Whole-genome analysis on seven selected strains confirmed the presence of numerous gene clusters with PGP and biocontrol functions and of novel secondary-metabolite biosynthetic genes, which could exert beneficial impacts on plant growth and protection. The predicted biocontrol potential was confirmed in dual culture assays against several phytopathogenic fungi and bacteria. Interestingly, the presence of predicted gene clusters with known biocontrol functions in some of the isolates was not predictive of the in vitro results, supporting the need of combining laboratory assays and genome mining in PGPB identification for future applications.Massive application of chemical fertilizers and pesticides has been the main strategy used to cope with the rising crop demands in the last decades. The indiscriminate use of chemicals while providing a temporary solution to food demand has led to a decrease in crop productivity and an increase in the environmental impact of modern agriculture. A sustainable alternative to the use of agrochemicals is the use of microorganisms naturally capable of enhancing plant growth and protecting crops from pests known as Plant-Growth-Promoting Bacteria (PGPB). Aim of the present study was to isolate and characterize PGPB from salt-pans sand samples with activities associated to plant fitness increase. To survive high salinity, salt-tolerant microbes produce a broad range of compounds with heterogeneous biological activities that are potentially beneficial for plant growth. A total of 20 halophilic spore-forming bacteria have been screened  In the past decades, social concern about the environmental effects of the uncontrolled use of chemical pesticides, fertilizers, and herbicides in the agricultural field has risen considerably. The use of chemicals for the protection and enhancement of crops has led to several negative consequences: the formation of stable phytopathogenic variants, the reduction in the number of beneficial microorganisms, and the accumulation of toxic substances in soils and aquatic ecosystems . Given tBacilli with potential applications as biofertilizers or biocontrol agents. For this purpose, samples from the rhizosphere of the nurse plants Juniperus sabina and nearby soils were collected from salt-pans , a phytohormone playing a key role in both root and shoot development. Additionally, both isolates showed a strong biocontrol activity, inhibiting the fungal phytopathogen Macrophomina phaseolina growth . Sand samples were heat-treated at 80°C, for 15min to kill vegetative cells and select for spore-forming bacteria, and 1g of sample was suspended in 9ml of TY broth  and 10-fold serial dilutions placed on TY plates 2, 10μM MnCl2, and 1μM FeSO4, Sigma-Aldrich, Germany) at 37°C for 30–48h, and the presence of spores was checked by light microscopy. Salt, pH, and temperature tolerance were determined as follows: about 50μl of culture of each isolate grown in TY broth for 6h at 37°C (107 cells/ml) were transferred to individual tubes containing 5ml of TY broth with different pH  or NaCl concentration  and left to grow at 37°C with agitation  placed in a sealed jar with bacteria streaked on TY agar plates and incubated at 37°C for 3days. To confirm the sporulation ability, the bacteria were grown in Difco sporulation medium  onto Pikovaskya’s agar medium  assay as described by 3 in 35% HClO4 solution), and the solution was vortexed and incubated at room temperature for 30min. The formation of pink color was considered a positive reaction . After an overnight incubation at 37°C, the swarm diameters were measured.Swarming motility was tested according to the method adopted by de novo genome assembly was carried out with SPAdes (version 3.7) via MicrobesNG. Genomes were annotated using Prokka  according to the manufacturer’s instructions. Genome sequencing was performed by MicrobesNG  with the genomic DNA library prepared using the Nextera XT library prep kit (Illumina) following the manufacturer’s protocol. Libraries were sequenced on the Illumina HiSeq using a 250bp paired-end protocol. Reads were adapted and trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15  and the g Prokka . BiosampAverage Nucleotide Identity (ANI) values between the sequenced genomes and the closest bacterial species identified from the 16S rRNA phylogenetic analysis (see below) were obtained using the OrthoANI algorithm of EZBioCloud . An ANI Clostridium difficile is used as an outgroup to root the tree.The 16S rRNA genes were extracted from the sequenced genomes using Anvi’o v2.3.3 . and comin vitro for growth inhibitory activity against phytopathogenic fungi and bacteria are listed in Stemphylium vesicarium. All the fungi were stored on Potato Dextrose Agar (PDA) in Petri dishes. Dual-culture plate method was carried out to detect the antifungal activity in accordance with −6CFU/ml were mixed with melted 0.8% TY agar medium before pouring plates. After solidification, 5μl of bacterial isolates solution (OD600=1.0) was spot inoculated onto the plates and incubated at 28°C for 48h, before measuring the diameters of inhibition halos. All experiments were performed in triplicate.Isolated bacterial strains were tested Obtained genomes were analyzed by antiSMASH 5.0  and BAGEJ. sabina, in salt-pans as described in the Materials and Methods section. Based on morphological characteristics, a total of 20 isolates were selected and preliminarily characterized for growth properties  usually associated with biocontrol activity  and the dependent glucose dehydrogenase (gdh) genes, involved in mineral phosphate solubilization as well as antifungal activities and systemic resistance induction. Interestingly, both isolates B. amyloliquefaciens RHF6 and RHFS18 did not carry the cofactor pqq gene cluster, suggesting that other mechanisms could co-exist , post-translationally modified peptides (RiPPs), hybrid lipopeptides  can be found in the article/RI: conceptualization, supervision, project administration, and funding acquisition. SC and CP: methodology. SC, CP, ML, and MS: validation and formal analysis. SC, CP, and DG: investigation. SC, CP, MS, AC, and RI: data curation. RI, SC, CP, and DG: writing original draft preparation. All authors have read and agreed to the published version of the manuscript.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Konservat-Lagerstätten—deposits with exceptionally preserved fossils—vary in abundance across geographic and stratigraphic space due to paleoenvironmental heterogeneity. While oceanic anoxic events (OAEs) may have promoted preservation of marine lagerstätten, the environmental controls on their taphonomy remain unclear. Here, we provide new data on the mineralization of fossils in three Lower Jurassic Lagerstätten—Strawberry Bank (UK), Ya Ha Tinda (Canada), and Posidonia Shale (Germany) —and test the hypothesis that they were preserved under similar conditions. Biostratigraphy indicates that all three Lagerstätten were deposited during the Toarcian OAE (TOAE), and scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) show that each deposit contains a variety of taxa preserved as phosphatized skeletons and tissues. Thus, despite their geographic and paleoenvironmental differences, all of these Lagerstätten were deposited in settings conducive to phosphatization, indicating that the TOAE fostered exceptional preservation in marine settings around the world. Phosphatization may have been fueled by phosphate delivery from climatically-driven sea level change and continental weathering, with anoxic basins acting as phosphorus traps. They form under circumstances where soft-tissues are mineralized faster than they are degraded4, and notably vary in abundance and facies across geographic and stratigraphic space, suggesting that their preservation potential depends on both overarching global controls and local variability in paleoenvironmental conditions6. Ocean redox conditions may represent a control on the exceptional fossil record. Most marine Lagerstätten were preserved prior to the middle Paleozoic, when oceanic oxygen levels were low, and exceptional preservation was comparatively rare in younger marine settings6, except during Jurassic and Cretaceous Oceanic Anoxic Events (OAEs)15. Although water column anoxia or hypoxia may promote preservation of soft tissues by limiting scavenging and bioturbation4, some Lagerstätten contain evidence of benthic life at various levels16. Furthermore, anaerobic processes can degrade soft-tissues as rapidly as aerobic decay17, and anoxic conditions can limit fossil mineralization18. For these reasons, the effects of water column anoxia on exceptional preservation remain debated.Konservat-Lagerstätten19. The Karoo-Ferrar Large Igneous Provinces injected greenhouse gases into the atmosphere, causing carbon dioxide levels to increase from ~ 500 ppmv to ~ 1000 ppmv during the TOAE and subsequent global warming, with estimated temperature increases of 2–3.5 °C in the subtropics and 6–8 °C at higher latitudes24 Increased temperature and humidity exacerbated hydrological cycles, which led to high precipitation and continental runoff e.g. 26. Excessive nutrients in the ocean increased primary productivity, resulting in low oxygen conditions, global extinctions e.g.28, and organic-rich black shale deposition26.Toarcian Lagerstätten provide an opportunity to explore the effects of an OAE on exceptional preservation in disparate settings. The Early Jurassic was an interval of significant environmental changes and biotic crises, including the ~ 183 Ma Toarcian Oceanic Anoxic Event (TOAE) e.g.Dactylioceras tenuicostatum, Harpoceras serpentinum, Hildoceras bifrons, and equivalent (Dactylioceras kanense and Rarenodia planulata) biozones of the early Toarcian and exhibit signs of the negative isotope excursion that is a signature of the TOAE15  and Posidonia Shale (Posidonienschiefer Fm.) Lagerstätten record deeper marine settings, which intermittently developed bottom water anoxia29 between times of oxic and suboxic conditions30. These three Lagerstätten have similar fauna , Posidonia Shale (Germany), and Ya Ha Tinda (Canada) Lagerstätten Fig.  all occuE15 Fig. . The Struna Fig.  includin33. This Lagerstätte is located near the town of Ilminster, Somerset, and belongs to the Beacon Limestone Formation  of these fossils were preserved in carbonate concretions or nodules and collected from the ‘Saurian and Fish Zone,’ which is either a nodular limestone bed or thin unit of clay  ammonite biozone10, which in other locations, is corelated with the extreme negative values of the carbon isotope excursion caused by the TOAE45. Cephalopod and crustacean fossils support the interpretation that the nodular limestone was deposited in a setting influenced by marine life and processes. The abundance of insect fossils and relatively high clay content of the Beacon Limestone Formation at Ilminster suggest that the fossils were preserved in a transitional depositional environment near the paleocoastline10. Given that exceptional fossil preservation often occurs in restricted environments6 and the Beacon Limestone Formation exhibits great lithologic variation in Somerset, the Lagerstätte may have formed within a localized lagoon, mud flat, or similar quiet-water environment.Most of the fossils from this Lagerstätte were collected between 1840 and 1860 by Charles Moore and the original quarry has been backfilled and converted to farmlandlay Fig. 36. In ei46, most notably  from the Swabian Alb (Germany) and Switzerland (as the Rietheim Member of the Staffelegg Formation), the Franconian Alb (Mistelgau and Bad Staffelstein), and Lower Saxony (Schandelah). In general, this unit consists of black, microlaminated shales and mudstones intercalated with limestones47, which were deposited in the European epicontinental sea that existed during the Mesozoic48 , member II (εII), and member III (εIII)—which are further subdivided into beds and intervals that can be correlated across the region to varying degrees47 . With the exception of the Koblenzer layer, which is inconsistently laminated and contains faint evidence of bioturbation46, all of these units are microlaminated black shales with abundant but non-diverse benthic fauna16, including exceptionally preserved fossils47. The shales located higher in the middle member contain a number of limestone layers that are not present at all sites45. The lowermost of these limestone layers—the Unterer Stein—is a well-laminated and laterally extensive limestone, which serves as a marker bed for regional lithostratigraphic correlation46. The thin shale unit above the Unterer Stein—the Schieferklotz— is known for marine reptiles16. Above this layer, shales are interbedded with the following limestone layers (in ascending order): the Steinplatte, a non-laminated limestone that sometimes appears yellow in outcrop due to weathering; the Gelbe Platte, a similar limestone bed that is missing at some localities; and the Oberer Stein, another well-laminated and laterally extensive limestone that serves as a regional marker46. Above the Oberer Stein, the Posidonienschiefer exhibits a significant amount of regional variation in its lithology46. In general, the upper part of Member II consists of bituminous shales and mudstones intercalated with localized layers of limestone46. Notably, many of these shales and mudstones are irregularly laminated, show evidence of bioturbation, and include fossils of diverse benthic fauna. A number of these beds also exhibit evidence of sediment starvation and sedimentary condensation, such as ‘schlacken’ or time averaged accumulations of mollusk and vertebrate debris 16.The lowest member (Member I) generally consists of two or more intervals of non-laminated and highly bioturbated mudstone (e.g. the ‘Blaugraue Mergel’ and ‘Aschgraue Mergel’) separated by one or two layers of relatively hard yet bituminous black shale (e.g. ‘Tafelfleins’ and ‘Seegrassschiefer’), which are inconsistently laminated. The mudstones encompass a diversity of benthic body and trace fossils, whereas the shales contain few benthic body fossils and ichnofossils other than the subhorizontal branching burrow system, 16. Shell beds, schlacken, bioturbation, and assemblages of diverse benthic fauna are relatively common in the Wilder Schiefer46.Member III corresponds to the Wilder Schiefer, another unit of non-laminated calcareous mudstone and shale. It generally contains greater amounts of carbonate and lesser amounts of organic matter than the more bituminous rocks in the lower part of Member II48. Fish, marine reptiles, arthropods, echinoderms, bivalves, gastropods, ammonites, belemnites, and other coleoids occur through the succession; however, most of the exceptionally preserved fossils occur between the Koblenzer  to the middle of the falciferum/serpentinum biozone , and encompasses the negative carbon isotope excursion that was produced by the TOAE48.Given this succession of facies and the types of fossils present in the Posidonia Shale Lagerstätte, the organisms were most likely preserved in the deep water of an anoxic, stagnant marine basin during a slow transgression48. In addition, the presence of pyrite framboids are indicative of anoxic conditions in the water column and/or sediment pore water , and sulfur levels at Dotternhausen indicate long-term anoxia/dysoxia with periods of oxygenation, which lasted for weeks to several years50 with most specimens collected from the Ya Ha Tinda Ranch in Alberta, Canada9. In the area around Ya Ha Tinda Ranch, the Red Deer Member overlies rocks of the Middle Triassic Sulphur Mountain Formation, and consists of grey to black platy calcareous shale interbedded with fine siltstone and fetid black limestone51. The overlying lower Toarcian Poker Chip Shale consists of finer-grained and predominantly poorly-cemented fissile black, calcareous shales and mudstone52. The Poker Chip Shale is overlain by the Highland Member of the Fernie Formation53. The predominance of finely laminated rocks lacking major sedimentary structures54 suggest that they were deposited below fair weather wave base in a deep-water, open marine setting55.The Ya Ha Tinda Lagerstätte belongs to the Red Deer and Poker Chip Shale members of the Fernie Formation e.g.kunae, carlottense and part of the kanense biozones of the late Pliensbachian and lowermost Toarcian  and the Poker Chip Shale was deposited in the kanense and planulata zones of the later Toarcian 52. Exceptionally preserved fossils occur throughout this interval at Ya Ha Tinda18.The Pliensbachian/Toarcian boundary occurs within the Red Deer Member, and is located 1 m below its contact with the Poker Chip Shale52. Chemostratigraphic data places the TOAE within the Poker Chip Shale Member, with the onset of the carbon isotope excursion just at the end of the Red Deer Member52.According to ammonite biostratigraphy, the Red Deer Member spans the 56, ichthyosaurs53, crinoids57, crustaceans59, brachiopods, coccolithophores, trace fossils, bivalves, gastropods, ammonites60, and coleoids like vampyropods62. Exceptionally preserved fossils include crustacean (shrimp and lobster) cuticles59; coleoid gladii with ink sacs and mantle muscle tissues62; crinoid calyxes57, articulated fish56, and ichthyosaurs53. Like the Posidonia Shale, geochemical data  suggest that parts of the Ya Ha Tinda Lagerstätte were deposited under anoxic and even euxinic conditions29. Nevertheless, it is important to note that several well bioturbated layers, benthic colonization events60, and phosphatized fossil specimens18 are also found within these units, which indicate that intervals of oxygenation were rare to common throughout the depositional history of the succession.The Red Deer and Poker Chip Shale members contain fish63. See supplementary information for detailed methods. Material from the Strawberry Bank, Posidonia Shale, and Ya Ha Tinda deposits are reposited at Bath Royal Literary and Scientific Institution (BRLSI), Bath, UK; Non-vertebrate Paleontology Laboratory (NPL), University of Texas at Austin, USA; and Royal Tyrrell Museum of Palaeontology (RTMP), Alberta, Canada, respectively. Fossils analyzed include articulated teleost fish, crustacean carapaces, and coleoid gladii and soft tissues from all deposits . These data indicate that fossils consist of phosphatic and carbonaceous material as well as auxiliary minerals, including barite, calcite, pyrite, sphalerite, and aluminosilicate minerals18; additional images are displayed in Supplementary Figs. 63, generally occurring as thin, dark (low average atomic number) layers on phosphatic material and as void fill within fossils18 , formed in response to local/regional processes, like sedimentation rate, pore water pH, sedimentary organic carbon content, reactive Fe (or Zn) availability, and sulfate levels72.The three Toarcian Lagerstätten come from different regions , which may be a terrestrial deposit, all of these 11 “Lagerstätten” belong to the Lias Group of Europe, and are represented almost entirely by insect fossils. By and large, these fossils are preserved in calcareous nodules/concretions within marine or restricted marine “clays” of limited exposure. Therefore, they resemble the Strawberry Bank in terms of geology and preservation7. Like Strawberry Bank, these other Lias deposits belong to the tenuicostatum and serpentinum biozones suggesting that they all occur within beds that can be correlated with the negative carbon isotope excursion of TOAE. Depending on the locality, the fossils may represent a single bed (like Strawberry Bank) or a range of strata . Fossils at Grimmen, Dobbertin, Braunschweig, and other places have been reported from multiple sub-biozones (the elegantulum and exaratum subzones), suggesting that the exceptional preservation occurred throughout the duration of the event at these localities. In any case, although the taphonomy of these Lagerstätten have not received much attention, reports suggest that fossiliferous bedding planes such as those at Dobbertin are covered with phosphatized algae and that insect tissues at Gimmen have been replaced with apatite minerals.Other Toarcian Lagerstätten may provide additional evidence of phosphatization during the TOAE73. In marine environments, natural sources of P include nutrient runoff (weathering) and oceanic upwelling oxic bottom water18.Phosphatization occurs in environments conducive to phosphogenesis, or precipitation of P as apatite minerals in sedimenting Fig. . Phospho18. Although anoxic water masses did not directly cause exceptional preservation during the Early Jurassic, they set the stage by delaying carcass disarticulation, inhibiting soft-tissue decay, releasing P from sediments, enhancing P from upwelling and erosion, and/or trapping nutrients in the water columns of basins18  and Tethys Ocean basins . This work suggests that pulses of ocean anoxia lead to widespread preservation of Lagerstätten irrespective of depositional environment. Thus, our results indicate that lagerstätten may occur in a multitude of OAE facies. Use of this search criterion may lead to discoveries of exceptionally preserved fossils in other OAE (e.g. Cretaceous) intervals and provide new insights into the history of marine life.Supplementary Information.


Onchocerca volvulus infection, in order to determine their potential as a novel vector control-based approach to fight onchocerciasis. An entomological survey of a collection of samples was performed in the Bafia health district, a historical endemic focus for onchocerciasis in Cameroon. A total of 1270 blackflies were dissected and the infection rate was 10.1%, indicative of ongoing transmission of onchocerciasis in the surveyed communities. Sequencing process of blackflies’ gut DNA for bacteria screening revealed 14 phyla and 123 genera, highlighting the diversity of gut blackflies bacterial communities. Eight bacteria formed the core of blackfly bacteriome and Wolbachia was the predominant genus with 73.4% of relative abundance of blackflies’ gut bacterial communities. Acidomonas and Roseanomas genera were significantly abundant among infected blackflies (p = 0.01), whereas other genera such as Brevibacterium and Fructobacillus were associated with the absence of infection (p = 0.0009). Differences in gut bacterial distribution of blackflies according to their infection status by the parasite suggest a causal relationship between the bacteriome composition and the onset of blackflies’ infection by O. volvulus or vice versa. Blackfly native bacteria are then potentially involved in infection by O. volvulus, either by facilitating or preventing the parasite infestation of the vector. These bacteria represent an interesting potential as a biological tool/target for a novel approach of vector control to fight onchocerciasis.Vector control using larvicides is the main alternative strategy to address limits of preventive chemotherapy using ivermectin for the control of onchocerciasis. However, it remains substantially limited by implementation difficulties, ecological concerns and the resistance of vector populations. Therefore, efficient and environmentally safe alternative control strategies are still needed. This study explores the composition of the blackfly bacteriome and its variability in the presence of  Onchocerca volvulus. The vectors are blackflies belonging to the genus Simulium; they are arthropods that breed in the oxygenated waters of fast-flowing rivers [O. volvulus, during a subsequent blood meal. The larvae migrate to the subcutaneous tissue where they form nodules and undergo successive molt until the adult stage, with an average lifespan estimated at 10–15 years [Onchocerciasis or river blindness is an infectious disease caused by the parasitic nematode, g rivers . Followig rivers ,2 and pe15 years . After m15 years . Indeed,O. volvulus infections worldwide: 14.6 million of the infected people had skin disease and 1.15 million had vision loss [The Global Burden of Disease Study estimated in 2017 that there were 20.9 million prevalent ion loss  and exceion loss  among peO. volvulus is microfilaricidal, and adult worms are lifelong, treatments should be repeated annually or multi-annually to expect transmission interruption. Preventive chemotherapies through the community-directed treatment with ivermectin (CDTI) strategy led to the interruption of the transmission of the disease in four of the six onchocerciasis foci in Latin America [O. volvulus to ivermectin [Ivermectin was registered for the control of onchocerciasis in 1987 ,12. Sinc America ,14. Howe America ,16. Inde America ,16. The  America ,18 (ii) ermectin , and  demonstrated the impact of their microbiome in the vector competence, as well as their promising role as effective tools/targets for new generations of vector control strategies ,29,30. Ieir host ,32. Whilprolixus ,33, whilasmodium .O. volvulus infection and thus assess their possible impact on onchocerciasis transmission.Hence, native bacteria of vectors can be targeted and/or manipulated in different ways for vector control, notably as chemotherapeutic target, immunological reinforcement or cytoplasmic incompatibility, by inducing through genetic manipulation, a disturbance of molecular interactions between the parasite and vector host ,35. The ; 95% CI: 14.3–18.4) were parous and screened for Onchocerca volvulus infection , followed by Bayomen  and Nyamongo , although the difference was not statistically significant (p = 0.405). Analysis of DNA samples from blackfly heads revealed five samples positive to O. volvulus, with an infectivity rate of 2.4% (95% CI: 1.0–5.5), and no significant differences in infectious blackflies distribution among geographic areas (p = 0.42).From the 1270 dissected blackflies, a total of 207 , were selected for sequencing. Sequencing of 16S rDNA from total DNA extracted from blackflies intestine using Illumina sequencing technology generated a total of 3,427,049 high quality sequence reads across the V3–V4 region. After filtering of contaminants, the average number of tags per sample for the V3–V4 region was 60,819 , with read length varying from 300 to 400 nucleotides. A rarefaction curve showed the saturation of most of the samples between 80,000 and 100,000 reads. This range corresponds to a minimum richness in Operational Taxonomic Units (OTUs) estimated at 100 for all samples .Euryarchaeota phylum). For further analysis, rare taxa were filtered by excluding those presenting with less than 0.05% of relative abundance across all samples and those present in less than 10% of sequenced samples to exclude potential residual contaminants. Using these filters, a total of 14 phyla were found fulfilling defined criteria and retained for further investigations. Among retained phyla, Proteobacteria represented the most predominant bacteria phylum with 77.1% of global relative abundance across the 42 samples and represented more than 90% in proportion for 55% of samples. Other phyla were unevenly distributed among the different samples.The taxonomic assignment of OTUs sequences allowed for the identification of 23 phyla, among which 22 belonged to the Bacteria kingdom and only one belonged to Archaea (Wolbachia with 73.4% of relative abundance (ranging from 1.46% (AN29) to 94.1% (AP19), respectively, as minimum and maximum relative abundance across all samples) was distinctly separated from the other genera. However, the map showed a slight contrast on these bacterial distribution with a more uniform color print at the right half of the heat map. This observation was strengthened by the hierarchical clustering of the samples on the basis of the relative abundance of considered bacterial genera, which allowed for discriminating between the two main clusters: cluster 1 including samples AN40 to AN35, and cluster 2 including AN27 to AP20  with relative abundance of 84.4% of the core bacteriome, followed by Gluconobacter and Flectobacillus genera, with relative abundance of 2.2% and 1.7% among OTUs, respectively. The less abundant bacterial genera were Deinococcus and Thermicanus, with a relative abundance of 0.3% and 0.2%, respectively.Eight bacterial species were systematically present in all samples . These bp = 0.349)  of sampled blackflies (= 0.349) .Wolbachia in the presence of parasite (infected blackflies) (p = 0.2), the proportion of the majority of the core bacteria genera was globally similar regarding the infection status of blackflies. Considering the overall bacterial composition of blackflies’ gut (including bacteriome core components), the presence of parasite (infected blackflies) was negatively associated with the bacteriome variability.Investigating the potential factors associated with the variability of the core bacteriome components  revealedThe investigation of potential relationships between the gut bacterial communities of blackflies and their infection status (infected vs. uninfected) was done to highlight potential complex associations between gut blackfly bacterial composition and the other covariate. A hierarchical clustering using the Bray–Curtis index did not discriminate unambiguously the different groups regarding the infection status . HoweverAcidomonas, Roseamomas, Cnuella and Serratia, whereas other bacterial genera were significantly associated with uninfected status, including Fructobacillus, Sanguibacter, Phaseolibacter and Brevibacterium. At the level of species, Unclassified Wolbachia was significantly associated with infected blackflies whereas Unclassified Micrococcus, Fructobacillus Unclassified Fructobacillus were significantly abundant in uninfected blackflies.In order to determine potential biomarker(s) with reference to bacterial genera associated with specific feature (infected/uninfected status) of sampled parous blackflies, we performed richness analysis of each taxonomic unit in different conditions using feature selection method . Table 2This study’s aim to characterize the whole bacterial community within the blackflies’ gut and to assess their potential associations with vector competence is, to our knowledge, pioneering in onchocerciasis. Nonetheless, this approach based the on successful outcomes of parallel studies on other vector-borne diseases is a fundamental prerequisite for application of vector control strategy-based on modified non-infestable blackflies to gradually reduce disease transmission in onchocerciasis endemic areas.The global infestation rate was 10.0% in the Bafia health district, suggesting that onchocerciasis transmission is still ongoing in this historically endemic area, despite almost three decades of community-directed treatment with ivermectin (CDTI). Our results confirm previous evidences supportive of ongoing transmission both within the human population   and in vAccording to both infestation and infectivity rates, Biatsota was considered as the most active community in terms of disease transmission; this situation is likely to be related to its geographical situation which is closer to the Mbam River as compared to other surveyed communities. Importantly, in this community, economic activities are more intense along the river, thus increasing the biting rate in contrast to Bayomen and Nyamongo communities where inhabitants are living less close to the river, hence contact with blackflies are less frequent.Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus using 453 pyrosequencing technique revealed that they were made up of less than six bacterial phyla [In this study, taxonomic assignment allowed us to identify a total of 14 phyla and 123 genera. This result highlights the diversity of gut blackflies bacterial communities, which seems significantly larger than the bacterial diversity reported in many recent studies on other arthropods of medical importance such as tsetse and mosquitoes. Indeed, studies on tsetse, using the same molecular approach, showed that the gut bacteria communities were made up of 14 phyla and 83 different bacteria genera ,32; meanal phyla .Wolbachia was the most important bacterial genus of blackflies’ gut bacteriome. This result is in agreement with previous estimates suggesting that Wolbachia infects more than 65% of all insect species [Wolbachia is also an important endosymbiont of numerous worms of medical importance such as the main filarial parasites  [Loa loa [Our findings revealed that Proteobacteria was the predominant phylum of the core bacteriome of blackfly. This phylum common to several insects ,32,35,40 species , though  species ,45. Beyoncrofti) ,46,47 ex[Loa loa ,49 whereAnopheles [Trypanosoma rhodesiense. However, differences could not be exclusively associated with the ecological differences of surveyed foci, but also with tsetse species  that are commonly found in different biotic and abiotic habitats. In this frame, even though Simulium damnosum complex is known as the important vector for O. volvulus in Cameroon [S. yahense and S. squamosum are associated with forest and forest–savannah transitional zones [Simulium genetics of these localities to ensure if the highlighted homogeneity of bacterial communities within captured blackflies is shared by a common Simulium species or if there are different Simulium species with similar bacterial communities.The analysis of bacterial taxa from blackflies’ gut did not show significant differences in bacterial composition on blackflies originating from the three surveyed communities. This could be explained by the fact that the three selected communities, Bayomen, Biatsota and Nyamongo, are located in the same geographical area , within ~20 km and share both the same bio-ecological  and environmental features. These observations were similar to those recorded on nopheles  where nonopheles ,32, demonopheles  who repoCameroon ,52,53, Sal zones ,52. FurtSerratia, Acidomonas, Roseamomas and Cnuella) and the presence of the parasite. These bacteria potentially improve the susceptibility of blackflies to Onchocerca volvulus infection. Among these biomarkers, only Serratia. sp has been described in other arthropod vectors and its role seems to be vector-dependent. In Mosquitoes, Serratia odorifera has been associated with the susceptibility of Aedes aegypti both to chikungunya virus [Serratia marcescens to produce some trypanolytic compounds that increase the refractoriness of Rhodnius prolixus to T. cruzi infection [Serratia has also been described as facultative bacteria in blackflies [Brevibacterium, were found to be significantly associated with the absence of infection among blackflies. This Gram-negative bacterium was not yet reported to play a biological role in any vector-borne disease, thus opening a potential research avenue with possible outcome of interest. Beside genera, Wolbachia Unclassified Wolbachia was the single bacteria species significantly associated with the presence of the parasite infection in blackflies. Wolbachia are commonly classified in different super groups showing an asymmetric distribution within a large host range including both arthropod and filarial hosts. Further studies need to be undertaken to fully describe Wolbachia Unclassified Wolbachia. However, the overwhelming presence of the genus Wolbachia as a result of the sequencing of the whole blackflies’ gut bacteriome could have lowered the efficiency of amplification process of low abundant or rare bacteria genera with potential biological implications, hence increasing the sequencing depth associated with exclusion of Wolbachia amplification could increase the probability of detecting other rare taxa with potential biological role of interest for vector control.The investigation of bacterial genera (biomarkers) specifically associated with the infection status of blackflies showed a significant association between the abundance of some genera  could be at the origin of the observed structuration. Thus, it appears necessary to explore, besides the possible involvement of intestinal bacteria in blackflies infection, such a hypothesis in further investigation in order to get a better insight into the complex interactions between the three partners—the blackfly, its intestinal bacteria and the parasite, which are together responsible for the transmission of onchocerciasis.In addition to these questions about the possible association between intestinal bacteria and the susceptibility/resistance of blackflies to infection with The main limitation of this study is the fact that the methodology did not allow us to determine the precise anatomic location of identified bacterial in the vector since the whole abdomen of blackfly was considered for sequencing. In addition, the number of uninfected blackflies since the bacteriome comparison did not take into account nulliparous blackflies to reinforce the postulate about the origin of some bacteria of interest . Nevertheless, the methodology (control of sources of contamination during sampling/samples processing and consideration of negative controls) and the data analysis (use of high filtering thresholds) were sufficiently rigorous to detect and exclude potential contaminants an improve integrity of the data generated.Although this study did not directly involve human subjects, samples (capture of blackflies) were collected using the human landing collection technique, which requires volunteers. Hence, an ethical clearance was obtained from the Centre Regional Ethics Committee for Human Health Research (N°1011/CRERSH/C/2020) and administrative authorizations were granted by the Centre Regional Delegate for Public Health and the Bafia District Medical Officer. Prior to the beginning of the entomological survey, the objectives and schedules of the study were explained to all the volunteers. Participation was entirely voluntary and each of them (aged 24 years and above) was free to opt out without fear of retaliation. The volunteers recruited lived in sampling sites, so they were not more exposed to fly bites than usual. Moreover, volunteers were trained to capture flies before being bitten. Finally, ivermectin was provided as preventive chemotherapy against onchocerciasis.This study was carried out in the Bafia health district, situated in Mbam and Inoubou Division, Centre Region, Cameroon. This health district is known for its historical endemicity to onchocerciasis and disease persistence despite two decades of ivermectin-based preventive chemotherapy. Communities of this health district are mainly watered by the Mbam River and its tributaries, whose falls and rapids promote and maintain blackfly breeding sites throughout the year. The phytogeography of this area shows a forest/savannah transition zone dominated by a peri-forest savannah with forest galleries along the rivers and important breeding sites favorable to the development of blackflies. Bafia is mainly dominated by the subequatorial climate with average temperature of 23.5 °C and bimodal rainfall regime marked by modest precipitations with average rainfall of 831.7 mm. Socio-economic activities are dominated by sand extraction in the Mbam river, as well as agriculture and trade on the shores of the latter.Entomological surveys were conducted in April 2019 in three communities of the Bafia health district, namely Bayomen , Biatsota  and Nyamongo  located between 5 and 10 km from the Mbam River . In eachSimulium damnosum complex [O. volvulus infection since they had already had at least one blood meal from potentially infected individuals, unlike nulliparous flies which are immature flies  and, therefore, could not be infected by the parasite. This latter group of flies was not considered for further analysis. Dissected parous blackflies were separated into four parts: gut, thorax, head and legs, which were individually transferred into well labelled 1.5 mL An Eppendorf tube containing 70° Ethanol was stored at −20 °C for further molecular analysis.From sample collection to statistical analysis of meta-taxonomic data, several measures have been taken into consideration to mitigate potential contaminant DNA and cross-contamination at each step of the process. Blackflies were collected using sterile tubes. Prior to the dissection process, captured blackflies were immersed for about 5 min in a saline solution supplemented with sodium hypochlorite (detergent) and individually isolated for dissection into a sterile drop of water. The dissection process was carried out in a wind-protected area to mitigate potential environmental contaminants and dissecting tools were systematically decontaminated after the dissection of each fly. Blackflies were described based on morphological criteria to ensure their belonging to  complex ,58 and pGenomic DNA was extracted from heads and guts samples and purified on MiniElute PCR purification columns using the QIAamp DNA Mini kit  and eluted in 50 µL molecular biology-grade water. DNA extraction processes were carried out in an isolated environment (laminar-flow hood), surfaces and equipment were decontaminated and exposed to UV light for 15 min prior experiments.O. volvulus by quantitative PCR (qPCR) using specific primers (Forward: 5′-GCTATTGGTAGGGGTTTGCAT-3′ and reverse: 5′-CCACGATAATCCTGTTGACCA-3′) targeting a DNA portion (128 bp) of ND5 O. volvulus gene and probe (5′-FAM-TAAGAGGTTAAGATGG NFQ-3′). Each well of the microtiter plate  was filled with 20 µL of final solution, containing 2 µL DNA template and 18 µL of PCR master mix made up of: 12 µL molecular biology-grade water, 2 µL of 10 × PCR buffer, 2.4 µL 50 × MgCl2 (50 mm), 0.1 µL dNTPs (10 mm), 0.6 µL forward primer (10 mm), 0.6 µL reverse primer (10 mm), 0.2 µL ND5 O. volvulus probe, and 0.1 µL HotStarq polymerase (5 U/µL). For each amplification process, negative and positive controls were used to ensure good interpretation of final results. Real-time PCR assays were performed on an Applied Biosystems Step One Plus real-time PCR machine  using the following cycling conditions: initial denaturation at 95 °C for 10 min, followed by 45 cycles, each including a denaturation step at 95 °C for 1 min, an annealing and elongation step at 60.1 °C for 30 s.DNA extracted from gut and head samples were processed for the detection of http://www.mrdnalab.com/shallowater (accessed on 21 December 2021), Shallowater, Texas, USA). PCR was performed using the HotStar Taq Plus Master Mix Kit  under the following conditions: 94 °C for 3 min for initial denaturation, followed by 30 cycles of successive steps: denaturation at 94 °C for 30 s, annealing at 53 °C for 40 s and elongation at 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. After amplification, PCR products were checked on 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple PCR products were pooled together in same proportions based on their molecular weight and DNA concentrations. Pooled PCR products were purified using calibrated Ampure XP beads (Details on Manufacturer). Then, the pooled and purified PCR product were used to prepare Illumina DNA library. Sequencing process was performed at MR DNA  (accessed on 21 December 2021), Shallowater, TX, USA) using a MiSeq following the manufacturer’s guidelines.The 16S rRNA gene V3-V4 variable region was amplified using specific primers designed in the scope of a previous study  to assesPCR blanks (where DNA free water was used instead of DNA template) were run as a control process for generated libraries. If PCR blanks showed any signal (likely contaminants), the experiment was repeated, as the PCR control (blank PCR) under 30–35 cycles for a library should not generate any signal.http://dx.doi.org/10.14806/ej.17.1.200) (accessed on 21 December 2021) with those primers to extract V3-V4 reference sequences from the SILVA SSU database (release 132). The generated sequences were deposited in the EMBL-EBI .Prior to running the metabarcoding pipeline, a specific reference file for the assignment step was generated. This was achieved by running CutAdapt v1.8 (https://dx.doi.org/10.7717%2Fpeerj.2584) (accessed on 21 December 2021) was used in combination with CutAdapt for the following series of tasks: (i) merging the forward and reverse reads of each sample; (ii) demultiplexing to obtain one fastq file per sample; (iii) clipping barcodes and primers; (iv) excluding sequences containing unknown bases; (v) calculating expected error rate; and (vi) performing sample-level dereplication. The remaining sequences were then pooled into a single FASTA file to allow VSearch to carry out a global dereplication, after which clustering was applied to remaining sequences using Swarm v2.2.2 (https://dx.doi.org/10.7717%2Fpeerj.1420) (accessed on 21 December 2021). VSearch was then used again to identify chimeric clusters.The first stage in the workflow consisted of filtering read quality using CutAdapt with a threshold value of 20. Then, VSearch v2.14 (https://github.com/frederic-mahe/stampa) (accessed on 21 December 2021) pipeline was then run for taxonomic assignment of representative OTU sequences based on the contents of the specific reference file generated from SILVA SSU records. This generated an OTU table to which the following filters were applied in order to retain targeted taxa at genus level: elimination of clusters with a high expected error (>0.0002) and elimination of small clusters (less than 3 sequences) observed in a single sample.The STAMPA (https://dx.doi.org/10.1093%2Fbioinformatics%2Fbtw725) (accessed on 21 December 2021), an online software dedicated to bacterial taxonomic analysis.Data obtained from bioinformatics analysis of raw data  was formO. volvulus parasites at the level of gut and head, respectively. Parity rate was calculated as the proportion of dissected blackflies that had already taken a blood meal at least once (parous blackflies). For all proportions, the exact (Clopper–Pearson) 95% confidence intervals (95% CI) were calculated as a dispersion parameter. Bacterial Relative richness was computed as the proportion of normalized operational taxonomic unit (OTU) among the overall OTUs identified, considering single, part or complete sequenced blackflies samples. Prior to data analysis, pre-filtering steps were performed to improve statistical analysis and counteracts sequencing errors. Filtering step included the removal of samples with less than 1000 sequence reads, removal of taxa having less than 0.05% of relative abundance across all samples. In addition, OTUs identified as contaminants (mainly associated with DNA extraction process) were filtered and excluded from the data set prior to data analysis. Total Sum Scaling (TSS) normalization combined with square root transformation (Hellinger transformation) was used to normalize count data by dividing feature read counts by the total number of reads in each sample. This method converted raw feature counts to relative abundance.Infection and infectivity rates were computed as the proportion of parous blackflies harboring Heat map was used for intuitive visualization of the bacterial genera distribution among samples. It highlighted the contrast in bacterial composition with associated clustering trends and relationships between relative richness and sample distribution. Color histogram and camembert plots were used to summarize the blackflies’ gut core bacteriome composition, according to geographical origin and the infection status of blackflies. A dendrogram was used to represent the random clustering of sample based on bacterial composition in order to highlight potential association with co-variables .A Chi-squared test was used to assess the difference of the prevalence of infection between geographical origins. Given that OTU associated relative abundance are typically over dispersed and therefore do not follow a normal distribution, the non-parametric Mann–Whitney U and Kruskal–Wallis tests were used to compared average bacterial abundance between infection status and geographical origin of samples, respectively. Shannon and Simpson Indexes dedicated to the measurement of bacteria community heterogeneity were used to compare the diversity of different groups of samples  using raw data . Shannon index was used to describe the disorder and uncertainty of individual genera; the uncertainty was proportional to the diversity, i.e., the diversity increased with the number of genera. Simpson diversity index was computed as the probability that two bacteria randomly sampled belong to different genera. The greater the Simpson index, the higher the diversity. For all analyses, the threshold of significance was set at 0.05.Wolbachia. Some bacteria genera are significantly associated with the presence of O. volvulus in blackflies while others are refractory to it, giving an insight of biomarkers with interesting potential as biological tool/target for developing of non-infestable blackflies.This study exploring the blackfly bacteriome is, to our knowledge, pioneering in onchocerciasis. It reveals the existence of a core bacteriome of blackfly dominated by the genus


Zootoca vivipara) occurrence records during the reproductive period and over the entire year.Parity mode (oviparity/viviparity) importantly affects the ecology, morphology, physiology, biogeography and evolution of organisms. The main hypotheses explaining the evolution and maintenance of viviparity are based on bioclimatic predictions and also state that the benefits of viviparity arise during the reproductive period. We identify the main climatic variables discriminating between viviparous and oviparous Eurasian common lizard . They suggest that during the reproductive period viviparity has an adaptive advantage over oviparity in less risky habitats thanks to the selfish behaviour of the mothers. Moreover, the results from both analyses stress that hypotheses about the evolution and maintenance of viviparity need to be tested during the reproductive period. Reproductive mode  is an important biological trait that may affect the ecology, morphology, physiology, biogeography and evolution of organisms –3. WhileBoth hypotheses exclusively explain the maintenance and evolution of viviparity by a positive effect on offspring fitness. However, selection favours strategies that increase an individuals’ rather than its offspring’s fitness , 13. ThuThe SSH predicts that females favour their own lifetime reproductive success and survival, rather than her offspring’s sucess . BecauseDespite the differences among the three hypotheses, they all predict that viviparity is an adaptation to the environmental conditions prevailing during the reproductive period, rather than those prevailing during the entire year e.g. , 23). St. St23]).Moreover, several studies highlighted that current distributions may not reflect the climate at the time and place where viviparity evolved , 22. ThiZootoca vivipara . Zootoca vivipara is a small lizard species with bimodal reproductive mode that exhibits the largest geographical and the northernmost distribution of terrestrial reptiles [Z. vivipara, niche comparison tests, and discrimination models of parity mode (oviparous versus viviparous) based on climatic data. According to the CCH [1). According to the MMH [1) or warmer locations . According to the SMH, we predicted that viviparous females inhabit locations in which they are less likely exposed to a trade-off between their own survival and fitness, and that of their offspring. Because viviparous Z. vivipara females take almost twice as long to lay their eggs , in which embryonic development is faster and egg laying is earlier [2 [Here we tested for differences in the inhabited climatic niche among oviparous and viviparous clades of the Eurasian common lizard reptiles . It consreptiles  and thusreptiles , Watson reptiles , and Felreptiles , most of the CCH , we pred the MMH , we pred , and/orl stages , 20) andultative , we pred earlier , 11. The earlier , 31). Morlier [2 , 33). Firlier [2 .Fig. 1SDuring the reproductive period, principal component analyses of the climatic variables rendered two axes, which accounted for 76.4% of the variation Fig. . The firDobs than expected by chance . In halCross-validation points to a tree of four leaves (three splits) and a correct classification rate of 82.7% Fig. . 54 out Dobs than expected by chance , the “mis” (CCH ), and/oris” (CCH ). In comis” (CCH ). Both cis” (CCH .1 [3) and/or with higher  or lower average temperature . The re(Table 1 ). Howeveble 1 [1 ). Riskieble 1 [1 , 11, 35 ble 1 [1 , and smaZ. vivipara had shorter telomere length than viviparous Z. vivipara [Z. vivipara may have an additional advantage when both reproductive modes are in contact. The finding that viviparous Z. vivipara inhabit less variable habitats during reproduction is thus congruent with the idea that viviparity provided an adaptive advantage over oviparity in less risky habitats.The higher climatic variability prevailing during the reproductive period in populations of oviparous common lizards may explain why viviparous lizards cannot expand their distributions southwards. In other words, why they cannot colonize habitat inhabited by clade A or B . In contvivipara  and telovivipara , suggestZ. vivipara’s distribution and has been suggested to be the most vulnerable clade to climate change [Clade A inhabits the Southern limit of e change . Northwae change ). The inThe correct classification rate of the classification tree including the reproductive period was higher than that including the annual period  exhibits two reproductive modes and it consists of six genetic lineages (clades A-F) that inhabit large parts of Eurasia . Matingribution , 44 and ribution . Time ofribution  and lastribution . In viviribution , 46, 47.ribution . In vivic signal . Gestatic signal , 49, andc signal , 36.Z. vivipara populations from [Z. vivipara lineages (clades A-F) and covering the majority of the known natural Eurasian distribution , since this metric performs better than other overlap indexes [D was computed  was calculated. For each pair of clades this procedure was repeated 1000 times to obtain the distribution of Dsim and to test whether Dobs is significantly smaller than expected by chance. The analyses were performed with the ecospat package [First, 20′000 pixels  were randomly selected within the area delimited by the convex hull, and a principal component analysis (PCA) was conducted on the environmental variables within the randomly selected pixels and those with occurrence records (20′000 + 185 = 20′185 pixels). Second, a grid of 100 × 100 cells was laid over the ordination space delimited by the two first PCA axes, and a kernel density function was used to create occurrence density plots for each clade. Third, overlaps in occurrence density between two clades were calculated for each pair of clades using the Schoener’s  indexes . Forth,  indexes  and Broe indexes . Brieflyed (Dsim ). This p package  for R [5 package .Recursive partitioning was performed to identify the main variables discriminating between occurrence records of oviparous and viviparous specimens. Classification trees  were run


Altererythrobacter, Aminobacter, Bacillus, Burkholderia, Lysinibacillus, Microvirga, Mycobacterium, Ochrobactrum, Paenibacillus, Pseudomonas, Pseudoxanthomonas, Rhizobiales and Sphingobium. These selected bacterial genera can be of particular interest for studying lignin degradation and utilization, as well as for lignin-related biotechnology applications.Lignin is nature’s largest source of phenolic compounds. Its recalcitrance to enzymatic conversion is still a limiting step to increase the value of lignin. Although bacteria are able to degrade lignin in nature, most studies have focused on lignin degradation by fungi. To understand which bacteria are able to use lignin as the sole carbon source, natural selection over time was used to obtain enriched microbial consortia over a 12-week period. The source of microorganisms to establish these microbial consortia were commercial and backyard compost soils. Cultivation occurred at two different temperatures, 30°C and 37°C, in defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the consortia at six timepoints (passages). The initial bacterial richness and diversity of backyard compost soil consortia was greater than that of commercial soil consortia, and both parameters decreased after the enrichment protocol, corroborating that selection was occurring. Bacterial consortia composition tended to stabilize from the fourth passage on. After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin extracted by alkaline method was used as a carbon source, whereas Proteobacteria were predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus level, visualized using NMDS plots, showed that the type of lignin used as a carbon source contributed more to differentiate the bacterial consortia than the variable temperature. The main known bacterial genera selected to use lignin as a carbon source were  The world is looking for petroleum substitutes, both for energy and for making products that are clean and renewable –4. LignoChemically, lignin is a phenolic heteropolymer formed by the dehydrogenation polymerization of p-hydroxyphenyl (H), guaiacil (G) and syringyl (S) units, derived from the monolignols coumaryl, coniferyl and synaphyl alcohol, respectively . Lignin’Bacteria from the phyla Phylobacteria, Firmicutes and Bacterioidetes are capable of producing enzymes to breakdown lignin . From a In this work, an enrichment strategy was applied to establish microbial consortia selected to use lignin as carbon source. To this end, two sources of microorganisms , two cultivation temperatures  and two sources of lignin  were used to establish eight microbial consortia in defined laboratory medium. Cultivation occurred over a period of 12 weeks, in which an aliquot of the consortia was inoculated into fresh medium every two weeks, totaling 6 subculturing cycles or passages. To understand the dynamics of microbial selection for the ability to utilize lignin as carbon source, we analyzed Illumina 16S rDNA gene sequence (iTag) phylogenetic profiling of bacteria data from these eight microbial consortia over six passages.2SO4 5 M was added until the pH of 7 was obtained. The solution was kept for 24 hours at 4°C, and then centrifuged at 10,000 x g for 30 min at 4°C. The supernatant was transferred to a second flask and centrifuged at 18,514 x g for 1 hour at 4°C; and the new supernatant was sterilized with 0.45 μm pore filter. A total of 50 mL of M9 minimal medium with trace elements [For the establishment of microbial consortia, two sources of microorganisms were used: Miracle Growth commercial substrate and backyard compost soil which was obtained from Alameda County . Commercial Kraft lignin and lignin from switchgrass extracted by alkaline method were used as carbon sources. The alkaline method for lignin extraction consisted in grinding 12.5 g of biomass and adding to it 250 mL of 0.5 M NaOH were added to 12.5 g of biomass and the solution was sterilized by autoclaving for 30 min at 121°C. Subsequently, Helements  was addeelements . At diffelements .5’GTG CCA GCM GCC GCG GTAA 3’ and RV (805R): 5’ GGA CTA CHV GGG TWT CTA AT 3’ primers were used [To identify the bacteria present in each consortium at each timepoint, DNA extraction was performed using All Prep DNA/RNA kit (QIagen) following the manufacturer’s instructions. DNA was then used as template in polymerase chain reactions (PCR) to amplify the copies of the 16S rDNA V4-V5 region. For this the FW (515F): ere used . The fraere used  on the Mere used .The 16S rDNA amplicons were grouped and classified into their respective operating taxonomic units (OTUs) using iTagger 2.0 program with the Usearch tool using 97% similarity by the Joint Genome institute (JGI) . Silva SAs shown in 16S rDNA sequencing data from the original soil (control) were used to estimate the diversity present in each consortium prior to the enrichment process for lignin utilization. As shown on To understand the dynamics during the enrichment process, taxonomic affiliation at family level of 16S rDNA amplicons to operational taxonomic units (OTUS) was performed using 97% sequence similarity . Figs 2 The main bacterial families with relative abundance above 1% in any of the passages were represented in taxonomy graphs Figs  Tables. A similar analysis was performed for backyard compost derived consortia . At the Other 120, an unknown genus of the Burkholderiales order. The consortia that used base-extracted lignin as carbon source  share three genera: Bacillus, Lysinibacillus and Paenibacillus; meanwhile, the consortia that used Kraft lignin  share four genera: Altererythrobacter, Camelimonas, Ochrobactrum and Pseudomonas. The genera Sphingobium was shared by the three consortia BE 37°C, Kraft 30°C and 37°C. The genera present exclusively at BE 30°C were Other 38 (Family: Flavobacteriaceae), Other 44 , Other 86 (Phylum: Other), Other 92  and Other 109 (Family: Rhodobacteraceae). The genera present exclusively at BE Lig 37°C were: Microvirga. The genera that appeared exclusively in Kraft 30°C were: Burkholderia, Devosia, Other 45 (Family: Sphingobacteriaceae), Other 100 (Class: Alphaproteobacteria), Other 111 , Other 124 (Class: Betaproteobacteria), Sphingopyxis, and Taibaiella. The genera that appeared exclusively in Kraft 37°C were: Aminobacter, Inquilinus, Isoptericola, Microbacterium, Roseomonas and Uncultured_2490 (Family: Hyphomicrobiaceae).The Venn diagram in Lysinibacillus, Other 42 (Family: Chitinophagaceae), Other 120 , Unknown_Genus_1000421 (Class: Thermomicrobia), Unknown_Genus_1001041 . The genera present exclusively at BE Lig 37°C were: Brevibacillus, Mesorhizobium, Other 52 (Class: Thermomicrobia), Other 53 (Class: Thermomicrobia), Other 86 (Phylum: Other), Pseudoxanthomonas, Sphingopyxis, Unknown_Genus_1000489 (Family: Paenibacillaceae). The genera that appeared exclusively in Kraft 30°C were Azospirillum, Burkholderia, Dokdonella, Kaistia, Microvirga, Terrimonas, uncultured_2499 (Family: Methylobacteriaceae). The genera that appeared exclusively in Kraft 37°C were Hyphomicrobium, Isoptericola, Mycobacterium, Other 106 (Family: Phyllobacteriaceae), Other 118 , and Rhodococcus. There was no genus present in common for the four conditions . Only 2 genera were present when base-extracted lignin was used as carbon source at both cultivation temperatures (Bacillus and Paenibacillus). On the other hand, 8 genera were present when Kraft lignin was used as carbon source at both cultivation temperatures .The Venn diagram in To evaluate how temperature and type of lignin  influenced the composition of microbial consortia during the enrichment process, genus level data were used for Bray-Curtis Non-Metric Multidimensional Scaling (NMDS) matrix dissimilarity analysis . NMDS gr2 of 0.32559 was obtained . Furthermore, temperature, passage and soil presented R2 values of 0.12246, 0.08914 and 0.05114, respectively. Bacillus and Lysinibacillus, the family Paenibacillaceae, and order Gemmatimonadales, , two different temperatures  and two types of lignin  were used to maximize the different kinds of bacteria selected during enrichment. At the end of the protocol, eight microbial consortia enriched for the ability to degrade lignin were obtained. The use of enriched microbial consortia has been considered as a promising approach to identify microorganisms involved in lignin degradation and conversion , 27, 30.Similar results of decreased diversity were also observed by Moraes et al. (2018) . when usConsortia started from backyard compost soil showed higher initial richness and diversity in comparison to consortia derived from MG soil, a commercial product. Furthermore, there is a legacy effect for the initial inoculum as the higher bacterial richness and diversity for backyard compost derived consortia persisted at the sixth passage after successive cycles of selection. Therefore, in future similar experiments if the intention is to maximize different taxa at the end of the selection process, it may be important to choose a highly diverse source of microorganisms for the initial inoculum.Our data also show influence of the type of carbon source on richness and diversity indexes. At the end of the enrichment process, consortia that used lignin extracted by alkaline method as carbon source had a higher number of observed OTUs, a higher PD whole tree index and a higher Chao 1 richness index, when compared to the sixth passage of the consortia that used Kraft lignin. It is likely that the lignin extracted by the alkaline method from plant biomass presented some contamination with carbohydrates , which would act as an alternative carbon source. This alternative carbon source may have allowed the presence of bacterial species that do not use lignin in the environment as an exclusive carbon source, and thus may have contributed to a higher richness and diversity.In this study, sequencing data were analyzed down to the genus level, as it was not possible to perform taxonomic affiliation at the level of species. Analysis of the consortia bacterial composition based on the amplification and sequencing of 16S rDNA are dependent on the sequences already deposited in public databases . At the Taxonomic profile graphs at family level revealed the bacterial composition and dynamics of consortia across the six passages Figs  and 3. BLysinibacillus, Paenibacillus and Bacillus from the phylum Firmicutes act on the degradation of Kraft lignin in the presence of 1% glucose and 0.5% peptone as an alternative source of carbon and nitrogen [When observing the family composition of each consortium, it is noticeable that each consortium tends to acquire its own specific profile. The divergence in bacterial family composition among consortia indicates that the variables used during the enrichment process, such as temperature and substrate, changed the composition of initial bacteria in the consortia. Our results indicate that the type of lignin used as a carbon source in the enrichment experiment, Kraft lignin or lignin extracted by alkaline method, favored distinct groups of bacteria. At passage sixth, Kraft lignin favored families of the Proteobacteria phylum, while base-extracted lignin favored the Firmicutes phylum. Kraft lignin is a highly pure commercial lignin. As previously mentioned, during the manual process of lignin extraction by alkaline method, small amounts of carbohydrates may remain, and act as an alternative source, acting as a “start-up” carbon source for the degradation of polymers . Certainnitrogen –52. TherThe phyla Bacteroidetes and Actinobacteria have also been identified in microbial consortia enriched to degrade lignin , 30, 53.Paenibacillus from the phylum Firmicutes was one of the main genera identified. In our work, the genus Paenibacillus is one of the most abundant in the sixth passage of the enriched consortia that used lignin extracted by alkaline method as carbon source, but its relative abundance was less than 0.5% in the sixth passage of consortia enriched using Kraft lignin.Some of the genera and families found in this work have previously been identified by their ligninolytic activity. In the enriched Lig Met consortium that used Kraft lignin isolated from black liquor obtained from delignification of sugarcane bagass, reported by Moraes et al. (2018) , it was Another interesting fact is that the Flavobacteriaceae family of the order Flavobacteriales, Pseudomonadaceae of the order Pseudomonadales, Sphingobacteriaceae of the order Sphingobacteriales also have a great representation in the sixth passage of consortia inoculated with both soils. The Sphingobacteriales and Pseudomonadaceae were present in the sixth passage in three of four consortia inoculated with Miracle Growth . Previous works demonstrate that species of this genus present ligninolytic enzymes and pathways to catabolize the compounds generated in the degradation of lignin [Venn diagrams  were usef lignin , 61. In Microvirga represents 1% of the abundance of the MG-soil derived bacterial consortium in the sixth passage of that used base-extracted lignin and was grown at 37°C  and this genus is also present in the sixth passage of the backyard compost soil derived consortia that used Kraft lignin and was grown at 30°C . A species in this genus isolated from nodules of the plant Vicia alpestris, Microvirga ossetia, had its genome completely sequenced and this allowed identification of a laccase [The genus  laccase . This en laccase , 21.Aminobacter genus of the Phyllobacteriaceae family is present in the microbial consortia MG Kraft 37°C and BY Kraft 37°C in the sixth passage  of the order Burkholderiales appeared in all MG soil derived consortia having the greatest abundance in the consortium enriched to degrade Kraft lignin and grown at 37°C  appeared in all BY soil derived consortia as well, and had the highest abundance in the consortium BE Lig 37°C . High-throughput 16S rRNA gene sequencing was used to assess the bacterial community structure during the enrichment process. Consistent with a selection process, diversity indexes showed a decrease in bacterial richness as well as diversity in consortia at the end of six enrichment cycles. Analysis of bacterial composition over 2-week enrichment cycles indicated that stability was achieved beyond the fourth cycle. Although the focus of the present work was bacteria, the presence of ciliates in the consortia may have influenced the dynamics of the bacterial community in the microbial consortia. The main known bacterial genera selected to use lignin as a carbon source were S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table5’ GAA CGC AGC RAA IIG YGA 3’ and RV (ITS4): 5’ TCC TCC GCT TAT TGA TAT GC 3’ [Data based on sequencing of the ITS region*. *Primers used for amplification were FW (ITS9): AT GC 3’ .(DOCX)Click here for additional data file.S4 Table30°C and 37°C. For the families classified as Other and Unknown, we were unable to obtain the taxonomic affiliation at the family level, so the closest previous hierarchical level is provided.(DOCX)Click here for additional data file.S5 Table30°C and 37°C. For the families classified as Other and Unknown, we were unable to obtain the taxonomic affiliation at the family level, so the closest previous hierarchical level is provided.(DOCX)Click here for additional data file.S1 FileBray Curtis matrix PERMANOVA analysis was performed using the adonis function of vegan package in R.(DOCX)Click here for additional data file.S2 FileVectors were found using the envfit function of vegan package in R performing 10,000 permutations.(XLSX)Click here for additional data file.S1 Data(DOCX)Click here for additional data file.


Hierarchical Data Format version 5) hdf5 files with homography matrices between every pair of sequential images per tissue rows.This article presents a dataset of raw microscopic images of the prefrontal cortex from wistar rat tissues, after an induced stroke, stained with NeuN antibody. The raw images were captured using a microscope equipped with a digital camera. The dataset is useful for testing techniques for the improvement, registration, and stitching to generate a high-resolution image with a full reconstruction of tissues. Besides, this dataset can be used to assess the neuronal brain after an ischemic event. The dataset contains 1370 microscope images with 20x magnification and 36 ( Specifications Table•This dataset presents microscope images of microscope capture and the homography matrices between each pair of images. These data are useful to evaluate and develop computer vision algorithms as register and stitching algorithms.•The dataset can be used in pattern recognition and computer vision researchers to develop, improve, and test methods for detection, description, matching or segmentation techniques.•The homography matrices included in this dataset can be used to evaluate errors in registration processes focused on microscope images.•Medical researchers can be allowed to evaluate NeuN immunoreactive changes that may indicate changes in connectivity in any area of the brain, including injured focus, exofocal or any area in the high-resolution image of the whole tissue .1The dataset contains sequential raw images of the prefrontal cortex from rat tissue stained with NeuN antibody tissue in glass slides. There are 36 rows of images captured from right to the left direction, as shown in 22.1Twenty-eight adult, male, Wistar rats were subjected to focal injury in the right hemisphere. The specimens weighted between 240 and 320 g (average 280 g), and were aged between 120 and 180 days. It was followed the protocol to avoid unnecessary suffering.The technique used was the experimental model of focal ischemic injury through intraluminal suture of the middle cerebral artery. Analyses were made for the five groups: and after the lesion (control), at 24 h, 96 h, 10 days, and 20 days.At the corresponding time, the specimens of each group were anesthetized and transcardially perfused through the left ventricle with 200 mL of 0.9% saline, followed by 200 mL of a mixture of paraformaldehyde (4.0%), lysine and sodium periodate in 0.1 M phosphate buffer (pH 7.4). Then, the brains were removed from the braincase and left in the fixative. Later, 10 coronal sections of 30 μm thickness were performed in the vibratome of liquid medium. The sections were incubated with the primary antibody NeuN.Exofocal neuronal damage was inferred from neuronal immunoreactivity changes to NeuN.2.2•Leica ICC50HD  and a vertical overlap of 36% (~0.12 mm between consecutive rows) using a Leica ICC50HD camera and DM500 microscope with 20x magnification. The capture protocol consisted of moving the slide, as shown in The homography matrices were calculated between every consecutive pair of images per row as affine transformations. The homographies were computed using “The experimental protocol was assessed and approved by the ethics committee of the Universidad Libre , to comply with the current regulations of the European Economic Community for the use and care of animals used for experimental and other scientific purposes , and the Colombian law  on ethics, care and control of animals for experimental purposes”.This work does not perform any human or animal experiment, however, images captured from glass slides of tissue from the experiment perform by Bladimir Salas-Quinchucua: Conceptualization, Methodology, investigation, Software; Writing - Original draft preparation, Data curation; Jean P. Díaz-Paz: Conceptualization, Software, Data curation; Humberto Loaiza-Correa: Supervision, Reviewing, Conceptualization, Reviewing and Editing; John Umbarila-Prieto: Data Curation (glass slides), Validation, Reviewing and Editing.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L−1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco ( Endophytes are a class of endosymbiotic microorganisms that internally inhabit plant tissues . Being p−1 [Agrobacterium tumefaciens bacterium-to-plant DNA transfer machinery as an instrument in plant genetic transformation [Agrobacterium growth upon plant tissue culture transformation. Left untreated, Agrobacterium, the causal agent of crown gall disease, shows excessive cell proliferation, which elicits a defense response in plant cells, leading to a detrimental effect on plant tissue growth and efficiency of transformation [Agrobacterium are commonly used [−1 [−1 [To eliminate contamination of endophytic origin, pretreatment with high dose  or mediu−1  and coul−1 . Anotherormation . Althougormation , antibioormation ,24,25. Cnly used ,27,28. Inly used . Howevernly used . Previounly used , and it used [−1  to 500 m [−1 [−1 , sometim [−1 [−1 .Nicotiana tabacum L.) has well established in vitro culture conditions [Bacillus genus were isolated from tobacco leaves [Cultivated tobacco  of the control tobacco shoots (TC) was estimated to be 164.7 ± 3.3 mg (p = 1.5 10−7) compared to the TC shoots.Supplementation of growth medium with 250 mg L± 3.3 mg Table A1−1 FW. The increased MDA accumulation in the control shoots was likely a consequence of the combined effect of stress associated with tissue senescence and shoot injury during the transfer to the fresh medium, which was followed by adaptation and active growth on the fresh medium. This resulted in an ~1.3-fold (p = 4 × 10−6) decrease in MDA concentration at the end of the first week of cultivation (day 7) and a subsequent gradual increase in MDA concentration during the course of culture senescence over the remaining two weeks of the propagation cycle. However, the TA shoots maintained on the antibiotic supplemented medium did not follow this cycle and showed 1.2–1.6-fold (p < 0.014) higher levels of MDA than the TC shoots over the entire period of the propagation cycle  concentration during the three weeks of the propagation cycle Table A1on cycle , which cp = 0.013) compared to TC (p < 0.033) higher compared to the control group over the remaining two weeks of the propagation cycle.Interestingly, after transfer to the medium without antibiotic (PA), a consistent residual negative effect of antibiotic treatment on shoot growth vigor and stress level was observed for at least several passages used in the experiments. Cumulative data from several of these experiments revealed that the growth vigor of the shoots was only partially restored, and that the accumulation of biomass remained significantly lower  but varied up to ~3.5-fold for different DNA preparation methods and primer pairs specific to distinct 16S rRNA gene regions Table A2The proportion of sequences assigned to the bacterial and tobacco plastid or mitochondrial 16S rRNA also varied depending on the PCR amplification region and/or specificity. Two primer pairs specific to the V4 and V8 regions generated the largest number of reads mapped as bacterial OTUs and the largest proportion of the bacterial OTUs compared to the overall number of reads b. MeanwhMycobacteriaceae family was included or excluded from the analysis, respectively. No singleton families were detected using V2-specific primers and 26  singleton families were detected by the remaining four primer pairs.The 16S rRNA region-specific primer propensity for specific taxonomic groups was assessed using family-level data to avoid bias due to the limited accuracy of genus or species identification using short-read sequences which could lead to underestimation of primer specificity. The association among the OTUs obtained using different primer pairs was mapped on the UpSet plot using cumulative data from both experimental groups . Among tMycobacteriaceae family was represented by 37%, 18%, and 39% of the total number of reads using V4, V6–7 and V8 primers, while only 6% and <1% of the total number of reads were assigned to the family for the V2 and V3 primer data sets, respectively. In another instance, the distribution of reads assigned to the Paenibacillaceae family among the V3, V4, and V8 data sets was 15%, 38%, and 44%, respectively, while V2 and V6–7 represented only 3% and <1% of the total number of reads, respectively. Regardless of the apparent taxa-specific amplification efficiency among the primer pairs, the hierarchical cluster analysis of the data did not reveal a consistent distribution of taxonomic groups among the primer data sets  upon antibiotic treatment.Among the six phyla detected in the tobacco shoot microbiome, Bacteriodetes, mainly represented by Sphingobacteriaceae, Chitinophagaceae, and Flavobacteriaceae, but all of them were undetectable in the antibiotic-treated sample. Among the Proteobacteria including 8% and 0.5% of mapped bacterial reads of the TC and PA, respectively, class Alpha-proteobacteria included 10 families of order Rhizobiales representing many well-known beneficial plant-associated bacteria [Caulobacteraceae and Sphingomonadaceae families including common environmental bacteria [Caulobacteraceae and Bradyrhizobiaceae were detected at 4- and 10-fold reduced abundance levels compared to control shoots, respectively. Interestingly, the families Kopriimonadaceae, Rhodobacteraceae, and Rhodospirillaceae, which includes species of common environmental and aquatic bacteria, were detected at low abundance only in the antibiotic-treated shoots.In the control shoots, 1.6% of mapped reads were assigned to five families of bacteria  and Caulbacteria ,49. In tDesulfovibrionaceae family of class Delta-proteobacteria was detected at low but similar abundance in both TC and PA shoots. However, more proliferous Beta-proteobacteria and Gamma-proteobacteria  were also largely reduced upon antibiotic treatment. Among the Beta-proteobacteria were notable Alcaligenaceae and Methylophilaceae which include nitrifying and methylotrophic environmental bacteria [Enterobacteriaceae and Pseudomonadaceae families of Gamma-proteobacteria represent numerous plant endophytes or pathogens [Planctomycetes and Verrucomicrobia, which include common soil and plant root-associated bacteria [The bacteria ,51. Meanathogens . In addibacteria  were botActinobacteria dominant in the in vitro tobacco shoot culture, an extract of antibiotic-treated shoots was plated on Loewenstein–Jensen growth medium. The isolate obtained from bright yellow colonies formed after 6–8 weeks of incubation showed 98% identity to the M. cookii strain ATCC 49103 (GenBank accession NR_114661.1) [−1) and chloramphenicol at 30 mg L−1   based on0 mg L−1 ; howeverOur study confirmed that application of multivariable region-based approach for 16S rRNA gene high-throughput sequencing can provide higher taxonomic resolution of microbial diversity compared to results obtained using individual regions. However, five primer pairs of the 16S Metagenomics Kit used in the analysis showed different specificity for bacterial and tobacco plastid or mitochondrial 16S rRNA sequences. Despite the fact that the V3 region was previously shown to efficiently represent the diversity of microbial communities of fecal or sewage samples ,56, our Proteobacteria but also Actinobacteria, Firmicutes and several less abundant phyla) and their introduction to in vitro shoot culture was demonstrated. The occurrence of endophytic bacteria in seeds is also well documented [Enterobacteriaceae was detected as the predominant species of the endophyte community of tobacco seeds of the four distinct tobacco cultivars, and a genotype-specific signature was observed for Alpha-proteobacteria [Bacillales, Rhizobiales, Burkholderiales, or Enterobacteriales could include plant endophytic bacteria adapted for vertical transmission and capable of inhabiting seed tissues.An abundant microbial community of the control tobacco shoots was revealed by the 16S rRNA gene high-throughput sequencing analysis, and 153 detected OTUs were assigned to 59 families of the Bacteria domain . Bactericumented ,59. Sincbacteria . In our Mycobacterium spp. which form resilient endospores [Sphingomonadaceae are widely distributed in nature, have been isolated from many different land and water habitats, as well as from plant root systems, and can form resilient biofilms that are often capable of surviving sterilization conditions [Alternatively, endospore- or biofilm-forming bacteria are adapted to survive in extreme environments, and many of the bacteria show resilience to sterilizing agents and would endure seed sterilization procedures. Such a route could be adequate for soil bacteria such as dospores , are capdospores ,62. Siminditions .Mycobacteriaceae, and the isolate closely related to Mycobacterium cookii was likely among the dominant species. Mycobacteria are widely distributed in water and soil [Mycobacterium comprises nearly 200 species, to date, research has mainly focused on obligate pathogens and information about plant-associated mycobacteria is relatively scarce. Mycobacterium spp. have been isolated from the rhizosphere of tomato [Mycobacterium isolated from orchid plants was shown to stimulate seed germination and plant growth [Mycobacterium spp. have not previously been described as tobacco endophytes, they could be, particularly depending on cultivation conditions. Specifically, endophytic, or epiphytic colonization would be plausible for the plants cultivated on peat substrate considering that mycobacteria are abundant in peat as they are common inhabitants of sphagnum vegetation [M. cookii [Mycobacterium spp. have been detected as dominant species in sweet cherry in vitro shoots [The control tobacco shoot microbiome was largely dominated by and soil ; howeverf tomato  and ricef tomato . They haf tomato , wheat rf tomato , shoots f tomato , and budf tomato . Endophyt growth ,71,72. Agetation , which w. cookii . It is no shoots  and tisso shoots .Mycobacteriaceae and Microbacteriaceae families  in vitro shoot culture was derived from seeds. Tobacco shoots cultivated in vitro for over five years were used as a control (TC) and were maintained on solid Murashige–Skoog (MS) medium [−1 6-benzylaminopurine, 30 g L−1 sucrose, 0.8% agar at 25 ± 1 °C under 150 μmol m−2 s−1 intensity illumination with a 16 h photoperiod. Antibiotic-treated tobacco shoots (TA) were maintained as described for the TC, except the medium was supplemented with timentin at 250 mg L−1. To ascertain a stable and homogeneous physiological response in the shoot culture, the duration of the treatment was 6 months. Prolonged cultivation was also used to clear residual DNA from antibiotic-inactivated bacteria to avoid false-positive results during the DNA sequencing-based shoot microbiome analysis. After transfer to medium without antibiotic (PA), the shoots were maintained under the same conditions as TC and at least one culture passage (one-month duration) was used before collecting samples for the analysis.Cultivated tobacco  medium  supplemeSamples for shoot fresh weight (FW) and microbial composition analysis were collected three weeks after transfer to fresh medium, and oxidative stress injury was assessed at four distinct time points during the propagation cycle.g for 15 min at 4 °C. Equal amounts of tissue extract and 0.5% thiobarbituric acid in 20% trichloroacetic acid were mixed, heated at 95 °C for 30 min, cooled on ice, and centrifuged at 10,000× g for 5 min. The absorbance measured at 532 nm was corrected by subtracting the absorbance value at 600 nm and MDA concentration was estimated using ε = 155 mM−1 cm−1. The absence of interference from the absorbance of anthocyanins at 532 nm was verified using control samples without thiobarbituric acid.Oxidative injury of tobacco shoot cellular membranes was estimated based on quantitative analysis of the lipid peroxidation product malondialdehyde (MDA) using a previously described method ,84. Homo2 and stored at −70 °C. To assess which methods are most efficient for bacterial DNA extraction from the plastid and mitochondrial DNA-rich plant material, four different DNA extraction methods described by [The samples from the TC and PA experimental groups were fresh frozen in liquid Nribed by ,41,85,86ribed by  were useribed by . Equal v−1), chloramphenicol (30 mg L−1), or rifampicin (25 mg L−1).Tobacco shoots were homogenized with a razor blade in MS medium, the homogenate was applied to Loewenstein–Jensen medium prepared from TB-Medium base  and incubated at room temperature for 6–8 weeks. The bacterial isolate was maintained on Actinobacteria Isolation Agar (AIA). Bacterial DNA was isolated using the GeneJET Genomic DNA Purification kit . The 16S rRNA gene fragment was amplified using universal primers as described previously  and the Statistically significant differences in shoot FW and MDA accumulation were assessed by ANOVA analysis and Tukey’s post hoc test using IBM SPSS Statistics v.21 . Data are presented as the mean of at least 3 independent experiments and standard error of the mean.The 16S rRNA gene sequencing data were rarefied to the minimum library size and total sum normalization was applied to taxonomic count data by dividing feature read counts by the total number of reads in each sample. Nonmetric multidimensional scaling (NMDS) was performed using the Microbiome Analyst  and HeatMycobacteriaceae family, which potentially could originate from seeds used to initiate the in vitro culture and might include endophytic species closely associated with the plant host as well as contamination of resilient environmental bacteria. The antibiotic treatment-induced decline in taxonomic diversity and increased dominance of Mycobacteriaceae and several other families was likely associated with antibiotic resistance traits of the bacteria. It was proposed that antibiotic-induced perturbation of shoot microbiome composition and/or interactions might contribute to the reduced adaptive capacity and impede the growth of tobacco shoots, leading to the reduced efficiency of in vitro culture propagation. Therefore, further insights into the specific role of the members and interactions of the tobacco shoot microbiome would ensure a better understanding of the antibiotic effect on the in vitro tissue and would potentially provide solutions required to improve the efficacy of the in vitro tissue culture.Our study revealed that the antibiotic timentin-induced suppression of growth and elevated stress level of tobacco in vitro shoots was followed by an enduring residual negative effect after shoot transfer to medium without antibiotics. The analysis of the control tobacco shoot microbiome detected an extensive bacterial community dominated by the


Early overfishing in the Gulf of Mexico highlights vulnerability of one of the world’s most productive marine fishery areas. 34S, δ13C, and δ15N) and zooarchaeological evidence from Gulf of Mexico fisheries to assess cultural, demographic, and technological changes affecting sheepshead  populations and fishing practices in Louisiana, USA. Concurrent with human population growth, average sizes of sheepshead caught decreased from the 1720s to 1830s. The size of fish caught after the 1830s increased to pre-1720 levels at the same time that isotopic compositions of fish bone collagen show that fish were being caught from a more diverse range of ecosystems, including distant seagrass beds. Our findings provide the first evidence for large-scale depressions of historical sheepshead populations and the processes driving them, including rapid human population growth and sustained harvesting pressure.Fisheries encompass complex interplays between social, economic, and environmental factors, but limitations on historical fisheries data can hamper efforts to identify and contextualize the long-term spatiotemporal patterns that shape them. We integrate 2500 years of stable isotope (δ Of the global challenges facing our oceans, overfishing is the most impactful on both marine biota and the human societies that depend on them , a marine fish cosmopolitan to coastal eastern North America, is emblematic of the fishing down the food web phenomenon in the Atlantic United States and Gulf of Mexico. Sheepshead are highly adaptable, allowing them to thrive in a wide range of coastal habitats, from freshwater to salt water. While sheepshead have always been fished by communities living across this vast coastal region  were ±0.11‰, ±0.07‰, and ±0.20‰, respectively; random errors [uRw) 300  coupled via continuous flow to a Nu Horizon isotope ratio mass spectrometer  in the Water Quality Centre at Trent University  and on 1.5 mg of collagen using a Carlo Erba NA Series EA  coupled to a Thermo Delta V IRMS  at the Center for Applied Isotope Studies at the University of Georgia . Stable sulfur isotope analyses were performed on 6.5 mg of collagen along with 10 mg of a combustion enhancer  Archaeology Laboratory following standard zooarchaeological procedures  and total area  with the SIBER package in R 3.0.3  Version 3.22 (