Nestor notabilis). Kea have undergone a long-lasting persecution between the late 1800s to 1970s where an estimated 150,000 kea were culled under a governmental bounty scheme. Kea now number 1,000–5,000 individuals in the wild and it is likely that the recent population decline may have reduced the genetic diversity of the species. Comparison of contemporary (n = 410), historical (n = 15) and fossil samples (n = 4) showed a loss of mitochondrial diversity since the end of the last glaciation  but no loss of overall genetic diversity associated with the cull. Microsatellite data indicated a recent bottleneck for only one population and a range-wide decline in eN dating back some 300 – 6,000 years ago, a period predating European arrival in NZ. These results suggest that despite a recent human persecution, kea might have experienced a large population decline before stabilizing in numbers prior to human settlement of New Zealand in response to Holocene changes in habitat distribution. Our study therefore highlights the need to understand the respective effects of climate change and human activities on endangered species dynamics when proposing conservation guidelines.The impact of population bottlenecks is an important factor to consider when assessing species survival. Population declines can considerably limit the evolutionary potential of species and make them more susceptible to stochastic events. New Zealand has a well documented history of decline of endemic avifauna related to human colonization. Here, we investigate the genetic effects of a recent population decline in the endangered kea ( Ovibos moschatus) were best explained by climate and associated habitat change [Mammuthus primigenius) appears to have been caused by the concomitant effect of over-hunting and climate change [Genetic diversity is an essential component of both the short and long-term persistence of species. It can be regarded as the basis for evolutionary potential  and favot change  while the change . It is tIn order to distinguish anthropogenic from natural drivers of population decline, one can refer to pre-human fossil records of the distribution of species. However, such data is often lacking or incomplete, leading to a potential bias in the estimation of population size and oversimplification of population dynamics, as critically addressed by Rawlence et al.  and AlleDNA-based approaches using contemporary samples only are routinely used to detect population declines –16 but cNestor notabilis), an endemic alpine parrot prehistorically present in both the North and South Islands of New Zealand [The avifauna of New Zealand evolved in the absence of mammalian predators , 21 and  Zealand –32 and n Zealand . Its inq Zealand , 35, opp Zealand –38 and m Zealand –41 promp Zealand  while a  Zealand  but it i Zealand . However Zealand . The estPrevious results have shown that despite its strong flying capabilities, the kea is subdivided into three distinct genetic clusters and that this population structure is most likely the result of postglacial recolonization processes but not a recent human-induced fragmentation . Kea popHere we test whether kea have experienced pre-human population declines or if the species started to decline only recently because of human persecution. We first assess changes in genetic diversity over time using microsatellite and mitochondrial data comparing contemporary, historical and fossil samples. Second, we look for a signature of ancient and recent bottlenecks using conventional methods for microsatellite data. We also look for a signature of population decline using coalescent-based Bayesian methods and estimate its timing and magnitude. Due to the magnitude of the reported recent population collapse , we expect to find extinct haplotypes and alleles among historical and fossil samples that are not present in contemporary samples and also a significant signature of a recent bottleneck.Fieldwork was conducted under the approval of the University of Otago Ethics Committee (No. 74/09) and DoC . No permits were required to sample fossil material from museums. Permission to sample museum specimens was obtained by NJR from individual institutions.We utilized a contemporary dataset from birds caught between 2010 and 2012 comprising 91 mitochondrial (mtDNA) control region (CR) sequences and 410 nuclear microsatellite genotypes (17 microsatellite loci) as described in Dussex et al. . AdditioAll ancient DNA (aDNA) extractions and PCR set up was carried out at the University of Otago in a purpose built aDNA laboratory physically isolated from other molecular laboratories . Strict Historical samples were genotyped for 17 microsatellite loci  using cyThe mtDNA CR from historical skins was amplified and sequenced using four primer pairs (producing ∼250 bp fragments including primers) , S1 Fig.2 (Life Technologies), 1 x Gold Buffer II (Life Technologies), 2.5 mM dNTPs (Bioline), 250 nM each primer, 1.25 U of AmpliTaq Gold DNA Polymerase (Life Technologies), and 2 µL DNA. Unsuccessful PCRs were repeated with 2 U AmpliTaq Gold DNA Polymerase and 4 µL DNA or 2 µL 1:10 DNA. PCR thermocycling conditions consisted of 94°C 9 min, 60 cycles 94°C 30 s, 50°C 45 s, 72°C 1 min, followed by a final extension of 72°C 10 min.Fossil samples were amplified and sequenced using seven primer pairs (∼150 bp fragments including primers) , S1 Fig.PCR products were run on a 2% 1 x TAE agarose gel. PCR products were purified  by incubation at 37°C for 30 min and 80°C for 15 min), and sequenced bi-directionally from independent PCR products using Big Dye Terminator technology and an ABI 3730xl. When an inconsistency between sequences from an individual was observed due to DNA damage (C-T and G-A transitions), additional PCRs and bi-directional sequencing were conducted, and a majority rule consensus applied to the four independent PCRs .Consensus sequences were obtained from the alignment of fragments made in Geneious 5.6.3 .P-value was determined using a Markov chain method following the permutation algorithm of Guo and Thompson [For microsatellite markers, we checked for scoring error due to stuttering, large allele drop-out, and null alleles using MICRO-CHECKER 2.2.3 . Tests fThompson . The sigThompson .Indices of genetic diversity were calculated for both contemporary and museum samples. To assess the level of genetic variation in microsatellites, FSTAT was used to calculate means of allelic richness corrected for sample size , observeh), nucleotide diversity  were computed in ARLEQUIN 3.5 [Mitochondrial diversity indices, such as number of haplotypes , numberQUIN 3.5  for contWe then constructed a temporal network to look at mitochondrial variation in kea through time using the R script TempNet v1.4. .K was estimated from 25 iterations of K values ranging from 2 to 4 (as the most likely K selected was 2 and 3 for contemporary data). The results obtained with STRUCTURE 2.2 were used as input in STRUCTURE HARVESTER [K-value the individuals‚ assignment coefficient (q) for each genetic cluster were averaged over the 10 runs with the lowest posterior probability using CLUMPP software [In order to compare historical and contemporary population structure, we examined the assignment of historical samples to contemporary populations. The rationale was that if the contemporary structure corresponded to the historical one, museum samples should cluster with samples of the same geographical origin. On the other hand, if the contemporary structure is the result of genetic drift caused by the recent population decline, historical samples should not cluster well with individuals of the same geographical origin or have more admixed membership. We first used STRUCTURE 2.2 , 66 to eARVESTER  to produsoftware . Finallysoftware .For all analyses described below, we first considered the three genetic clusters  identified in Dussex et al.  Fig. 1aFig. 1a aM-ratio test [M-P-Val and Critical_M [sp = 0.9 and sp = 0.78, 3.5 steps for multi-step mutations and pre-bottleneck eN values ranging from 500 and 5000. The mutation rate remained constant at μ of 5 × 10-4 [M-ratio was also calculated using only polymorphic loci to account for the upward bias in the M-ratio estimate, as implemented in ARLEQUIN 3.11 [eN through time, we used the coalescent-based Bayesian method of Storz and Beaumont [HN at T years in the past to the current CN, where the data are allele frequencies for unlinked microsatellites. These loci are assumed to have independent genealogies and to be evolving according to the strict single-step mutation model. Given the demographic parameters  and the mutation rate (μ), the genealogical history of the sampled data can be described using coalescent theory, and the posterior probability distribution of the genealogical and demographic parameters is estimated using Markov chain Monte Carlo (MCMC) simulations. We performed five simulations per dataset  with 2 × 109 iterations with parameter values recorded every 1 × 105 iterations resulting in 20,000 records. Prior distributions were set to the parameters of simulation 1  the heterozygosity excess method implemented in the software BOTTLENECK v. 1.2.02 . This aptio test  using M-itical_M , which i5 × 10-4 . The M-rUIN 3.11 . SecondlBeaumont  implemenlation 1 . After ceN (HN) was uniformly distributed between 10 and 2 × 105, the upper bound encompassing 150,000 kea reported being killed between the 1870s and 1970s [eN (CN) was uniformly distributed between 10 and 104. These values included the current and conservative population estimate of 1,000 to 5,000 birds [eN was uniformly distributed through time for 0 to 1,000 generations and bounded between 10 and 2 × 105 individuals. For the two models, a generalized stepwise mutation model [-5 and 1.00 × 10-3 substitutions/generations [Finally, two different demographic scenarios were compared within an ABC framework  using DInd 1970s . The pri00 birds  and alsoon model  was implerations –88. The erations , 90. TheeN through time and demographic scenarios are available as Further details on tests of bottleneck detection using moment-based methods, variations of For museum samples, two microsatellite loci (Nmed31 and Strhab43) failed to amplify. After Bonferonni correction, no significant departure from Hardy-Weinberg equilibrium was observed and no tests for linkage disequilibrium were significant. Signs of null alleles were detected in single loci in the Kahurangi (Strhab43), Arthurs Pass (Nnot8) and Mt Cook (Nnot38) populations.When comparing microsatellite genetic diversity among our historical and contemporary datasets, no extinct alleles were identified within the 15 skin samples . AllelicConsensus sequences for the mtDNA CR for historical and fossil samples varied between 245 and 616 bp . Three nq > 0.85) to their putative cluster of origin when considering two distinct genetic clusters . Individual NMW 12.211  showed again shared membership with q-values of 0.44, 0.28 and 0.28 for the north, central and south cluster respectively. Samples NMW 12.208 and NMW 49.760, putatively originating from the north cluster, also had admixed origins with q-values of 0.64 and 0.57 to the central cluster. However, those two samples had very high q-values of 0.94 and 0.88 to their cluster of origin when considering a scenario of two clusters. It is worth noting that because the distinction between the north and central cluster is not very strong and as gene flow is marked between these two clusters, shared membership between those two clusters is perhaps not surprising.When comparing historical and contemporary population structure using the Bayesian algorithm implemented in STRUCTURE, 14 individuals out of 15 clustered well with contemporary individuals from the same sampling locations. Of the 15 historical samples, 13 had high membership coefficients . Depending on the parameter set used, M-ratio values ranged from 0.76 to 0.86 (CM) that represents the threshold under which a bottleneck is supported. We only found evidence of a bottleneck when using a θ value of 1, corresponding to a pre-decline eN of 500 and only for the north and central clusters.When using the  to 0.86 . These vHN) than contemporary (CN) effective population sizes for both demographic models . Bayes factor values were >10 for all clusters and models indicating strong evidence for a population decline (CN/HN) clearly supported a population decline. Using an exponential model of population size change, the 90% highest posterior density (HPD) of the ratio r was of 0.0019–0.052, 0.053–0.068, 0.019–0.031 and of 0.0003–0.017 for Historical Canterbury, the north, central and south cluster samples respectively. These r values indicated that the contemporary eN is less than 10% of the historical HN.Historical Canterbury and all three contemporary genetic clusters examined showed larger historical ( decline . The ratHN) for the exponential model were historical-CanterburyN = 3,995, northN = 4,493, centralN = 3,823, and southN = 4,620 compared to modal values for contemporary effective population sizes (CN) of 131, 279, 106, and 28 respectively   , Fig. 5.HN and CN for the linear model were similar to the exponential model  in contemporary samples was probably due to the paucity of samples available to our study with robust locality data. However, aDNA data shows two haplotypes and three new substitution sites found among fossil samples suggesting that a loss of genetic diversity might have occurred prior to the cull of the late 1800s. A larger sample size representing a wider time period between the Otiran Glacial to European arrival in New Zealand would be required to estimate the timing of such a decline and the extent of genetic diversity loss with more accuracy.It is possible that our historical samples were collected after the peak of the government cull had already occurred. This is particularly true when considering the nature of the bottleneck. If for instance, the cull was rapid, of great intensity and resulted in populations being extirpated locally, it is quite likely that our historical sampling was not able to capture the effect of the bottleneck. If on the other hand, the cull lasted over a longer time span (> 10 years), we would be more likely to detect a loss of genetic diversity with our samples, as the commonly accepted date for the start of the government cull is 1860 . HoweverEven if we cannot exclude the possibility of populations being extirpated locally in areas of high conflict with farmers, it is likely that some other populations could have persisted in more remote areas without farming and remained unaffected by the cull, thus maintaining historical levels of genetic diversity. Although this has never been reported, because of the dispersive , 96 and Assignments of historical samples scored at 15 microsatellite loci did not suggest a different historical population structure, with most historical samples assigning well to their putative cluster of origin. This gives further support to the contemporary structure not resulting from genetic drift caused by the recent human-led population decline .The demographic histories of endangered and fluctuating populations is often complex. While conservationists often focus on the effect of human pressures on natural populations, the identification of a single decline event can be difficult, as the genetic variation of natural populations is influenced by multiple events varying in timing and nature. In this respect, kea is a good example as it has presumably been influenced by Pleistocene glaciations, climate change at the onset of the Holocene (11.6 kya), human colonization of New Zealand and by recent European persecution as well. Also, the usual rarity of kea fossils throughout New Zealand contrasting with a high abundance of remains in some sites  possiblyM-ratio method did not detect any bottleneck while a signature of a bottleneck was detected in the central cluster using the heterozygote excess method and the most appropriate TPM model with 90% stepwise mutations [Moment-based methods of bottleneck detection yielded contrasting results. The utations . As theoutations . Decreasutations , 98. Morutations . Often, utations . Thus, putations ), an aveThe detection of a bottleneck signal in the central cluster is consistent with the fact that the McKenzie area was an easily accessible area and one of the first regions where intensive farming occurred –102. TheThe modeling approaches implemented in MSVAR and DIYABC were the only methods that consistently identified population declines in the historical and contemporary samples examined. While mode estimates were slightly different, posterior distribution of parameters were overlapping among the two methods. However, the timing of this decline was not consistent with the reported cull of the late 1800s. Rather, our analysis identified a decline dating back a few hundreds of years ago (south cluster) to up to 6,000 years ago  and suggests that kea could have experienced a population decline prior to human arrival in the northern part of its range at least. The finding of kea fossils from the Pleistocene in some areas of the South Island where kea are now absent such as central and eastern Otago , coastalPachyornis australis) changed altitudinal, longitudinal and latitudinal ranges through the Late Quaternary in response to alterations in the distribution of suitable habitat [Climate warming at the end of the last glacial period  induced a change in environment in the South Island of New Zealand with the expansion of podocarp forest first , 109 and habitat . The fin habitat  and appa habitat , 113 may habitat  and newl habitat  suggest  habitat , 116. HoAnother important aspect to consider is the possible impact of pre-European Maori on kea population dynamics. Estimates of the timing of decline predate Maori arrival in New Zealand circa 1280 A.D., ) for the0 A.D.,  Arctocephalus gazella) during the 1800’s suggests that ABC approaches are powerful enough to detect recent demographic declines [Our results do not exclude the possibility of a recent 1800s to 1970s) human-induced population decline in kea and are consistent with the conclusion of Girod et al.  and the declines . The que70s humandeclines , 131–136First of all, the detection of a bottleneck is highly dependent on the amount of genetic diversity present before the decline. If kea populations declined before human arrival as supported by the results of MSVAR and DIYABC, it is very likely that the inherent paucity in genetic variation following this ancient decline  could have precluded further genetic loss , making eN (10- to 1000-fold) have been evaluated . If the estates , it is p estates , with ne estates , 141. Su estates , 143.Thirdly, it is plausible that the cull was not homogeneous over the whole range of the species and that population density could have been significantly reduced mainly close to sheep stations, while populations in remote areas might have survived the cull . Also, bFinally, as has been suggested for long-lived species such as white-tailed eagle , HawaiiaAlthough genetic signatures of historical contraction were detected, no recent bottleneck consistent with the report of an important cull was noted. This suggests that the cull was not strong enough to leave a genetic signature or that a combination of the factors discussed above reduced the probability of bottleneck detection. The discovery of additional mitochondrial diversity among fossil samples supports an ancient and possibly non-anthropogenic decline in kea, but further study of fossil samples is required to determine with more certainty the importance of habitat change during the Holocene on kea population dynamics. In a context of global climate change, our findings suggest that kea abundance could be further reduced by increasing temperatures either through associated habitat change, or through the effects of increased predator pressure in alpine habitats. While we cannot rule out the possibility that kea was once present in the lowlands and that it is now an alpine relict species because of habitat loss, persecution and predation, our study highlights the need to examine histories of endangered species and try and understand the respective anthropogenic and non-anthropogenic effects on population dynamics when proposing conservation guidelines.The lack of detection of a recent genetic bottleneck associated with a documented human-induced demographic decline further highlights the need to interpret results of tests for population bottleneck with great caution. As failure to detect a genetic bottleneck does not exclude the possibility of a recent population contraction, our results further highlight the need to consider historical and fossil genetic diversity and effective population size estimates to develop efficient conservation recommendations for species of conservation concern.While the species has been fully protected since 1986 , the intS1 Supporting Information(PDF)Click here for additional data file.S1 FigThe left hand side of the figure represents the 5‚ end of the sequence.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableeN (CN), historical eN (HN), mutation rate scaled in terms of contemporary population size (θ), and time (T). scale.Columns 3–6 show the starting values for the mean and variance of the prior distributions. Columns 7–10 show the means and variances (and their means and variances) of the hyperprior distributions. Parameters listed are generation interval (g), contemporary (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableIndividuals with unusual assignments and referred to in the text are in bold.(PDF)Click here for additional data file.S1 Data(TXT)Click here for additional data file.S2 Data(TXT)Click here for additional data file.


RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments (‘tanks’) formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24), compared to only 14 phyla in the other (Wg25). Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size , and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2). Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length), genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts. Because epiphytic bromeliads virtually lack absorptive roots, these tanks provide the plants with water and nutrients released by heterotrophic decomposition of impounded debris Due to their small size, defined boundaries, and relatively simple faunal communities, bromeliad catchments are ideal for the investigation of whole ecosystem complexity, a notoriously difficult task for lakes and other large, more complex freshwater habitats With a new appreciation for the intertwined worlds of metazoans and microbes, it is an exciting prospect to explore the possibility of examining the entire community (from viruses to eukaryotes) with a single technique. The current state of knowledge with regard to plant-associated aquatic habitats is based on the counting of animals or protozoa or, in the case of bacteria and archaea, based on 16S rRNA ribosomal surveys ex. . There h22.1Werauhia gladioliflora specimens in La Selva Biological station, a wet  lowland neotropical reserve located in northeastern Costa Rica . Samples were collected in June 2012, from two plants, neither of which was in bloom, growing at heights of ~ 2.0 m from the ground. The bromeliads had tanks of ~ 10 cm depth , and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall. Since previous studies revealed strong correlations between bacterial diversity and bromeliad tank pH, which can range from pH 3 to 7 Bromeliad tank debris and water were collected from two 2.2Zap Wipes (Ambion). The RNA PowerSoil Total RNA Isolation Kit (MoBio Laboratories) was used for bulk extraction of RNA, with slight protocol modifications to extract sufficient quantities of nucleic acid for metatranscriptomic analysis (~ 90–120 ng μl− 1). Modifications included the use of freshly prepared low-pH phenol:chloroform:isoamyl alcohol  for nucleic acid extraction (PowerSoil Step 5), a room temperature incubation for secondary debris precipitation (PowerSoil Step 9), gentle encouragement of fluid flow through the RNA capture column using a syringe (PowerSoil Steps 16–19), and an overnight nucleic acid precipitation step (PowerSoil Step 20). Successful nucleic acid extraction was verified by NanoDrop UV spectrophotometry, ensuring sufficient RNA concentrations and purity. RNA extraction was immediately followed by removal of contaminant DNA from the extracted RNA, using TURBO DNA-free DNase Treatment and Removal Reagents (Ambion). A treatment of 4 Units TURBO DNase per 100 μl RNA was applied, with a 20 min 37 °C incubation for sufficient DNA contaminant removal.Nucleic acid extraction was performed at Occidental College, using RNase-free materials (tubes and pipette tips) on workstations treated with RNase2.3Total extracted RNA was submitted to Otogenetics Corporation  for RNA-Seq analysis. Briefly, 9–12 μg of total RNA was subjected to rRNA depletion using the RiboZero Meta-Bacteria kit (Epicentre Biotechnologies) and cDNA was generated from the depleted RNA using the NEBNext mRNA Sample Prep kit (New England Biolabs). Illumina libraries were created from the cDNA using NEBNext reagents (New England Biolabs). The quality, quantity and size distribution of the Illumina library products were determined using an Agilent Bioanalyzer 2100. The libraries were then submitted for Illumina HiSeq2000 sequencing according to standard operations.2.4− 5 against the Integrated Microbial Genomes (IMG) system v.4.0 http://github.com/minillinim/sam2fpkg; Paired-end 100 nucleotide (nt) reads were generated and checked for data quality using FASTQC (Babraham Institute). For removal of ribosomal RNA sequences  that were not subtracted via RiboZero depletion, SortMeRNA software 3Poplar biomass reactors, enrichment cultures), and associated with eukaryotes  was performed on the water catchments formed by tropical bromeliads from a Costa Rican rainforest. The community associated with Costa Rican tropical bromeliads was deduced through affiliation of non-ribosomal transcript sequences with particular taxa in public databases. Overall results from two bromeliad tanks indicate the presence of genes from 25 bacterial phyla (including 4 classes within the Proteobacteria), 7 archaeal orders, 21 metazoan classes (including 6 orders of insect), 8 fungal classes, as well as viruses and transcripts related to those recovered from environments, including plant biomass reactors, freshwater lakes, and the rhizosphere , Table 3Oophaga pumilio, which commonly uses bromeliad tanks as nurseries Bromeliad eukaryote communities are abundant and diverse, typically dominated by annelids and larvae of aquatic insects, including many species of Coleoptera and Diptera Transcripts from numerous bacterial phyla (including 4 classes within the Proteobacteria), were recovered from the bromeliad tanks, comprising 25 phyla in Wg24 and a subset of these (~ 14 phyla) from Wg25 . Both taThe metatranscriptome data for archaeal presence and diversity also followed the same pattern as recovered from previous DNA-based studies. Transcripts with archaeal taxonomic assignments were present at similar levels in both bromeliad tanks, comprising ~ 7% of the transcript reads. Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25 , Table 242−) and hydrogen peroxide metabolites, and small heat shock proteins, were almost exclusively detected in Wg24. Together, such differences in genes relevant to cellular defense and response mechanisms perhaps reflect the high-stress, low-pH environment in this particular bromeliad catchment.Bacterial communities play essential roles in energy and matter flow in aquatic microcosms, through detrital mineralization and nutrient release. Of particular interest is the microbial role in nitrogen and carbon-cycling in bromeliad tanks, due to the abundance of carbon-based leaf litter, animal carcasses, and feces that constitute the organic detritus. Despite their status as one of the most abundant aquatic ecosystems in the world, and their classification as true microcosms , remarkably little is known about the function of bromeliad-associated microbial communities 52 on the planet could result in the loss of biodiversity within bromeliad tanks over time. In order to confirm this hypothesis, it will be necessary to conduct a more thorough analysis of the structure and function of organisms in these habitats.Freshwater tropical ecosystems, including bromeliad tanks, support disproportionate levels of biodiversity, especially microorganisms and invertebrates, relative to their spatial coverage, and are expected to be highly vulnerable to habitat perturbations resulting from climate change Number of bacterial species represented in metatranscriptomic libraries from two bromeliad tanks. For Wg25, with a pH of 6.2, species number for each phylum was multiplied by 1.3 from  to accouThe following is the supplementary data related to this article.


Important genotypic variations were observed both at the plant and fruit levels and variations in fruit and leaf traits were found not to be correlated. Overall, the WD treatments were at the origin of important osmotic regulations, reduction of leaf growth, acclimation of photosynthetic functioning, notably through an increase in the chlorophyll content and in the quantum yield of the electron transport flux until PSI acceptors (J0RE1/JABS). The effects on fruit sugar, acid, carotenoid and AsA contents on a dry matter basis ranged from negative to positive to nil depending on genotypes and stress intensity. Three small fruit size accessions were richer in AsA on a fresh matter basis, due to concentration effects. So, fruit quality was improved under WD mainly through concentration effects. On the whole, two accessions, LA1420 and Criollo appeared as interesting genetic resources, cumulating adaptive traits both at the leaf and fruit levels. Our observations show that the complexity involved in plant responses, when considering a broad range of physiological traits and the variability of genotypic effects, represent a true challenge for upcoming studies aiming at taking advantage of, not just dealing with WD.Episodes of water deficit (WD) during the crop cycle of tomato may negatively impact plant growth and fruit yield, but they may also improve fruit quality. Moreover, a moderate WD may induce a plant “memory effect” which is known to stimulate plant acclimation and defenses for upcoming stress episodes. The objective of this study was to analyze the positive and negative impacts of repeated episodes of WD at the plant and fruit levels. Three episodes of WD  followed by three periods of recovery (“WD treatments”), were applied to the eight parents of the Multi-Parent Advanced Generation Inter-Cross population which offers the largest allelic variability observed in tomato. Predawn and midday water potentials, chlorophyll  S. lycopersicum L. have been selected for their higher rate of SNP differences among 360 others tomato accessions (Table 1). RIL populations have been developed from the MAGIC TOM population; they are used for genetic studies and breeding programs , WD has been shown to improve fruit sweetness, flavor and fruit size when applied during the stage of cell division and rapid endocarp hardening , the impact of WD may differ according to the stage of fruit development at the time of WD  were assessed before and after each WD period. Fruit quality  was measured on two batches of fruits which developed at different periods of the WD treatments.In the present study, our objectives were: (i) to provide an overview of the beneficial and detrimental impacts of WD treatments at the plant and fruit levels, (ii) to assess the genetic variability of these responses, and (iii) to reveal interesting traits of plant acclimation to WD, which could be used in future breeding programs. The work was performed on the eight parents of the MAGIC TOM population. Plant and fruit responses were measured during three successive periods of WD of increasing intensity followed by RP (“WD treatments”), which is clearly an original feature of the present study. Moderate WD was achieved by reducing the supply of water by about 38% during the first WD period, 45% during the second WD period and 55% during the last WD period when compared to control plants. Predawn water potential, stem water potential at midday, chlorophyll content and chlorophyll Table 1) encompass four large-fruit accessions , and four cherry accessions . All are indeterminate tomatoes. LA1420 seeds were provided by the Tomato Genetics Resource Centre, Davis, CA, USA. Cervil and Levovil seeds were provided by Vilmorin Seed Company. The other accessions were supplied by the Tomato Genetic Resource Centre of INRA, Avignon, France . The control irrigation met the evaporative demand. The WD treatments corresponded to three phases of WD of increasing intensity  followed by three RP . Each WD and RP period lasted approximately 15 days. During recovery, WD was first reduced by half for 2 days and then brought back to the control level. Climate conditions  in the glasshouse were recorded every minute and data were averaged hourly throughout the experiment. Average day and night temperatures were stable until 12 June, i.e., until RP2 . Temperatures increased during WD3 and RP3  due to seasonal effects. At the same time, the daily light integral increased in the glasshouse (from 5 to 11 MJ m-2 day-1), whereas average day and night humidity decreased (from 57 to 37% at daytime and from 80 to 60% at night) from WD1 to RP3. Plants were grown in 4 L pots filled with compost  distributed in two rows (control and stressed plants) of 80 plants each (10 plants per genotype) surrounded by border plants. The density was 1.3 plant m-2.The experiment was conducted during spring and summer 2012 in a glasshouse located near Avignon, France. Irrigation was calculated according to daily ETP , taking soil) was measured daily with Watermark probes  placed at the opposite of the drippers . One probe per treatment per genotype was used and connected to a data logging system . Results were converted into MPa using equation 8 given by soil was rather stable until the WD3 period (-0.04 ± 0.01 MPa) and decreased during RP3 due to plant development (–0.05 ± 0.01 MPa). On the contrary, the soil water potential decreased to –0.09 ± 0.02, –0.29 ± 0.06, and –0.43 ± 0.04 MPa during WD1, WD2, and WD3, respectively. Nutrients were applied daily using a commercial solution .The soil water potential (ΨTable 1) in order to obtain comparable levels of competition among fruits for all genotypes . No chemical treatment was applied and Macrolophus caliginosus were released throughout the culture to protect plants from whiteflies.Flowers were pollinated three times a week using an electrical bee. Trusses were pruned according to final fruit weight (predawn and Ψmidday) at the end of each WD and RP period . Leaves were bagged the day before, at nightfall.Stem water potential was measured using a pressure chamber  at predawn and at solar noon (Ψa was measured on dark adapted leaves (30 min.) using a fluorimeter . Dark-adaptation allowed the PSII electron acceptor pool to be gradually re-oxidized to a point where all PSII reaction centers are capable of undertaking photochemistry. Measurements were carried out with an induction period of 1 s and leaves were illuminated to a light level of 3000 μmol photons m-2 s-1. The measurements were carried out on non-senescent mature leaves, at around 11 a.m. during the last three days of each period . The maximum photochemical efficiency of light harvesting of PSII (FV/FM), the PI of J0RE1/JABS; Fluorescence of chlorophyll n ≥ 5). The last day of the WD3 period, two non-senescent mature leaves, which were initiated during the WD treatments, were harvested on each plant and their specific leaf area  was measured. Leaf area was measured with a Planimeter  and leaf dry weight was measured after seven days at 70° C in a ventilated oven.Plant leaf number and leaf length were measured at the end of each period , immediately frozen in liquid nitrogen and stored at –80°C, for biochemical analysis.Figure 1A). Fruit setting and abortion were recorded on the first eight trusses of each plant.The dates of anthesis of the successive trusses were recorded on all plants during the whole experiment. Thus, fruits could be pooled according to the developmental stage at the time of the WD treatments. The first pool of fruits (S1) was harvested during RP2, whereas WD1 occurred during the cell division period and WD2 during the cell expansion period. For the second pool of fruits (S2), WD2 occurred during the cell division period and WD3 during the cell expansion and ripening periods (All measurements were performed on red mature fruits (breaker stage plus at least five days) harvested on five plants per genotype and per treatment . Fruits were harvested at 11 a.m., avoiding the first proximal and the last distal fruits of each truss. Fruit diameter and fresh weight of all fruits were measured. Then fruits were frozen in liquid nitrogen and kept at –80°C prior to biochemical analysis of pericarp soluble sugars, organic acids, carotenoids, AsA, starch (only for Cervil), and DM contents. For biochemical analyses, fruits were pooled into five batches of three to five fruits for each treatment and genotype.Soluble sugars and organic acids were extracted according to Assays of total, reduced and oxidized AsA content were carried out on ground powder conserved at –80°C using microplates and a plate reader, as previously described by P threshold < 0.001; GGMselect, GeneNet, and igraph packages; All statistical analyses were performed using R3.1.0 . The evopredawn, Ψmidday, DM content, soluble sugars, organic acids, starch content, chlorophyll content, the maximum efficiency at which light absorbed by PSII is used for reduction of QA (FV/FM), the quantum yield of the electron transport flux until PSI acceptors J0RE1/JABS and the PI index (Table 2). AUCs represent the cumulated response from the onset of the WD treatments until the end of WD3 . RP3 was discarded because healthy non-senescent mature leaves were rare at this time.In order to evaluate the global plant response to the WD treatments, AUCs were calculated for ΨFV/FM index decreased in response to the WD treatments in all genotypes except in Criollo. AUCs of PI index also significantly decreased for four genotypes . On the contrary, J0RE1/JABS increased in several genotypes  as well as the relative chlorophyll content . Then, leaf DM content (Table 2) increased due to the WD treatments (except in Cervil and Stupicke) as well as the contents in malic  and quinic acids, in glucose (except LA1420 and Ferum), in fructose , and in starch (except Cervil and LA1420). The plant leaf area, assessed through leaf size and leaf number, significantly decreased in all genotypes except Cervil, LA1420, and Stupicke.The Figure 2).The specific leaf surface area measured at the end of the experiment on non-senescent mature leaves varied by a factor three among genotypes and it significantly decreased in response to the WD treatments in LA1420 (–26%), Stupicke (–26%), LA0147 (–15%) and Levovil (–36%) while it increased in Cervil (+21%), . Clusters indicate fruit traits that co-varied under a given condition and highlight the differences in metabolite concentrations among genotypes. For both conditions, total soluble sugars, total organic acids, lutein, and β-carotene could be pooled together, as could be pooled together AsA and phytoene contents, on the one hand, and lycopene and total carotenoids contents, on the other hand. Figure 3 highlights contrasted composition among genotypes in the control. For instance Cervil fruits which had the highest DM content, had the lowest content in total sugars, acids, carotenoids, and lycopene, but the highest content in total AsA on a DM basis. Similarly, LA1420 fruits were poor in all compounds except acids and β-carotene. On the contrary, Stupicke fruits were the richest in all compounds except phytoene. Similar results were observed in control fruits of the S2 fruits (data not shown).A hierarchical clustering analysis, based on fruit composition variations (on a DM basis) among genotypes, is presented in Tables 3 and 4, respectively). Though not significant, the decrease in fruit size and fresh mass was more pronounced in S2 fruits than in S1 fruits. For instance, on Levovil, the fruit fresh mass decreased by 42.8% in S2 fruits and by 13% in S1 fruits. Cervil S1 fruits were the less sensitive (Table 3). The number of set fruits measured on the eight first trusses was not significantly different between control and WD plants since inflorescences were pruned (data not shown).Variations in fruit composition in response to the WD treatments are presented on a DM basis for S1 and S2 fruits . In the case of Cervil fruit, an increase in DM content (+8.9%), total soluble sugars (+20.3%), and starch content (+56.3%) was observed. Acid contents dropped in LA1420 fruits (–19.8% citric acid) and in LA0147 fruits . DM content was higher in Stupicke fruits (+12.7%), without any change in DM composition. In Levovil fruits, the β-carotene content was significantly reduced (–20.4%). Interestingly the contents in lycopene and carotenoids increased in four genotypes , whereas total AsA content decreased in all genotypes except LA1420. However, these variations were not significant. Consistent results were observed in S2 fruits (Table 4) except for LA0147 whose contents in lycopene, carotenoids and total AsA were hardly affected. The fruit DM content increased in all genotypes (except Ferum) and to a larger extend in Cervil (+21%), LA1420 (+22.3%), and Levovil (+33.7%). Among soluble sugars, the sucrose content was more affected than glucose or fructose contents (+47.7% in Cervil and +61.8% in Stupicke).On a DM basis, the variations in fruit composition were significant for five genotypes . So, metabolic and concentration effects added up for the compounds that increased both on a dry and fresh matter basis (mainly sugars and acids), whereas the negative effects of WD observed on a DM basis were mitigated by concentration effects, resulting in fruit quality homeostasis.On a fresh matter basis, sugars and quinic acid contents were significantly higher under WD in Cervil  and in Levovil . In Stupicke only the quinic acid content was higher under WD . For the S2 fruits, reduced and total AsA contents were higher in Cervil, LA1420, and Plovdiv . Interestingly no leaf trait correlated to any fruit traits under both conditions. In control conditions, four independent leaf clusters emerged . Positive correlations existed between leaf starch content and leaf DM content, between leaf malic acid content and Ψpredawn, and between leaf fructose and glucose contents. Then PI correlated with J0RE1/JABS and FV/FM. Concerning fruit traits, five independent clusters were found under control conditions. Fructose, glucose, and quinic acid contents were positively correlated one to each other, as well as lycopene and phytoene. Finally, fruit citric acid content, fruit malic acid content, and fruit lutein content were positively correlated one to each other while fruit fresh mass was negatively correlated with the fruit β-carotene content.A partial correlation network was built based on the AUCs calculated for the different leaf and fruit traits  suggesting that physiological acclimation processes were at play. At the leaf level, J0RE1/JABS did not correlate any more with PI and FV/FM indexes. This observation suggests a regulation of the functioning of the photosynthetic machinery (Table 2). Leaf sugars  constituted an independent cluster. Leaf malic acid did not correlate anymore with Ψpredawn but with leaf citric acid, due to the increase in organic acid contents (Table 2). Leaf DM content was positively correlated to leaf sucrose content instead of starch content. At the fruit level, the fruit DM content negatively correlated with the total ASA content, while the phytoene content was positively correlated with the lycopene content, as well as the lutein and β-carotene contents, the citric and quinic acid contents, and the glucose and fructose contents.A different network was observed under the WD treatments when compared to the control  and WD  plants. Four clusters emerged for the control plants. LA1420 and Criollo were clustered according to their high fruit citric acid content and low leaf chlorophyll content. Levovil, Stupicke, and LA0147 stand out due to their similar malic acid and phytoene contents in fruits and by their low leaf glucose content. Ferum and Plovdiv were clustered due to their high PI index value and their low fruit AsA content. The cherry tomato Cervil constituted its own cluster due to its high starch, glucose, fructose, and malic acid contents in leaves and its high DM and low quinic acid contents in fruits. Similarly, four clusters emerged for the WD plants. Clustered genotypes did not necessarily respond to the WD treatments in the same way (Table 2). The first cluster includes Levovil, Stupicke, and Criollo, which have high fruit β-carotene content. LA1420, LA0147 and Ferum were clustered due to similar Euclidean distances without emergent traits. Plovdiv and Cervil constituted their own single cluster due to high glucose and low citric acid contents in leaves for Plovdiv, and to high DM content in fruits, high sucrose and fructose contents in leaves, and low Ψpredawn and Ψmidday values for Cervil.Clustering of the leaf and fruit traits measured in the experiment was realized for control , a reduction of leaf growth, and osmotic regulations (midday except in Criollo and Ferum (Table 2). The absence of significant decrease in Ψmidday may be explained by an increase in turgor. Indeed, when the elasticity modulus decreases, reflecting a decrease in cell wall rigidity, the decrease in turgor is mitigated during dehydration  could be explained by a return of electrons from PSI to PSII named the cyclic electron flow , water supply has been shown to negatively correlate with organic acid content in ripe fruits, but in grapes Vitis vinifera L., nectarines Prunus persica var. nucipersica L. , and phenolic compounds, are the largest group along with AsA. Of all of the environmental factors that play a stimulating role in the synthesis and accumulation of useful phytochemicals in fruits, moderate stress, and more specifically, controlled drought may influence the metabolism of these phytochemicals via at least two major mechanisms that are not mutually exclusive and that may even interact . Firstlya fluorescence measurements and leaf composition, we may hypothesize that for some genotypes the cyclic electron flow (extrapolated from J0RE1/JABS) and quinic acid content were involved in energy dissipation and regulation of oxidative stress during the WD treatments. Negative effects on fruit fresh weight were dependent on stress intensity, while beneficial effects on fruit taste (sugars and acids) and nutritional value were weak or even negative. Interestingly, high starch accumulation in fruit could be a potential asset to sustain fruit growth under WD. Considering a large range of plant and fruit traits, our observations clearly show that responses to drought are highly variable and that they strongly depend on genotypic effects and on the stage of development at the time WD is applied. On their whole, the present results demonstrate that drought could be exploited positively, and that repeated cycles of WD and recovery may be used to improve fruit taste and at the same time minimize fruit size reduction. A strategy for breeding would be to stack in one single genotype adaptive traits at the leaf and fruit levels. To this end, small-fruit genotypes, in particular LA1420 and Criollo, represent an interesting potential source of traits of interest, as far as acclimation is concerned. However, our capacity to take full advantage of drought events or controlled WD is clearly conditioned by a shift in our way of thinking. We need to explore the full variability of genotypic responses by taking into account a much broader range of crop performance criteria than the ones that are usually considered and by systematically including observations made at different stages of development. The complexity revealed by our observations clearly suggests that exploring the variability of genotypic responses represents a difficult task, but then it is our belief that this is how the issue of drought on crop performance should be addressed from now on, and that the reward will come up to the challenge.In the present study, the WD treatments, which consisted in three successive cycles of moderate WD and recovery during the plant reproductive period, resulted in independent responses at the leaf and fruit levels. Considering parameters derived from chlorophyll This work is part of the Ph.D. thesis of JR, who significantly contributed to the experiment, the biochemical analyses, the statistical analyses, and the redaction of the article. Original idea of this project was developed by NB and LU, who contributed to the experimental protocol, the redaction of the article, and to the mentoring and training of JRThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes . Compared to adults, prepubertal oocytes showed higher parthenogenesis  and polispermy , lower cleavage rates  and blastocyst output . Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development. Oocyte quality is currently regarded as the key limiting factor in female fertility, and it is referred to as its intrinsic developmental potential. It depends upon the biochemical and molecular state that allows a mature oocyte to be fertilized and develop to an embryo, which upon transfer will enable healthy development to term. In accordance with this, poor oocyte quality results in either aneuploidy, epigenetic disorders, arrested embryonic development or spontaneous abortion –3 One ofThe best test to determine oocyte quality is the in vivo transfer of the obtained embryo, followed by gestation, birth, and continuing health of the resulting offspring. Obviously, ethical problems in human reproductive technologies restrict these experimental approaches to animal models. However, these experiments are cost prohibitive, take a great deal of time to achieve results, and require large animal numbers to obtain data that can be statistically analyzed. Thus, other parameters can be used to assess oocyte quality. Endpoints that are usually used for this purpose include nuclear maturation success (to MII), fertilization rates, kinetic of embryonic development, development to the blastocyst stage, blastocyst total cell number, inner cell mass and trophectoderm cell numbers, oocyte and embryo metabolism, ATP content, mRNA and proteins storage, and mitochondrial activity and distribution. Examination of all of these parameters will assist in discovering the complex mechanisms that synchronously act to impart oocyte quality.Cytoplasmic maturation is associated with a considerable increase in storage of foodstuffs and informational macromolecules such as mRNAs, proteins and transcription factors. At this time, the oocyte undergoes a regional redistribution of cytoplasmic organelles which is functional to the acquisition of meiotic competence that ultimately will influence the basic properties of the future embryo , 5.Increasing evidence shows the role of mitochondria as determinants of developmental competence for mammalian oocytes, due to their function as energy suppliers . The quaIn addition, it has been postulated that kinetics of cell cycle could be used as an indicator of oocyte and embryo quality. Cleavage kinetics has been related to the quality of blastocysts in several species  unless stated otherwise.Determination of prepubertal and adult oocytes meiotic and developmental competence: prepubertal and adult oocytes were incorporated into an in vitro embryo production system in order to compare their meiotic and developmental competence, as evaluated by maturation and fertilization rates, embryo developmental kinetics and total blastocyst output.Evaluation of the kinetic of meiotic progression: timing of meiosis progression is considered as a marker of oocyte quality . TherefoDetermination of protein electrophoretic pattern during meiosis: it has been proposed that protein synthesis and secretion are related to developmental competence of mammalian oocytes . We compQuantification of ATP intracellular content: oocyte energetic level has been regarded as a suitable marker of its quality since during meiotic progression an increase in ATP content is required for subsequent fertilization and embryo development , 8, 29. Submicroscopical evaluation of mitochondria and organelles by light and transmission electron microscopy: cumulus oocyte complexes (COCs) collected in three replicates from prepubertal and adult ovaries were processed at 0, 7, 19 and 24 hrs of IVM.Evaluation of active mitochondrial distribution patterns: COCs collected in three replicated experiments from prepubertal and adult ovine ovaries were processed at collection (0 hrs) and after 24 hrs of IVM to evaluate mitochondrial distribution, mitochondrial activity and chromosomal configuration and then analyzed by confocal laser scanning microscopy (CLSM).Oocytes from prepubertal (30–40 days old) and adult (4–6 years old) ewes were obtained from ovaries collected at a commercial slaughterhouse. Thereafter, the following experiments were performed:2 in air at 39°C.Collected ovaries were transported from the commercial slaughterhouse to the laboratory within 1–2 h in Dulbecco Phosphate Buffered Saline (PBS) with antibiotics at 27°C. After being washed in PBS fresh medium, the ovaries were sliced using a micro-blade and the follicle content was released in medium TCM199  supplemented with 25 mmol HEPES, 0.1 g/L penicillin, 0.1 g/L streptomycin and 0.1% (w/v) polyvinylalcohol (PVA). Cumulus—oocyte complexes (COCs) with 4–10 layers of granulosa cells, oocytes with a uniform cytoplasm, homogenous distribution of lipid droplets in the cytoplasm and outer diameter of about 90 μm (mean) were selected for these experiments. COCs selected for in vitro culture were washed three times in the same fresh medium and matured in vitro in TCM 199 supplemented with 10% heat-treated oestrus sheep serum (OSS), 1 IU/mL of FSH/LH, 8 mg/mL pyruvate and 100 μM cysteamine. 40–45 COCs were put in 500 μL of maturation medium in four-well Petri dishes , layered with 300 μL of mineral oil and cultured for 24 h in 5% COMeiotic competence was determined in 4 replicated experiments in prepubertal (n = 1357) and adult (n = 970) oocytes. After 24 hrs of in vitro maturation COCS were denuded of cumulus cells using a narrow bore glass capillary, fixed for 24 h in acetic acid ethanol solution (1:3 vol/vol), and stained with 1% (wt/vol) of Lacmoid. Chromatin configuration was evaluated under a phase contrast microscope at 200 to 400 x.2, 5% O2 and 90% N2 atmosphere in four-well Petri dishes (Nunclon).Fertilization potential was computed in 3 replicates using 1070 prepubertal and 766 adult in vitro matured oocytes. COCs were partially stripped of the granulosa cells and fertilized in vitro at 39°C and 5% CO6 spermatozoa/mL concentration, layered with mineral oil. After 14 hrs oocytes were washed from spermatozoa and fixed with acetolacmoid as described above. Oocytes were classified as fertilized when showed 2 pronuclei, as parthenogenetic when showed only one pronucleus and as polyspermic when presented more than 2 pronuclei or one pronucleus and 2 or more decondensed sperm nuclei.Frozen thawed spermatozoa from the same ejaculate of a single ram were used for all experimental procedures. The IVF system was composed of 300 μL of synthetic oviductal fluid (SOF) medium supplemented with 2% heat-treated ovine oestrus serum (OSS) and swim-up derived motile spermatozoa, at 1x102, 5% CO2. Newly formed blastocysts were recorded daily, starting from the 6th day of culture. Total blastocysts computed those obtained within 9 days post insemination.Developmental competence was determined in 5 replicated experiments after in vitro maturation of prepubertal (n = 2110) and adult (n = 1522) oocytes. Fertilization was carried out as above described and at 24, 26, 32 hrs post-insemination the number of cleaved oocytes, showing two distinct blastomeres, was recorded. Total cleavage computed all oocytes which cleaved within 32 hrs post insemination. The cleaved embryos were transferred to a culture system composed by SOF medium supplemented with 4 mg/mL of bovine serum albumin and essential and non-essential amino acids at oviductal concentration, and kept in maximum humidified atmosphere, at 39°C, 5% OOocyte in vitro maturation was performed as above described. In three replicates, at each time point , oocytes were decumulated by gentle pipetting using a narrow bore glass capillary, fixed in ice cold methanol, and incubated with 10μg/mL Hoechst 33342 in ice-cold methanol for 15 min. Stained oocytes were mounted into a small droplet of glycerol on a glass slide and examined under an epifluorescence inverted microscope .Here and thereafter, 0 hrs of maturation indicate GV stage, 7 hrs indicate MI stage and 22 or 24 hrs indicate MII stage.Prepubertal and adult COCs were matured as described above, retrieved from the culture system at different time points , denuded by gentle pipetting and stored at -80°C until analysis. At thawing, oocytes  were diluted to 300 μl with rehydration buffer  and used to rehydrate a pH 3–10 linear gradient IEF Immobiline Dry Strip . Isoelectric focusing (IEF) was performed using a Protean IEF Cell (Biorad) for 12 hrs at 20°C, followed by 500V for 1,000 Vhr, 1,000V for 2,000 Vhr, and 8,000V for 22,000 Vhr.IPG strips were incubated in equilibration buffer 1  for 15 min, rinsed with equilibration buffer 2  for 10 min. Strips were rinsed in SDS—gel running buffer, transferred into 12% SDS gels and sealed with 0.5% (w/v) agarose solution before running the second dimension (50 mA/gel) in a 15°C termostated Protean II electrophoretic cell (Biorad). After electrophoresis, gels were silver stained  and dried (Biorad Gel dryer). Experiments were repeated three times and differences between gel images were analyzed using the PdQuest software (Biorad). This software allows us to compare gels obtained from the two experimental groups at the same maturation time, in order to identify the spots present in gels of only one experimental group , and to determine differences in intensity (quantitative differences) between spots present in gels of both groups at the same maturation time.2CO3. After 3 minutes centrifugation in a Eppendorf Microfuge at 10000 rpm, the supernatant was derivatized before capillary electrophoresis. Ten μL of sample or standard were mixed with 40 μL of 1.8 mol/L 1-ethyl-3--carbodiimide hydrochloride  and 5 μL of 27 mmol/L Bodipy FL EDA  and incubated for 25 h at 37°C in the dark. A P/ACE 5510 CE system equipped with Laser Induced Fluorescence  was used. The dimension of the uncoated fused silica capillary was 75 mm ID and 57 cm length (50 cm to the detection window). Analysis was performed applying 21 nl of sample under nitrogen pressure (0.5 psi) for 3 s using a 10 mmol/L sodium phosphate buffer, pH 11.4. The separating conditions  were reached in 20 s and held at a constant voltage for 8 min. All separations were carried out at 40°C.ATP was quantified in oocytes using capillary electrophoresis according to Zinellu et al. . After bPrepubertal (n = 82) and adult (n = 68) COCs were fixed at sampling (0 hrs), and at different time intervals during IVM , and processed for light microscopy (LM) and transmission electron microscopy (TEM) analysis as previously described . COCs fi2 of the oocyte area. Mitochondrial counting was performed through collection of low-magnification TEM microphotographs of the same oocytes, on 3 equatorial sections per oocyte. Images were further enlarged on the PC screen, in order to easily recognize and count mitochondria. Values were expressed in number of mitochondria per 100 μm2 of the oocyte area. The number of mitochondria per each cluster was extrapolated from an integrated evaluation of the above data.The number of mitochondrial clusters was evaluated at the LM level in 5 oocytes per group, on at least 3 equatorial sections per oocyte (distance between the sections: 3–4 μm), and values were expressed in number of clusters per 100 μm2XRos , hereafter called MT-Red, a mitochondrial-specific fluorescent and cell-permeant probe and Hoechst 33342, to stain chromosomes. Reduced MT-Red is a derivative of dihydro-X-rodamine and is readily sequestered only by actively respiring organelles, depending upon their oxidative activity. This reduced probe does not fluoresce until it enters live cells, where it is oxidized to the corresponding fluorescent mitochondrion-selective probe and then sequestered in the mitochondria.COCs collected from prepubertal (n = 45) and adult (n = 41) ewes ovaries in three replicate experiments were processed at collection (0 hrs) and after 24 hrs of IVM to evaluate the mitochondrial distribution, mitochondrial activity and chromosomal configuration and then analyzed by confocal laser scanning microscopy (CLSM). COCs were subjected to a double staining with MitoTracker Red CM-HImmature germinal vesicle (GV) stage oocytes were retrieved at 0h IVM and used as controls. Following IVM, COCs were incubated for further 30 min at 38.5°C in TCM-199 plus 10% FCS supplemented with 1 IU/mL FSH,1IU/mL LH, 100 μM cysteamine, 8 mg/mL pyruvate; 500 nM MT-Red (stock solution: 100 μM in DMSO).After exposure of COCs to the probes, cumulus cells were mechanically removed from the oocytes by repeated pipetting. Oocytes were then washed three times in fresh, pre-warmed 0.1% PVA in PBS/ and fixed in 2.5% glutaraldehyde/PBS for at least 15 min. After fixation, oocytes were washed three times in PBS/PVA and mounted on glass slides with 2.5 μg/mL Hoechst 33342 3:1 (v/v) in PBS and glycerol solution. Slides were kept at 4°C in darkness until evaluation.2XRos . Each oocyte was examined along the z-axis by means of about 30 serial confocal planes (each 3 μm thick), going from the top to the bottom of the cell. Microscope adjustments and photomultiplier settings were kept constant for all experiments. Acquisition, storage and image analysis were made with the LAS lite 170 Image software. To evaluate quantitatively the fluorescence intensity in the confocal microscopic sections an image analysis was performed using ImageJ software .Maturation, fertilization and developmental rates, kinetics of maturation and development and active mitochondria phenotypes were analyzed using chi square test or Fisher exact test when appropriate. ATP concentrations, 2D electrophoretic protein spots during meiotic progression, morphometric data on semithin and ultrathin sections and fluorescence intensity data obtained by confocal microscopy were analysed by ANOVA test after analysis for homogeneity of variance by Levene’s test.Statistical analyses were performed using the statistical software program Statgraphic Centurion XV  and a probability of P ≤ 0.05 was considered to be the minimum level of significance.As showed in In addition, the kinetic of embryo development was delayed in prepubertal oocytes compared to adult ones. At 26 hrs post-fertilization, adult fertilized oocytes underwent the first cleavage at higher rate compared to prepubertal ones . Therefore, the first cell cycle was longer in prepubertal than in adult fertilized oocytes. This delay grew as embryo development progressed and reached 24 hrs at the blastocyst stage.The kinetic of maturation differs between adult and prepubertal oocytes. As showed in As summarized in In prepubertal oocytes, a similar pattern was observed in the first 7 hrs of culture, but ATP intracellular concentration started to rise again only after 19 hrs of culture and did not reach values higher than the initial ones after 24 hrs of culture.According to Khalili and coworkers , type ancristae were easily discernible  PattIn prepubertal and adult GV oocytes CLSM analysis revealed that the trans-zonal cytoplasmic projections penetrating through the zona pellucida the oocyte from the surrounding granulosa cells, appeared intensely labeled by MT-Red. Moreover, an evident thin layer of active mitochondria was present under the plasma membrane in GV-stage oocytes. On the contrary, MII-stage oocytes from prepubertal and adult animals showed a more homogeneous distribution of mitochondria in the cytoplasm.In the present work we showed that ATP concentration and protein pattern fluctuations in low quality oocyte is altered compared to high quality ones. We demonstrated that in GV stage of low and high quality oocyte active mitochondria have a fine or granular homogeneous distribution while only in high quality oocytes most of MII display functional mitochondria aggregated in a clustered structure. The number of mitochondria grows from GV to MII in adult oocytes but not in prepubertals, increasing the number of mitochondria/cluster. We evidence that the prompt recover of ATP production after the transition GV-MI is fundamental to oocyte progression toward meiosis and determines the kinetic of the meiotic cycle.Present data confirm a previous work  showing Prepubertal oocytes reach MI stage 1 hr later than adult ones. This delay grows as the first meiotic division proceeds. After 19 hrs of IVM, MII rates in adult oocytes are higher than in prepubertal ones, which reach comparable rates only after 21 hrs of culture. The delay is amplified at the blastocyst stage where the difference in development kinetic between prepubertal and adult embryos reaches almost 24 hrs. Embryonic development occurs in the absence de novo transcription till the oocyte-to-embryo transition and is dependent of mRNA and/or proteins stored into the oocyte during oogenesis .Proteins appear to be of basic importance for meiotic resumption  but alsoEnergy in the form of ATP is critical for nuclear/cytoplasmic maturation events and for developmental potential of the embryo , 42, 43.TEM observations revealed that the mitochondria number and its cytoplasmic distribution change during IVM, reflecting fluctuations in ATP intracellular contents. We showed an increment in the number of mitochondria in adult oocytes at MII compared to GV while in prepubertal this number did not change.Mitochondria distribution is modified during in vitro maturation. While at 0 hrs of IVM prepubertal and adult oocytes showed a similar arrangement of mitochondria, with big cortical clusters often in close proximity to elements of the SER or Golgi apparatus, at 7 hrs such aggregations appeared smaller in adults than in prepubertal oocytes. After 19 hrs of culture a sort of reduction/dispersion of mitochondrial clusters was evidenced, with the presence of numerous isolated elements spread throughout the ooplasm, especially in prepubertal oocytes. At the end of the culture period (IVM 24 hrs), big clusters were newly visible in adult but not in prepubertal oocytes, where small aggregations were still present. In all, numerous isolated mitochondria were scattered in the cytoplasm.Our data are in agreement with previous studies on mammalian oocytes revealing that mitochondria are redistributed during IVM  and those that remain exhibit a granular distribution (Pattern B), while in most of adult oocytes (54.5%) active mitochondria are distributed in big clusters (Pattern C), indicating that most mitochondria of big clusters seen in TEM were active. A schematic drawing  depicts In conclusion our work evidenced the importance of the quality of oocyte cytoplasm to determine the fate of an embryo and contributed to clarify the biochemical mechanisms associated with the meiotic progression and acquisition of developmental competence. We demonstrated that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The cytoplasmic ATP content promotes all the energy requiring processes which determine the timing of the cell cycle and the acquisition of developmental competence. The oocytes with low developmental competence have a slowed down energetic metabolism which delay physiological times of later development.


ARB SILVA database and Qiime.Today there are more than 2 billion alcohol users and about 1.3 billion tobacco users worldwide. The chronic and heavy use of these two substances is at the heart of numerous diseases and may wreak havoc on the human oral microbiome. This study delves into the changes that alcohol and tobacco may cause on biofilms of the human oral microbiome. To do so, we used swabs to sample the oral biofilm of 22 subjects; including 9 control-individuals with no or very low consumption of alcohol and no consumption of tobacco, 7 who were chronic and heavy users of both substances and 6 active smokers that reported no significant alcohol consumption. DNA was extracted from swabs and the V1 region of the 16S rRNA gene was PCR amplified and sequenced using the Ion Torrent PGM platform, generating 3.7 million high quality reads. DNA sequences were clustered and OTUs were assigned using the Neisseria abundance was significantly decreased in both groups when compared to controls. Smokers had significant increases in Prevotella and Capnocytophaga and reductions in Granulicatella, Staphylococcus, Peptostreptococcus and Gemella when compared to the two other groups. Controls showed higher abundance of Aggregibacter, whilst smokers/drinkers had lower abundances of Fusobacteria. Samples from only smokers clustered closer together than to controls and smokers/drinkers, and also had a significant reduction in inter-group dissimilarity distances, indicating a more homogenous group than controls.We found no differences in species diversity and evenness among the groups. However, we found a significant decrease in species richness in only smokers and in smokers/drinkers when compared to controls. We found that Our results indicate that the continued use of tobacco or alcohol plus tobacco significantly reduces bacterial richness, which apparently leads to a reduction in inter-group variability, turning the respective biofilms into a more homogenous microenvironment in terms of bacterial community composition, with possible consequences for human oral diseases.The online version of this article (doi:10.1186/s12866-014-0250-2) contains supplementary material, which is available to authorized users. Each day nearly 4,000 people younger than 18 years old smoke their first cigarette, a quarter of which will become addicted . CigaretNeisseria and gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumonia [The use of cigarettes by adults, as well as passive smoke exposure in children, has been associated with increased carriage of pathogenic organisms in the upper airways . It has neumonia . Howeverin vitro [The human oral cavity harbors more than 700 different bacterial taxa, possessing relevant quantitative  and qualitative  differences between individuals –22. Alsoin vitro ,26, commIn the present study we investigate the effects of the chronic use of alcohol and tobacco over the oral microbiome, in terms of diversity and composition, using the 16S rRNA gene as a target and the Ion Torrent Personal Genome Machine (PGM) platform for high throughput sequencing. To our knowledge, this is the first large-scale study focused on evaluating the effects that chronic use of alcohol and tobacco have over the microbiome of human oral biofilms.A total of twenty-two subjects were recruited and signed an informed consent form prior to sample collection. A questionnaire was given to all subjects to evaluate general characteristics including oral health, oral hygiene habits, alcohol and tobacco use and others Table . Subject.Smokers/Drinkers (SD) (n = 7): subjects belonging to this group were recruited at the Alcohol and Tobacco Abuse Group at the Institute and Department of Psychiatry, part of the São Paulo Medical School (FMUSP), in São Paulo, Brazil. All individuals in this group reported the use of at least 20 cigarettes/day with a regular smoking history of at least 10 years. These individuals also reported daily drinking habits  and a regular drinking history of at least 10 yearsControls (C) (n = 9): All subjects belonging to this group were healthy individuals recruited at the cancer screening campaigns at the AC Camargo Cancer Center, also in São Paulo, Brazil. Individuals from this group never smoked cigarettes or any other tobacco-related derivatives, reported no or very low consumption of alcohol, defined as: participants reporting the consumption of only low-ethanol beverages (<5% ethanol), in small doses (maximum of 1 drink/occasion), with a drinking history inferior to 3 years and absence of daily drinking during this period. None of these individuals used alcohol-containing mouthwash solutions.. Regarding ethanol consumption as an excluding aspect, the same criteria as the control group was adopted, including mouthwash use.Smokers (S) (n = 6): All subjects belonging to this group were recruited at the cancer screening campaigns at the AC Camargo Cancer Center. These individuals reported a regular smoking history of at least 20 cigarettes/day for the last 10 years and maintained their smoking habits prior to sample collection1) subjects diagnosed with cancer; 2) subjects reporting the use of antibiotics within the last 3 months prior to sample collection; 3) subjects reporting residence outside of the city of São Paulo in the past 5 years; 4) presence of comorbidities such as HIV, diabetes and ischaemic heart disease; 5) individuals younger than forty years of age and 6) presence of malignant or potentially pre-malignant oral lesions, such as leukoplakia or erythroplakia.Additional exclusion criteria for all subjects were as follows: This study was approved by the ethics in research committees of AC Camargo Cancer Center (protocol 1459/10) and University of São Paulo (protocol 0895/11).2 each) from different areas of each card and then eluted each one in 30 μl of sterile water by incubating for 25 mins at 95°C, according to the manufacturer’s recommendations. These four elutions from a single subject were pooled together and used as template DNA for subsequent procedures.In order to sample bacteria from the oral biofilm, we swabbed the following oral mucosal surfaces of each individual: the dorsum and the laterals of the tongue, the floor of the mouth and the buccal mucosa. Bacterial samples were collected from these areas using Sterile Foam Tipped Applicators and transferred to FTA elute cards, both from Whatman , where DNA was preserved after chemical lysis of the cells. In order to obtain a better representation of the whole oral mucosa in each sample, we collected four perforations  software package (Version 1.6.0) [Sequences processed by the latest version of the Ion Torrent server (version 3.6.2) were used as input into the n 1.6.0) . We firsQiime interface to perform BLAST [After primer- and quality-filtering, we clustered the remaining sequences with 97% identity using UPARSE (implemented in USEARCH v7) , and picrm BLAST  analysisrm BLAST  for taxoTo investigate differences in phyla and genera abundance between the three groups, raw counts were normalized using the following normalization method :\documenQiime. Observed species richness and richness estimators Chao1 [To investigate species diversity, richness and evenness, we used the same sequence sampling depth for all samples by rarefying the OTU table. Rarefaction curves, the Shannon-Weaver diversity index  and equirs Chao1  and ACE rs Chao1  were calrs Chao1 .Qiime.The Euclidean distance matrix, the coordinates for the Principal Coordinate Analysis (PCoA), and the intra- and inter-dissimilarity indexes were calculated using Kruskal-Wallis tests were performed to compare mean similarities between controls, smokers/drinkers and only smokers for phyla and genera log abundances. P-values were corrected for multiple testing  using:  and 3 females (one in each group). The composition of the groups showed no statistical differences in terms of age, gender, weight and height. Whereas the self-reports of general oral health, oral hygiene habits, gingival bleeding, annual visits to the dentist and others were also similar among the groups, as expected, differences in alcohol and tobacco consumption showed to be statistically significant. Table After size, quality and primer trimming, a total of 3,843,174 sequences remained, with an average of 174,689 sequences/sample and a mean sequence length of 72 ± 4.9 nt (standard deviation) after primer trimming and barcode removal.When all individuals were considered, a total of 2,931 clusters were obtained. Twenty-one (0.7%) of these were removed after being identified as chimeras by UCHIME, and 112 (3.8%) had no assigned taxonomy. After filtering to remove OTU clusters with less than 3 sequences and not present in at least 25% of all samples, 1,849 OTUs remained.Using oral wash and broncheoalveolar lavage samples, a previous study found significant differences in bacterial communities when comparing smokers and non-smokers, suggesting that tobacco may significantly affect the oral microbiome . In our vs inter-group dissimilarity analysis. Figure We then used the binary Euclidean metric to investigate if bacterial communities were more similar within each sample group or between groups, i.e. an intra-group A recent study showed that even with the considerable intra- and interpersonal variation in the human microbiome, this variation could not be partitioned into community types that are predictive of each other, and were probably the result of life-history characteristics , which mIn order to measure species richness in the three groups we used the number of distinct OTUs observed in each sample when the same sequence sampling depth was applied at two different OTU-abundance thresholds Figure . SaturatC: 0.58; mean SD: 0.55; mean S: 0.55, where 1 indicates complete equitability) and found no statistical difference in species evenness between these three groups (p > 0.05)  Table . This suC: 5.19, SD: 4.76 and S: 4.59). This suggests that the key difference in alpha diversity between controls, smokers/drinkers and only smokers lies solely in species richness. A previous study found a more diverse microbiome in the oropharynx of smokers than non-smokers [Clostridium difficile-associated diarrhea [Also, we evaluated species diversity using the Shannon-Weaver index and found no significant differences in diversity between each of the possible group pairs, but did see an increase in controls , but after correcting for multiple taxa testing, this decrease was no longer significant (p=0.06). A reduction in the relative abundance of this phylum in broncheoalveolar-lavages of smokers when compared to non-smokers has been reported [The most abundant phyla found in all groups were (in decreasing order of abundance): s Figure A. At thearound 1%,54 and uaround 1% of the oaround 1%,56. In oreported , pointinGenera log abundances. At the genus level, 47 out of the 279 (16.8%) identified genera were found to have significant differential log abundances between the three groups . However, after multiple taxa correction, the p-value was no longer significant . Subsequent reports using 16S rRNA sequence profiling of subgingival plaque identified an increase in several disease-associated organisms in smokers, including Parvimonas, Fusobacterium, Campylobacter, Bacteroides, Dialister, and Treponema spp. and a decrease in potential health-promoting taxa from the Veillonella, Neisseria, Streptococcus, and Capnocytophaga genera [Aggregibacter levels were higher in controls, whilst smokers/drinkers had lower log abundances of Fusobacteria compared to C and S .Only smokers had significant increases in d before , suggestd before . Anotheris genus . In our  smokers ,43 and t smokers ,62, was a genera . AggregiIn silico analysis indicated that, whereas this primer pair covers about 54.5% of the eubacterial phyla present in the ARB SILVA database, certain phyla are clearly under-represented, including Fusobacteria and Bacteroidetes. Also, whereas the DNA extraction protocol used here does not include the use of physical lysis (such as a bead beating step), which may improve the representation of certain bacteria , and whilst PCR/sequencing artifacts may still persist, all these effects should be homogeneous for all groups and should not greatly affect our conclusions. A previous study [The study presented here has certain limitations that need to be considered. First we would like to point out that the sample size was relatively small (N=22) and limited to a set of individuals living in the city of São Paulo, Brazil. The study of larger cohorts, and a cohort composed of individuals that consume alcohol but do not smoke would be important for a better understanding of the individual effects of these substances. The study of individuals who have quit using alcohol and tobacco, as well as recent users, would be important to reveal how long a ‘normal’ microbiome persists and how long it takes to recover after the use of alcohol/tobacco. Besides the overall evaluation of oral hygiene habits and oral health status Table , we did us study  reportedus study  showed tTaking these limitations into consideration, we found a reduced bacterial richness in both only smokers and smokers/drinkers when compared to controls. To our knowledge, no studies have evaluated, up to now, if alcohol and tobacco affect the microbial composition of the human oral mucosa. In this study we found that bacterial communities from subjects who chronically use tobacco – together or not with alcohol – cluster together and possess reduced species richness, suggesting that this substance may play a key role in reducing the oral cavity’s bacterial richness. This reduced richness might have contributed to the significant decrease in dissimilarity observed between subjects of the only smokers group, showing to be the most homogenous in terms of bacterial communities. Species diversity and species evenness were not significantly different between any of the group pairs. Although, independent of the metrics used, all analyses indicated a higher species-richness in controls.Firmicutes and TM7 among others and significant increases in Fusobacteria and Actinobacteria.Our data indicates the presence of over 300 distinct bacterial OTUs in the human oral mucosa (Figure Neisseria abundance in both only smokers and smokers/drinkers, reinforcing evidence that points to a modulation of this genus’s abundance by tobacco smoke. We found that two other genera, Gemella and Peptostreptococcus, were exclusively decreased in only smokers.We found a significant decrease in A recent study showed that modern dietary habits introduced in the early Neolithic period, including carbohydrate ingestion, affected the oral microbiome composition and diversity . The autNucleic acid sequences are available at NCBI/GenBank/SRA under accession number “SRP044345”.