNA via Fenton chemistry. The ionic radius of Mg2+ is small, the charge density is high, the coordination geometry is strictly octahedral, and the hydration enthalpy is large and negative 2+ has a greater affinity for phosphate oxygens 2+ is an excellent Mg2+ mimic in the absence of O2, readily substituting for Mg2+ in RNA folding and catalysis.Here we use computation and experiment to address the question of whether Fe2+ on RNA structure and function could provide important links between the geological record and the RNA world. It is believed that life originated with RNA-based genetic and metabolic systems, i.e. the RNA world 2+, either instead of or in combination with Mg2+, seems to be a possible partner of RNA in the biology of the pre-photosynthesis anoxic earth.Our primary motivation is to study RNA under plausible early earth conditions. Understanding the influence of Fe2+ of the early earth was oxidized and sequestered. Iron was deposited in banded iron formations (BIFs) With the rise in free oxygen, a product of photosynthesis, the Fe2+ is replaced by Fe2+ in first shell RNA-metal complexes. We focused here on an RNA-Mg2+ clamp 2+  calculations show that RNA conformation and coordination geometry are conserved when Mgclamp 2+ . A compl2+ clamp and an RNA-Fe2+ clamp are nearly identical, as determined by Density Functional Theory (DFT) 2+ over Mg2+ by 1.3 kcal/mol in continuum solvent as indicated by DFT calculations. Natural Bond Orbital analysis 2+ (0.43 e-) than from phosphate to Mg2+ (0.29 e-). This difference implies that compared to Mg2+, Fe2+ better activates the phosphorous of RNA to nucleophilic attack. The increase in activation is attributable to the accessibility of the d-orbitals of Fe2+.The conformations of an RNA-MgSelective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful RNA foot-printing technique that provides structural information at single-nucleotide resolution T. thermophila Group I intron P4–P6 domain was assayed by SHAPE in the presence of Na+ alone, giving a reaction pattern consistent with the known secondary structure The secondary structure of the 2+  specific magnesium binding, (ii) diffuse interactions of Mg2+, and (iii) RNA-RNA tertiary interactions facilitated by Mg2+, as previously demonstrated for tRNA 2+ observed here is nearly identical to that described previously for the same RNA in the presence of Mg2+ by Cech and coworkers (using N-methylisatoic anhydride instead of BzCN) We probed the structure of the P4–P6 domain RNA in presence of Mg2+ . The fol2+ is replaced by Fe2+ under anaerobic conditions. 2+ are identical, within the accuracy of the experiment, to those in presence of 2.5 mM Mg2+. These results suggest that tertiary interactions and even the ‘ion core’ of the P4–P6 domain are recapitulated by Fe2+ in the absence of oxygen. As expected, if Fe2+ is added to the RNA in the presence of atmospheric free oxygen, the RNA is quickly degraded (not shown).The pattern of SHAPE reactivity for P4–P6 domain RNA is conserved when Mg2+, we tested the catalytic activity of the L1 ribozyme ligase in the presence of Mg2+ or Fe2+ (in the absence of oxygen). This ligase catalyzes formation of a phosphodiester linkage. The 3′-hydroxyl group of an RNA substrate attacks the α-phosphorus of the ribozyme 5′-triphosphate in vitro in the presence of high [Mg2+] (60 mM) by Robertson and Ellington, and has been described as Mg2+-dependent 2+ is 1.4×10−6 min−1, while the initial rate of ligation in 100 µM Fe2+ is 3.5×10−5 min−1, which is 25-fold higher .The hammerhead ribozyme was also assayed for activity in the presence of Fed higher . The max2+ was an important metallo-cofactor for RNA. In this model, Fe2+ was replaced over time by Mg2+, by processes driven at least in part by photosynthesis and the Great Oxidation Event.The results here support a model of early evolution in which Fe2+ in the early Archean would have been available, soluble and non-toxic. Our observations here of Fe2+-mediated RNA folding and catalysis, in combination with paleogeological information, suggest that RNA could have originated and evolved in association with Fe2+. The RNA-Fe2+ complexes recently observed in extant biology 2+ into RNA World models opens broad new possibilities for ancient biochemistry. RNA and Fe2+ could, in principle, support an array of RNA structures and catalytic functions far more diverse than RNA with Mg2+ alone. Complexes of RNA with Fe2+ offer the prospect of redox chemistry and electron transfer reactions for ancient ribozymes.The RNA world is hypothesized to have occurred during the early Archean eon, prior to the Great Oxidation Event. Fe2+ and Fe2+ are used as cofactors in a broad class of superoxide dismutases (Fe2+/Mn2+ SODs) 2+ and Fe2+ while the coordination geometry, amino acid sequence and global fold of the protein are conserved. The discrimination between Fe2+ and Mn2+ in Fe2+/Mn2+ SODs in vivo is determined by species, organelle, protein isozyme, protein expression level and metal bioavailability. Metal substitution appears to be a useful biological strategy in nutrient-limited environments 2+ was appropriated into some Fe2+ dependent enzymes 2+ was converted to Mg2+. It has been suggested, based on sites of Fenton cleavage (using iron/O2), that Fe2+ and Mg2+ compete for common sites in RNA in vitro.The conversion of one metal to another is facile in some protein enzymes. In just one example, Mn2+ and Fe2+ complexes  in the atmosphere (Fe2+ precipitates as siderite: Fe2+CO3−). Early earth PCO2 is inferred using estimates of greenhouse effects, sun luminosity, earth albedo and temperatures required to maintain liquid oceans. A variety of recent results challenge high PCO2 models CO2 was low, [Fe2+]marine could have been as high as 100–1000 µM HS (atmospheric pressure of HS) would also have been an important influence on [Fe2+]marine due to precipitation of FeS It seems very likely that the early Archean earth provided a variety of Fe2+]marine in the early Archean is also uncertain. Although the models are tentative, it has been suggested that [Mg2+]marine during this time was attenuated by submarine hydrothermal systems associated with higher heat flow 2+ delivery to the oceans by smaller continental landmasses or from the weathering of peridotite in the sea floor +2]marine of early oceans in comparison to today.The  is increased from 100 µM  to 625 µM . The rate of the reaction in 1 mM Mg2+ (RH panel) is roughly equivalent to that in 100 µM Fe2+ (LH panel).(TIF)Click here for additional data file.Table S1Electronic energies, interaction energies and the corresponding counterpoise-corrected interaction energies calculated at the (U)B3LYP/6–311++G level of theory within the framework of IEFPCM in water.(DOCX)Click here for additional data file.Table S22+ and Fe2+ in the RNA2− -Mg2+(H2O)4 and RNA2− -Fe2+(H2O)4 complexes as revealed by the NBO at the (U)B3LYP/6–31G level of theory.Electronic configurations of Mg(DOCX)Click here for additional data file.Text S1RNA Synthesis and Purification.(DOCX)Click here for additional data file.


However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders. Gene expression profiling investigations involving postmortem brain tissue of cases with neuropsychiatric disorders such as autism have been limited due to tissue availability and tissue quality -3. Such The cDNA-mediated annealing, selection, and ligation (DASL) gene expression assay has been shown to produce highly reliable results when applied to formalin-fixed, paraffin-embedded tissues ,8. To ovThe DASL-based methodology has already been applied in the study of human liver, esophagus, breast, prostate, ovarian, and other biopsy and autopsy tissues ,10-13. IIn this study, we investigated the utility of the standard IVT- and DASL- based genome-wide expression profiling assays in the context of a clinically important neuropsychiatric disorder, autism. Our objectives were to: 1) compare the quality of microarray data from IVT-based and DASL-based platforms on artificially degraded reference RNA; and 2) compare the quality of microarray data from these two RNA profiling platforms on postmortem frozen and formalin-fixed brain tissue.in vivo. The more time that RNA is subjected to heating, the more fragmented the RNA becomes , 10, 30, and 60 minutes  and tissue preservation  analysis . MultiplIn a small sample of cases with available RIN (N = 4) and assayed on both IVT- and DASL-based assays, RIN also explained a large percentage of variability in the expression data. However, the influence of RIN on the IVT-processed samples  in single regression MDMR was greater than that on the DASL-processed samples . These results confirm that the IVT-based assay is more greatly influenced by differences in RNA degradation than the DASL-based assay.QPCR validation of the DASL-based microarray data was performed on a subset of genes. We compared the log2 fold changes of the qPCR data with the log2 transformed, quantile normalized data. Using a Spearman's rank correlation, the log2 fold changes of these 19 genes  to visualize levels of degradation  and from the University of Miami/University of Maryland Brain and Tissue Bank ,All cases were deceased, and were deidentified by the brain banks where tissue was obtained. However, the same human protections procedures were employed as for live subjects. Research procedures employed in this study were approved by the institutional review board of the University of California, San Diego (protocol number 091205).Due to documented variability of gene expression in neighboring brain areas ,20, it i® kit from Ambion according to manufacturer's instructions (http://www.ambion.com). Extraction of total RNA from 5-10 mg of formalin-fixed tissue sections was performed using the Roche® High Pure FFPE RNA Micro Kit. Select RNA samples were analyzed with BioAnalyzer® (Agilent) according to the manufacturer's protocol for quality control and quantification, and available RNA Integrity Numbers (RIN) are reported in Table ® spectrophotometer.Extraction of total RNA from 5-10 mg of frozen tissue from both grey and white matter, with as many layers of cortex as possible, was performed using MELTTotal RNA from reference samples, frozen, and formalin-fixed cases underwent cDNA synthesis, and cDNA-mediated annealing, selection, and ligation (DASL)-based labeling, hybridization to Illumina HumanRef8 v3 and Human 12K microarrays (DASL assay on reference RNA samples only), and scanning on two separate occasions as described previously . Both bihttp://www.ncbi.nlm.nih.gov/geo/).Following extension and ligation, the ligated oligonucleotides were PCR amplified with a biotinylated and a fluorophore-labeled universal primer, and captured using streptavidin paramagnetic beads. Finally, the single-stranded PCR products were eluted and hybridized to the BeadChips at 58°C for 16 hours. A BeadArray Reader was used to scan array images and extract fluorescence intensities, and all data were uploaded into BeadStudio software without normalization or background subtraction for quality control and processing. All raw data is available on the NCBI Gene Expression Omnibus under accession number GSE28475  according to manufacturer's protocols. Following RNA extraction, an IVT reaction for biotinylated cRNA was performed overnight (~16 h). 750 ng cRNA were hybridized on the beadchip at 58°C overnight and detected with Cyanine3-streptavidin. Arrays were again scanned with the Illumina BeadArray Reader and read into Illumina GenomeStudio software without normalization or background subtraction.All data analyzed were raw and unprocessed. Probe detection and signal information was directly output from GenomeStudio. Probe concordance and self-reproducibility were calculated based on technical replicates in each category .All plots were generated using the R/Bioconductor package Lumi -23 and Mhttp://polymorphism.scripps.edu/~cabney/cgi-bin/mmr.cgi). Variables of interest that were related to the expression profiles reflected in the distance matrices included batch, RNA source , assay type (DASL or IVT), gender, diagnosis, and age of cases from which we sampled. We leveraged both single independent variable and multiple independent MDMR results. Case data analyzed by MDMR as predictors  were compiled by a clinical psychologist (C.C.B).To assess the variance within the dataset attributable to a set of variables before and after manipulating and pre-processing the expression assay results (e.g. batch correction), multivariate distance matrix regression  with 10RNA from 1 male autistic and 1 male control case of 31 years was analyzed using SYBR green RT-PCR to validate the intensity values detected by microarray. 19 genes were chosen with a wide range of fold change values (positive and negative), and are listed in Table One microgram of total RNA was used for cDNA synthesis using random hexamers and AMV reverse transcriptase. An equivalent of 50 ng of RNA was processed by qPCR using Roche's LightCycler rapid thermal cycler system  according to the manufacturer's instructions, in a 96-well, 10-uL format using standard PCR conditions. 1 μL of cDNA template, 250 nM of forward and reverse primer, and 5 μL of qPCR Master Mix (Roche) were mixed for each reaction.According to Vandesompele et al.,  we took MLC participated in designing and conducting experiments, data analysis, interpretation of results, and constructing the manuscript. HRL participated in designing and conducting experiments, and revising the manuscript. MEW participated in data analysis. CCB performed clinical assessments on autopsy reports to collect case data for data analysis and revised the manuscript. CA and JBF participated in microarray processing. JBF also revised the manuscript. AWB, XDF and EC designed and supervised experiments. NJS designed and supervised experiments, interpreted results, and revised the manuscript. Each author has given final approval to the manuscript for publication.BioAnalyzer analysis showing reference RNA fragmentation and RIN values for each sample. Artificially degraded brain and pooled reference RNA was visualized using Agilent BioAnalyzer. B0 and P0 samples show two ribosomal RNA bands, while samples heated at 95°C showed smaller RNA fragments. The longer the duration of heating, the smaller the fragments became. B0 = intact reference brain RNA; B10 = 10 minute heating at 95°C; B30 = 30 minute heating at 95°C; B60 = 60 minute heating at 95°C; P0 = intact reference pooled RNA; P10 = 10 minute heating at 95°C; P30 = 30 minute heating at 95°C; P60 = 60 minute heating at 95°C.Click here for fileBrain Reference RNA assay performance. Scatterplots of reference RNA samples with increasing levels of artificial degradation, histogram of data distribution, correlation, and number of up- and downregulated genes differing between samples in IVT- (left) and DASL- (right) based platforms are shown. Degraded brain reference RNA profiled on IVT-based assays show lower correlations with intact RNA than on DASL-based assays. Intensity values also cluster at background levels for increasing RNA degradation for IVT-based samples but not DASL-based samples. For example, correlations between intact RNA and RNA degraded for 60 minutes (B60) differed greatly between two assays (four corner boxes on the two plots). IVT_B60 had a correlation of 0.5 (lower left corner box) with the intact sample (IVT_B0). In contrast, DASL_B60 had a correlation of 0.82 with the intact sample (DASL_B0). In addition, the histogram of IVT_B60 shows most intensity values at background levels, but that of DASL_B60 showed a similar distribution to that of DASL_B0. In addition, the scatterplot comparing IVT_B0 and IVT_B60 (top right corner box) shows skewing from the y = x line, reflecting the clustering of IVT_B60 intensities at background values. The scatterplot comparing DASL_B0 and DASL_B60, however, shows better adherence to the diagonal despite some dispersion. B0 = intact reference brain RNA; B10 = 10 minute heating at 95°C; B30 = 30 minute heating at 95°C; B60 = 60 minute heating at 95°C.Click here for fileClustering of samples with similar levels of RNA degradation evident in results from both IVT and DASL assays. Average hierarchical clustering was applied to the Euclidean distance between intact and artificially degraded brain and pooled reference RNA samples. Thus the more degraded the sample is regardless of assay, the less similar it is to the intact sample. Labels indicate assay type and time of degradation. B0 = intact reference brain RNA; B10 = 10 minute heating at 95°C; B30 = 30 minute heating at 95°C; B60 = 60 minute heating at 95°C; P0 = intact reference pooled RNA; P10 = 10 minute heating at 95°C; P30 = 30 minute heating at 95°C; P60 = 60 minute heating at 95°C; B75P25 = 75% brain reference RNA and 25% pooled reference RNA combination; B25P75 = 25% brain reference RNA and 75% pooled reference RNA combination; rep = technical replicates.Click here for fileIntensity distributions vary with RNA degradation in IVT- but not DASL-based assays. Boxplot showing the amplitude of genome-wide intensity distributions detected for intact and artificially degraded reference brain and pooled RNA samples processed by IVT- and DASL-based platforms. In the IVT-processed samples, increasing RNA degradation correlated with a smaller range of intensity values clustered at low intensities. In the DASL-processed samples, this confound was not observed. B0 = intact reference brain RNA; B10 = 10 minute heating at 95°C; B30 = 30 minute heating at 95°C; B60 = 60 minute heating at 95°C; P0 = intact reference pooled RNA; P10 = 10 minute heating at 95°C; P30 = 30 minute heating at 95°C; P60 = 60 minute heating at 95°C; B75P25 = 75% brain reference RNA and 25% pooled reference RNA combination; B25P75 = 25% brain reference RNA and 75% pooled reference RNA combination; rep = technical replicate.Click here for fileAssay performance on formalin-fixed tissue. Correlations between fixed tissue technical replicates and between samples on IVT- (left) and DASL- (right) based platforms are shown. Scatterplots of genome-wide profiling results from 2 formalin-fixed samples (BTB3859 and UMB1670) processed by the two assays, histogram of data distribution, correlation, and number of up and downregulated genes show low reliability in profiling RNA from these samples in both assays. Most intensity values tended to cluster at background levels, particularly for the IVT-processed samples. For example, technical replicates of BTB3859 (four top left boxes on two plots) show 0.83 correlation on the IVT-based assay, and 0.77 correlation on the DASL assay. However, the histogram and scatterplots show that this correlation on the IVT-based assay (left plot) is due to detection of most probes at low background levels. In contrast, the histogram of the DASL-based assay results for this case (right plot) shows a subset of probes that showed some reliability, with intensity values above baseline. Rep = technical replicate.Click here for fileRNA degradation in degraded frozen-tissue based RNA affects IVT greater than DASL processing. Boxplots show genome-wide intensity distribution of frozen brain-extracted RNA samples from four cases and replicates processed by IVT- and DASL-based assays. The IVT-processed samples clearly show distributions clustered at background intensities especially for samples with low RIN (2.2 for UMB4670 and 2.4 for UMB4721). The distribution of DASL-processed samples for UMB4670 and UMB4721 did not appear significantly different from samples with higher RIN (6.3 for UMB4849 and 4.9 for UMB1649). Rep = replicate.Click here for fileLow correlations between IVT- and DASL-based assays on the same three frozen tissue-extracted RNA samples. Scatterplots of 3 samples processed by the two assays, histogram of data distribution, correlation, and number of up and downregulated genes show low concordance between the same tissue sample processed by two platforms, and differences in intensity distribution between platforms. For example, the correlation between case UMB1349 processed by IVT- and DASL-based assays  was 0.76. Histograms for this case show a similar distribution for both assays, but the scatterplot shows many probes diverging from the y = x line.Click here for fileLow correlation between processed samples of UMB1796. Frozen and fixed samples taken from case UMB1796 were processed by both IVT- and DASL-based methods. Genome-wide results are plotted to compare correlations and distributions. Scatterplots of UMB1796 replicates, histogram of data distribution, correlation, and number of up- and downregulated genes differing between samples show low correlations between brain tissue profiled using IVT- and DASL-based approaches from the same case in all except DASL technical replicates. Labels indicate assay type (DASL/IVT), preservation method (Frozen/Fixed), sample code, and replicate number (if applicable).Click here for fileLog2 Fold Change Correlations of selected genes detected by microarray and RTPCR. Log2 Fold change detected by RTPCR is depicted on the x axis, and microarray on the y-axis of 19 up- and down-regulated genes in the dataset. Spearman's rank correlation detected an R = 0.78  correlation between microarray and qPCR detection of fold change.Click here for file


Fish assemblages of unconsolidated sedimentary habitats on continental shelves are poorly described when compared to those of hard substrata. This lack of data restricts the objective management of these extensive benthic habitats. In the context of protecting representative areas of all community types, one important question is the nature of the transition from reefal to sedimentary fish assemblages. We addressed this question using Baited Remote Underwater Videos (BRUVs) to assess fish assemblages of sedimentary habitats at six distances from rocky reefs  at four sites in subtropical eastern Australia. Distance from reef was important in determining fish assemblage structure, and there was no overlap between reef sites and sedimentary sites 400 m from reef. While there was a gradient in assemblage structure at intermediate distances, this was not consistent across sites. All sites, however, supported a mixed ‘halo’ assemblage comprising both reef and sediment species at sampling stations close to reef. BRUVs used in conjunction with high-resolution bathymetric and backscatter spatial data can resolve differences in assemblage structure at small spatial scales (10s to 100s of metres), and has further application in unconsolidated habitats. Unless a ‘reef halo’ assemblage is being examined, a minimum of 200 m but preferably 400 m distance from any hard substrate is recommended when designing broader-scale assessments of fish assemblages of sedimentary habitats. Despite the fact that sedimentary environments are the most extensive subtidal habitat of the world’s oceans The lack of available data on unconsolidated habitats and associated biotic patterns restricts the objective management of these habitat types. Marine Protected Areas have been developed worldwide with conservation of biodiversity as a primary objective In New South Wales (NSW) marine parks, a Habitat Classification Scheme (HCS) is employed as a surrogate for biodiversity, and uses depth and habitat type (such as ‘rocky reef’ and ‘unconsolidated sediment’) as primary categories. While the HCS has been tested for effectiveness against patterns of fish assemblage structure on rocky reefs Many species found on unconsolidated habitats are morphologically and behaviourally adapted to this environment, posing problems for visual surveys. For example, fish such as those from the family Platycephalidae bury themselves in the sediment to ambush prey Baited Remote Underwater Video (BRUV) has been developed to reduce constraints involved in fish assemblage research by SCUBA methods Not only are data lacking for fish assemblages of unconsolidated habitats in general, but also relatively little is known about linkages between unconsolidated and reefal habitats but see . ‘Top doGradients in assemblage structure have been inferred by studies demonstrating a reduction in predators The focus of this research was, therefore, to investigate how reef proximity influences fish assemblages of unconsolidated substrata. We deployed replicated BRUVs in two sites at each of two locations and a fixed depth range (20–30 m) to: 1) quantify changes in fish assemblage structure with increasing distance from reef, and 2) identify the species that drive differences in assemblage structure across this gradient. We also tested the spatial consistency of these patterns between and within two locations at the scale of 100s of metres to kilometres.The Solitary Islands Marine Park (SIMP) is situated on the north coast of New South Wales, in the Tweed-Moreton Bioregion, and covers an area of approximately 71,000 hectares. The region lies within a broad ecotone between tropical and temperate assemblages, supporting high diversity with consequently high conservation status e.g. . The SIMA three-way, partially nested design was used to assess demersal fish assemblages at two locations within the SIMP using BRUVs. Sites were selected using existing high-resolution bathymetric and backscatter spatial data Sardinops neopilchardus) was used to attract fish to the field of view  for analysis using Adobe Premiere Elements , at a suitable resolution for fish identification (720×576). Files were then analysed using the program Eventmeasure . The identity of each fish species and an index of its relative abundance (MaxN) were recorded. MaxN is the maximum number of individuals of each species within the field of view at any one instant during the 30-min recording. This removed the possibility of double counts of individual fish. As counts reflected relative abundance and not density, we expected data to be robust to variability in estimating this field of view, and all video analysis was performed by the same observer (AS) A range of multivariate and univariate analyses were applied using procedures in the PRIMER 6.0 software package Where PERMANOVA revealed a statistically significant result, SIMPER analysis was used to determine the species most responsible for differences in assemblage structure Pagrus auratus , the schooling planktivore Atypichthys strigatus  and the piscivore Platycephalus longispinis . Twenty-four species were recorded only once, which is consistent with studies of reef fish in the SIMP A total of 76 species from 37 families was observed. Eight species of chondrichthyans were recorded, four sharks and four rays. Osteichthyans were from various trophic groups including planktivores, herbivores and predators. The families Labridae and Platycephalidae were the most speciose (six species each). A total of 1640 individuals were recorded by combining all MaxN data. The most abundant species were the predator There was an obvious gradient in community structure with increasing distance from reef . Howeverpost-hoc pairwise contrasts were performed where the effect of distance was significant  . Howevernificant .P. auratus, A. strigatus, P. longispinis and Sillago ciliata, appeared more than three times in pairwise comparisons between distances, so abundances of these were explored further.Having established that differences in assemblage structure were consistently found across distances at each site, SIMPER analysis was then used to determine which species were primarily responsible for these differences. Ten species were found to be important discriminators (accounting for the first 50% of assemblage differences) across all analyses. Of these, four species, post-hoc pairwise tests indicated significant differences between 0 vs 100 m (P = 0.001), 0 vs 200 m (P = 0.001), and 25 vs 400 m (P = 0.012).Trends in mean species richness were similar across three of the four sites, with the highest values at 0 or 25 m, a rapid decline to a distance of 100m, and similar values thereafter . Forty AA. strigatus dominated the combined abundance  and Split Bommie West (P = 0.016). Post-hoc pairwise contrasts were also performed for these two sites, but no consistent trends were evident  has been identified as an important contributor to biodiversity in this marine park A relatively high diversity of fish species was recorded when compared with previous studies in this marine park using BRUVs (76 species from 72 replicates). Malcolm et al. P. auratus and mado A. strigatus, which were abundant at reef sites, but less so in the halo and absent from sediment habitat; and whiting S. ciliata and longspine flathead P longispinis, which were abundant in sediment and halo habitats, but uncommon or absent at sites on or adjacent to reef .Despite the relatively high diversity recorded in this study, only a small percentage of species consistently contributed to the observed variation in assemblage structure, which is consistent with other studies A. strigatus, a schooling, reef-associated planktivore, was the most abundant species at 0 m but was highly variable between sites , which were two of the species most influential in discriminating between sedimentary and reef assemblages. However, this varied in relation to distance from reef. S. ciliata was recorded at 200 m at three sites, and at 100 m at two sites, but only in the absence of P. auratus. The reasons for this remain unclear but could include: the unknown presence of very small patches of reef (as previously discussed) and consequent interaction with reef fish assemblages; the aggressive nature and feeding behaviour of P. auratus, particularly when in higher densities.There was an inverse correlation between snapper Platycephalus arenarius, P. caeruleopunctatus, P. fuscus, P. longispinis, P. richardsonii, Ratabulus diversidens), none were recorded on reef sites, showing a steady increase in abundance with increasing distance from reef. We considered the possibility for platycephalids to concentrate predation effort closer to reef in pursuit of small reef-associated fish, but our theory was not supported by this study. Species from this family were highly responsive to BRUVs, and this method is likely to be suitable for further specific investigations of platycephalids in these habitats.There are no previous studies on the effects of reef proximity on abundance of flatheads . Of the six species recorded in this study (There have been few studies using BRUVs in unconsolidated shelf habitats, and most research on fish assemblages in Australian waters in these habitats has been through netting Within the SIMP, there are vast areas of unconsolidated sediments which, as they are below the depth of access by SCUBA, are poorly described in terms of biotic assemblages and ecology The influence of rocky reef on sedimentary fish assemblages in this region was strongest within 25 m of reef, diminishing rapidly thereafter. Between 100 and 200 m, there was an apparent halo effect, dominated by sedimentary fishes but still with some reef-associated species. By 400 m there was no distinguishable reef influence. Broader-scale studies targeting fish assemblages of unconsolidated habitats should, therefore, maintain a minimum distance of 400 m from any reef structures to minimise influence of reef species, unless the intention is to include the halo assemblage.This study extends current knowledge of demersal fish assemblages within this marine park, and provides important data for the design of further studies on fish assemblages of sedimentary habitats. High-resolution bathymetric and backscatter maps were effective aids to the design of the study, especially at the smallest spatial scales. In combination, these methods enable broader examination of sedimentary fishes at various spatial and temporal scales, including cross-shelf and depth-associated patterns, and the assessment of fidelity with different types of sediment. These data will facilitate the informed conservation management of this dominant habitat both regionally and more broadly.


Carcharhinus amblyrhynchos) observed by dive guides and the telemetry data at both daily and monthly intervals, suggesting that variation in relative abundance of sharks was detectable in datasets collected by dive guides in a similar manner to data derived from telemetry at these time scales. There was no correlation between the number or mean depth of sharks recorded by telemetry and the presence of tourist divers, suggesting that the behaviour of sharks was not affected by the presence of divers during our study. Data recorded by dive guides showed that current strength and temperature were important drivers of the relative abundance of sharks at monitored sites. Our study validates the use of datasets of shark abundance collected by professional dive guides in frequently-visited dive sites in Palau, and supports the participation of experienced recreational divers as contributors to long-term monitoring programs of shark populations.Citizen science is promoted as a simple and cost-effective alternative to traditional approaches for the monitoring of populations of marine megafauna. However, the reliability of datasets collected by these initiatives often remains poorly quantified. We compared datasets of shark counts collected by professional dive guides with acoustic telemetry data from tagged sharks collected at the same coral reef sites over a period of five years. There was a strong correlation between the number of grey reef sharks ( Many shark species are experiencing unsustainable rates of mortality due to fishing, a phenomenon that is driving population declines globally The assessment and monitoring of shark populations through fishery-independent techniques presents considerable challenges due to the naturally low population densities and relatively large home ranges common to most species Chelonia mydas and Eretmochelys imbricata), minke whales  and manta rays  Data collected by the public can provide a cost-effective means of monitoring populations of wild animals Rhincodon typus), manta rays and white sharks (Carcharodon carcharias), but is unsuitable for the many reef and pelagic sharks that generally lack any persistent features that might be used to distinguish individuals. For these species, counts by divers provide one of the simplest means to monitor numbers.Recreational divers and snorkelers have been used to collect data on elasmobranchs in two principal ways: firstly, by recording counts of animals seen during a dive Traditional approaches to underwater visual surveys involve standardized techniques that focus on quantifying the area sampled and the abundance and length of individuals within the sample space Acoustic telemetry can provide a means to address these issues and validate datasets generated by citizen science programmes. Arrays of acoustic receivers are now commonplace in many coastal marine environments that are inhabited by marine megafauna such as sharks In our study, we compared datasets of shark relative abundance collected by professional dive guides with tagging data generated by passive acoustic telemetry at the same sites on coral reefs in Palau, Micronesia. We aimed to determine if the observations reported by dive guides produced comparable estimates of relative abundances and temporal patterns in numbers of sharks as those obtained from presence/absence data derived from acoustic tagging and monitoring. We also used the telemetry data to investigate the effect of the presence of tourist divers (i.e. observer effect) on the relative abundance and depth use by sharks. Finally, we analysed the citizen science data in order to identify environmental correlates of patterns of relative abundance of sharks at dive sites.This project was conducted under the Republic of Palau Marine Research Permit no. RE-09-26 and the Koror State Marine Research Permit no. 10–204. Shark tagging in 2011 was also conducted under UWA animal ethics permit no. RA/3/100/975, in adherence to provisions contained within the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Participants of the shark counts were aware of the use of these data for research and provided written consent for the use of the data collected. This project has been assessed as exempt from ethics review by the Human Research Ethics Office of the University of Western Australia (protocol code: RA/4/1/6457).Carcharhinus amblyrhynchos) and whitetip reef sharks (Triaenodon obesus) Palau supports a dive industry consisting of approximately 20 tourism businesses that use mainly small speed boats to transport tourists to local reef sites for two to three dives per day. In 2010, it was estimated that approximately 41,000 tourists visited Palau to engage in dive activities, of which approximately 8,600 visited the country specifically to dive with sharks Our dataset consisted of counts of sharks sighted during dives by dive guides who worked as employees of a dive tourism business. Standard questionnaires were completed after the day trips by dive guides from October 2007 to November 2012. A total of 62 dive guides recorded information for 2,360 dives at 52 dive sites in Palau. Each questionnaire contained information on the dive site visited, date, species and counts of individual sharks sighted by the guide. Dive guides estimated the depth of the divers during the sightings, current strength , visibility (in meters), and recorded dive time and number of tourist divers in the group. Each completed questionnaire provided observations from a single dive guide.Most of the dive guides participating in the study were local residents familiar with the identification reef shark species in Palau. Guides were instructed to report the total number of individual sharks of each species observed during the entire dive. We also instructed the dive guides to be conservative with counts, observing features that could permit individual identification  and reduce the potential for repeated counts. An office staff member was responsible for the administration and management of the survey, encouraging dive guides to return questionnaires regularly. This staff member was also trained to enter data and maintain the dataset. To promote engagement and consistency in data collection, we also established an annual event to provide feedback to the participants, where the dive guides who collected data regularly would receive small rewards. During these events, the researchers involved in the project would also provide lectures to the dive community, where updates on relevant issues and results obtained from the dataset were presented.We used passive acoustic telemetry to monitor the attendance of resident grey reef sharks at four key dive sites , known to host predictable aggregations of sharks . An arra−1) of sharks observed by dive guides as the mean value of all dives in a given day at the same site. Given daily variation in diving activities, we also calculated a mean daily abundance for each calendar week.We limited our analysis to data from experienced dive guides, defined as guides who participated for at least three of the five years during which the study was undertaken, who had returned a minimum of 100 questionnaires. In order to obtain a reasonable representation of questionnaires for each calendar month, our analysis focused on the dive sites that also yielded a minimum of 100 questionnaires. We estimated the daily relative abundance , in which case we summed the number of tourist divers reported by each guide as a measure of the total potential influence on the sharks. We also used linear regression to analyse the relationship between the mean depth of the tagged sharks and 1) the number of tourist divers reported to be in the water during a dive and 2) the mean depth of these divers.t-test for slopes to compare the regressions of monthly observations by dive guides with the mean number of grey reef sharks detected using telemetry. Monthly estimates of relative abundance observed by dive guides and numbers of sharks detected by telemetry were calculated by averaging the daily values across a given month. For the regression, we included only telemetry values that had two or more corresponding observations by dive guides. The explanatory variable “month” was sine-transformed to account for cyclical variation in abundance of sharks. We also fitted circular regressions to investigate patterns of seasonality in the mean monthly relative abundance of sharks observed by dive guides at the selected study sites. This analysis was performed for all sharks combined but also separately for grey reef and whitetip reef sharks.We used circular regression ΔAIC (difference between AIC of a given model and best fitted model) and wAIC (weighted AIC) to select the models with best fit n = 148) from the dive guides.We used multiple linear regression to examine the influence of environmental factors on the relative abundance of sharks observed by dive guides. Our response variable was the log-transformed daily abundance of sharks  averaged per week. Explanatory variables included in the models were: year, current strength, temperature, visibility, number of tourist divers and moon phase. We applied the Akaike’s Information Criterion (AIC) test statistics including AIC, All analyses used the R statistical software Our final dataset, filtered to only include records of the most frequently visited dive sites and observations of selected guides (those returning more than 100 questionnaires), consisted of data for 1,252 dives (53%) that were collected by 24 dive guides (39%) at five dive sites (10%)  over a period of five years. These dive sites are known to be aggregations or hotspots of charismatic megafauna, including reef sharks. The total number of dives at each site varied from 118 at Siaes Corner to 388 at Blue Corner, with an overall mean dive time of 57±0.22 min and a mean dive depth of 16±0.18 m .Carcharhinus melanopterus) were also sighted frequently (14% of dives) but with relative abundance of 0.3±0.1 sharks per dive. The other species of observed sharks were recorded very infrequently (2% of dives) and in low numbers (0.02 sharks per dive)  . Shark rer dive) .Paired sets of abundance estimates provided from dive guides and acoustic detections were available for 406 dives . For the2R = 0.74, p<0.001) between the mean daily relative abundance of grey reef sharks observed by dive guides and the number of tagged individuals detected by telemetry , there was some divergence between the two data sets during March and April . Mean values varied from 6.7±0.3 sharks detected when up 10 tourist divers were present to 7.2±1.1 when 40 or more tourist divers were present. Similarly, there were no significant relationships between the mean depth of tagged grey reef sharks and the number of tourist divers present during the dive (p = 0.31) or the mean depth of the tourist divers (p = 0.44).The linear regressions showed no significant relationship between the number of grey reef sharks detected by telemetry and the number of tourist divers present during the dives (p<0.001), 64% of the variation in relative abundance for grey reef (p = 0.003) and 73% for whitetip reef sharks (p<0.001).Grey reef and whitetip reef sharks were present at the monitored sites throughout the year however, there was seasonal variation in the number of individuals of both species. Lower values of monthly relative abundance occurred in October and November, with means of 8.1±0.4 for grey reef sharks and 4.2±0.2 for whitetip reef sharks. Between March and April, the relative abundance of sharks was higher with monthly means of 12.4±1.2 and 6.5±0.7 for grey reef and whitetip reef sharks respectively . There w2R = 0.18, p<0.001; 2R = 0.14, p<0.001), grey reef  and whitetip  reef sharks, while temperature displayed a negative linear relationship with relative abundance of all sharks  and grey reef sharks   . Year, v = 0.03) .2R value indicated a strong positive relationship between the two metrics, with increases of daily relative abundance of grey reef sharks observed by dive guides matched by a corresponding linear increase in numbers of grey reef sharks detected by telemetry. Data generated by professional dive guides have great potential for providing estimates of relative abundance Our analysis of data generated by dive guides suggests that citizen science initiatives can provide estimates of the relative abundance of reef sharks that are consistent with the estimates from long-term telemetry. Patterns in the relative abundance of grey reef sharks as reported by dive guides and numbers detected by telemetry at the monitored sites followed very similar trends at both daily and monthly scales. The high Although there was a close correlation between dive guide counts and telemetry results, to some extent this may have been a function of the particular circumstances of our study. We used experienced dive guides to gather data and the addition of tourist divers and less experienced guides might have reduced the strength of the relationship. While there is some evidence that diving experience may not necessarily be positively correlated with count accuracy To be successful, monitoring programs need to have well defined objectives and standardised protocols that are effective in collecting accurate data of the target species. In our study, this was possible through data collection by recreational dive guides. However, alternative strategies might be necessary for species of sharks where diving conditions do not allow for underwater visual surveys by recreational divers, such as turbid coastal waters or waters below the limits of recreational diving depths. For such areas, programs designed to collect information from recreational catch and release fishers Our study suggests that programs that use dive guides to monitor shark relative abundance in coral reefs could be a cost-effective alternative to traditional science-based surveys. However, this is not to imply that the set up and maintenance of such monitoring programs do not involve considerable logistics. We found that the success of our long-term program relied in part on sufficient resources for personnel, training and management of datasets to ensure data quality. On-ground leadership, encouragement and feedback was required to ensure that participants remained engaged in the program and maintained regular sampling throughout the study, as has been highlighted in other studies Dive guide data revealed a seasonal cycle in relative abundance of sharks, with peaks from March to June and the lowest values recorded in October and November. Dive guides also recorded relatively low numbers of both grey and whitetip reef sharks in May, a pattern that was more pronounced for the former species. This decrease in relative abundance coincided with an increase in water temperatures from around 25°C in previous months to a peak of 29°C in May Short-term  changes in relative abundance of sharks at the monitored sites were correlated with current strength and temperature. Current strength appeared to be the more important driver of variations in relative abundance of whitetip reef sharks, with mean relative abundance steadily increasing with the current flow. For grey reef sharks, our models identified a combination of both current strength and temperature as the principal drivers of relative abundance. These sharks were three times more abundant during dives when currents were strong and relative abundance was also higher when water temperatures decreased from 30°C to 28°C. Earlier studies have noted the association of reef sharks with areas of strong current flow, typically around reef promontories, channels and passes http://oceantrackingnetwork.org/about/ocean). The development of arrays of listening stations by programs such as these offers the opportunity to validate citizen science initiatives, since both often target the charismatic megafauna that inhabit coastal systems. Such comparative studies will be necessary to ensure that the data that citizen science initiatives provide to management and conservation strategies in marine systems is credible, precise and reliable.Some scepticism surrounds the use of citizen science due to potential problems with the quality of data generated by untrained observers