Oomycete metabarcoding reveals the presence of Lagenidium spp. in phytotelmata
The oomycete genus Lagenidium, which includes the mosquito biocontrol agent L. giganteum, is composed of animal pathogens, yet is phylogenetically closely related to the well characterized plant pathogens Phytophthora and Pythium spp. These phylogenetic affinities were further supported by the identification of canonical oomycete effectors in the L. giganteum transcriptome. In this study, culture-independent, metabarcoding analyses aimed at detecting L. giganteum in bromeliad phytotelmata (a proven mosquito breeding ground) microbiomes were performed. Two independent and complementary microbial detection strategies based on the amplification of cox1 DNA barcodes were used and produced globally concordant outcomes revealing that two distinct Lagenidium phylotypes are present in phytotelmata. A total of 23,869 high quality reads were generated from four phytotelmata, with 52%, and 11.5% of these reads taxonomically associated to oomycetes, and Lagenidium spp., respectively. Newly designed Lagenidium-specific cox1 primers combined with cloning/Sanger sequencing produced only Lagenidium spp. sequences, with a majority of variants clustering with L. giganteum. High throughput sequencing based on a Single Molecule Real Time (SMRT) approach combined with broad range cox1 oomycete primers confirmed the presence of L. giganteum in phytotelmata, but indicated that a potentially novel Lagenidium phylotype (closely related to L. humanum) may represent one of the most prevalent oomycetes in these environments (along with Pythium spp.). Phylogenetic analyses demonstrated that all detected Lagenidium phylotype cox1 sequences clustered in a strongly supported, monophyletic clade that included both L. giganteum and L. humanum. Therefore, Lagenidium spp. are present in phytotelmata microbiomes. This observation provides a basis to investigate potential relationships between Lagenidium spp. and phytotelma-forming plants, and reveals phytotelmata as sources for the identification of novel Lagenidium isolates with potential as biocontrol agents against vector mosquitoes.
Leoro-Garzon et al. (2019) Oomycete metabarcoding reveals the presence of Lagenidium spp. in phytotelmata. PeerJ 7:e7903 https://doi.org/10.7717/peerj.7903
Artificial substrata increase pond farming density of grass carp (Ctenopharyngodon idella) by increasing the bacteria that participate in nitrogen and phosphorus cycles in pond water
Aquaculture has become a primary method to produce various aquatic products, and intensive aquaculture technologies have become commercially important. To improve the efficiency of intensive aquaculture per unit area without reducing the growth rate of cultured fish, the present study explored the potential of artificial substrata in ponds. Our results showed that the concentrations of total nitrogen (TN) and total phosphorous (TP) in the ponds with different stocking densities of grass carp were lower than those in the control group in most cases. Further, the feed conversion rate of grass carp was significantly reduced by introducing these artificial substrata, and the culture density could be significantly increased without reducing the growth rates of these fish. Artificial substrata also significantly enriched specific bacteria and changed the structure of the microbiota in pond water. The relative abundance of Proteobacteria was significantly increased, and bacteria closely related to N and P cycles, such as Hyphomicrobium, Chitinimonas, Legionella, Shewanella, Roseiflexus, and Planktothrix were significantly enhanced. These results showed that the artificial substratum could increase TN and TP removal in aquaculture pond water by enriching N and P cycle-related bacteria, thus significantly increasing the specific growth rate of grass carp and significantly reducing their feed conversion rate. Finally, the stocking density of grass carp and the yield per unit area of pond could be increased without reducing the growth rate.
Li et al. (2019) Artificial substrata increase pond farming density of grass carp (Ctenopharyngodon idella) by increasing the bacteria that participate in nitrogen and phosphorus cycles in pond water. PeerJ 7:e7906 https://doi.org/10.7717/peerj.7906
An integrative approach to discern the seed dispersal role of frugivorous guilds in a Mediterranean semiarid priority habitat
Seed dispersal is an essential process to maintain the viability of plant populations, and understanding this ecological process allows management strategies to be developed to conserve ecosystems. European Union priority habitat 5220* is defined as “Mediterranean arborescent shrubland with Ziziphus lotus” and it represents a favorable microclimate within the severe climatic conditions typical of the semiarid south-eastern region of the Iberian Peninsula. Therefore, the study of seed dispersal in this priority habitat by different frugivorous guilds, is a challenge for its conservation. In this study, we have characterized a mutualistic network of seed dispersal that is mediated by vertebrates (mammals and birds) in the protected habitat 5220*. The aims of this study were to: (i) identify the seed disperser community; (ii) analyze the relative role of key species in the dispersal process; and (iii) compare the functional ecology of the seed dispersal process between mammals and birds. As such, we collected animal faeces to determine seed dispersers taxonomy, identifying the mammals through the visual aspect of the faeces and the birds by DNA barcoding. In the case of birds, we also collected regurgitated seeds in which the disperser species was also identified through molecular techniques. This allowed us to build-up a mutualistic network and to identify the relative role of these animals in seed dispersal. Our results showed that mammals and birds fulfilled complementary roles in seed dispersal, with birds representing the main dispersers of key plants within the 5220* habitat, and mammals the main dispersers of human-cultivated plants. Herein, we provide a useful approach with relevant information that can be used to propose management policies that focus on restoring the threatened 5220* habitat, promoting the role of birds to disperse key species that structure plant communities of this priority habitat.
Acosta-Rojas et al. (2019) An integrative approach to discern the seed dispersal role of frugivorous guilds in a Mediterranean semiarid priority habitat. PeerJ 7:e7609 https://doi.org/10.7717/peerj.7609
Isolation and molecular characterization of the indigenous Staphylococcus aureus strain K1 with the ability to reduce hexavalent chromium for its application in bioremediation of metal-contaminated sites
Background Urbanization and industrialization are the main anthropogenic activities that are adding toxic heavy metals to the environment. Among these, chromium (in hexavalent: Cr+6 and/or trivalent Cr+3) is being released abundantly in wastewater due to its uses in different industrial processes. It becomes highly mutagenic and carcinogenic once it enters the cell through sulfate uptake pathways after interacting with cellular proteins and nucleic acids. However, Cr+6 can be bio-converted into more stable, less toxic and insoluble trivalent chromium using microbes. Hence in this study, we have made efforts to utilize chromium tolerant bacteria for bio-reduction of Cr+6 to Cr+3. Methods Bacterial isolate, K1, from metal contaminated industrial effluent from Kala Shah Kaku-Lahore Pakistan, which tolerated up to 22 mM of Cr6+ was evaluated for chromate reduction. It was further characterized biochemically and molecularly by VITEK®2 system and 16S rRNA gene sequencing respectively. Other factors affecting the reduction of chromium such as initial chromate ion concentration, pH, temperature, contact-time were also investigated. The role of cellular surface in sorption of Cr6+ ion was analyzed by FTIR spectroscopy. Results Both biochemical and phylogenetic analyses confirmed that strain K1 was Staphylococcusaureus that could reduce 99% of Cr6+ in 24 hours at 35 °C (pH = 8.0; initial Cr6+ concentration = 100 mg/L). FTIR results assumed that carboxyl, amino and phosphate groups of cell wall were involved in complexation with chromium. Our results suggested that Staphylococcusaureus K1 could be a promising gram-positive bacterium that might be utilized to remove chromium from metal polluted environments.
Environmental Contamination and Remediation
Tariq et al. (2019) Isolation and molecular characterization of the indigenous Staphylococcus aureus strain K1 with the ability to reduce hexavalent chromium for its application in bioremediation of metal-contaminated sites. PeerJ 7:e7726 https://doi.org/10.7717/peerj.7726
Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD)
Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.
Bayona-Vásquez et al. (2019) Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD). PeerJ 7:e7724 https://doi.org/10.7717/peerj.7724
The dopamine receptor D5 gene shows signs of independent erosion in toothed and baleen whales
To compare gene loci considering a phylogenetic framework is a promising approach to uncover the genetic basis of human diseases. Imbalance of dopaminergic systems is suspected to underlie some emerging neurological disorders. The physiological functions of dopamine are transduced via G-protein-coupled receptors, including DRD5 which displays a relatively higher affinity toward dopamine. Importantly, DRD5 knockout mice are hypertense, a condition emerging from an increase in sympathetic tone. We investigated the evolution of DRD5, a high affinity receptor for dopamine, in mammals. Surprisingly, among 124 investigated mammalian genomes, we found that Cetacea lineages (Mysticeti and Odontoceti) have independently lost this gene, as well as the burrowing Chrysochloris asiatica (Cape golden mole). We suggest that DRD5 inactivation parallels hypoxia-induced adaptations, such as peripheral vasoconstriction required for deep-diving in Cetacea, in accordance with the convergent evolution of vasoconstrictor genes in hypoxia-exposed animals. Our findings indicate that Cetacea are natural knockouts for DRD5 and might offer valuable insights into the mechanisms of some forms of vasoconstriction responses and hypertension in humans.
Alves et al. (2019) The dopamine receptor D5 gene shows signs of independent erosion in toothed and baleen whales. PeerJ 7:e7758 https://doi.org/10.7717/peerj.7758
Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)
Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.
Glenn et al. (2019) Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext). PeerJ 7:e7755 https://doi.org/10.7717/peerj.7755
Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)
Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.
Glenn et al. (2019) Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix). PeerJ 7:e7786 https://doi.org/10.7717/peerj.7786
Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador
Background Mimusops coriacea (A.DC.) Miq., (Sapotaceae), originated from Africa, were introduced to coastal areas in Ecuador where it is not extensively used as a traditional medicine to treat various human diseases. Different therapeutically uses of the species include: analgesic, antimicrobial, hypoglycemic, inflammation and pain relieve associated with bone and articulation-related diseases. Furthermore, Mimusops coriacea could be used as anti-oxidant agent. However, botanical, chemical or molecular barcode information related to this much used species is not available from Ecuador. In this study, morphological characterization was performed from leaves, stem and seeds. Furthermore, genetic characterization was performed using molecular barcodes for rbcL, matk, ITS1 and ITS2 using DNA extracted from leaves. Methods Macro-morphological description was performed on fresh leaves, stem and seeds. For anatomical evaluation, tissues were embedded in paraffin and transversal dissections were done following incubation with sodium hypochlorite and safranin for coloration and fixated later in glycerinated gelatin. DNA extraction was performed using a modified CTAB protocol from leaf tissues, while amplification by PCR was accomplished for the molecular barcodes rbcL, matK, ITS1 and ITS2. Sequence analysis was performed using blast in the GenBank. Phylogenetic analysis was performed with accessions queried in the GenBank belonging to the subfamily Sapotoideae. Results Leaf size was 13.56 ± 1.46 × 7.49 ± 0.65 cm; where is a macro-morphological description of the stem (see Methods). The peel of the seeds is dark brown. Sequence analysis revealed that amplicons were generated using the four barcodes selected. Phylogenetic analysis indicated that the barcodes rbcL and matK, were not discriminated between species within the same genus of the subfamily Sapotoideae. On the other hand, the ITS1 and ITS2 were discriminative at the level of genus and species of the Sapotoideae.
Bustamante et al. (2019) Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador. PeerJ 7:e7789 https://doi.org/10.7717/peerj.7789
Exogenous melatonin confers drought stress by promoting plant growth, photosynthetic capacity and antioxidant defense system of maize seedlings
Melatonin is an important biologically active hormone that plays a vital role in plant growth and development. In particular, it has been investigated for its roles in abiotic stress management. The current experiment was carried out to investigate the protective role of melatonin in photosynthetic traits and the antioxidant defense system of maize seedling under drought stress. Maize seedlings were subjected to drought stress (40–45% FC) after two weeks of seedling emergence, followed by a foliar spray (0, 25, 50, 75 and 100 µM) and soil drench of melatonin (0, 25, 50, 75 and 100 µM). Our results indicated that drought stress negatively affected maize seedling and decreased plant growth and development, biomass accumulation, reduced chlorophyll, and carotenoid content, and significantly declined photosynthetic rate and stomatal conductance. On the other hand, reactive oxygen species, soluble protein, and proline content increased under drought stress. However, the application of exogenous melatonin reduced the reactive oxygen species burst and enhanced the photosynthetic activity by protecting from damages through activation of various antioxidant enzymes under drought stress. Foliar application of 100 µM and soil drench of 50 µM melatonin was the most effective treatment concentrations under drought stress. Our current findings hereby confirmed the mitigating potential of melatonin application for drought stress by maintaining plant growth, improving the photosynthetic characteristics and activities of antioxidants enzymes.
Ahmad et al. (2019) Exogenous melatonin confers drought stress by promoting plant growth, photosynthetic capacity and antioxidant defense system of maize seedlings. PeerJ 7:e7793 https://doi.org/10.7717/peerj.7793