Caterpillar gut and host plant phylloplane mycobiomes differ: a new perspective on fungal involvement in insect guts

2020 ◽  
Vol 96 (9) ◽  
Author(s):  
Denisa višňovská ◽  
Petr Pyszko ◽  
Martin Šigut ◽  
Martin Kostovčík ◽  
Miroslav Kolařík ◽  
...  
Keyword(s):  
Jaccard Index ◽  
Herbivorous Insects ◽  
Rrna Gene ◽  
Its2 Rdna ◽  
Leaf Sample ◽  
Dna Metabarcoding ◽  
Genus Richness ◽  
Qualitative Similarity ◽  
Feeding Substrate ◽  
New Perspective

ABSTRACT Compared with the highly diverse microbiota of leaves, herbivorous insects exhibit impoverished gut microbial communities. Research to date has focused on the bacterial component of these gut microbiomes, neglecting the fungal component. As caterpillar gut bacterial microbiomes are derived strongly from their diet, we hypothesized that their mycobiomes would reflect the host leaf mycobiomes. Using the ITS2 rDNA and V5–V6 16S rRNA gene regions for DNA metabarcoding of caterpillar gut and host leaf sample pairs we compared their mycobiome genus diversity and compositions and identified genera associated with caterpillar guts. Leaves and caterpillar guts harbored different mycobiomes with quite low qualitative similarity (Jaccard index = 38.03%). The fungal genera most significantly associated with the caterpillar gut included Penicillium, Mucor and unidentified Saccharomycetales, whereas leaf-associated genera included Holtermanniella, Gibberella (teleomorph of Fusarium) and Seimatosporium. Although caterpillar gut and leaf mycobiomes had similar genus richness overall, this indicator was not correlated for individual duplets. Moreover, as more samples entered the analysis, mycobiome richness increased more rapidly in caterpillar guts than in leaves. The results suggest that the mycobiota of the caterpillar gut differs from that of their feeding substrate; further, the mycobiomes appear to be richer than the well-studied bacterial microbiotas.

scholarly journals Ichthyoplankton metabarcoding as a tool for studying fish reproductive dynamics

2021 ◽  
Vol 4 ◽  
Author(s):  
Daniel Teixeira ◽  
Heron Hilário ◽  
Gustavo Rosa ◽  
Guilherme Santos ◽  
Gilmar Santos ◽  
...  
Keyword(s):  
Fish Assemblages ◽  
Fish Larvae ◽  
Reference Sequence ◽  
12S Rrna ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Fish Eggs ◽  
Migration Routes ◽  
Dna Metabarcoding ◽  
Primer Sets

The study of ichthyoplankton composition, abundance and distribution is paramount to understand the reproductive dynamics of local fish assemblages. The analysis of these parameters allows the identification of spawning sites, nursery areas and migration routes. However, due to the lack of characters in early life stages, the morphological identification of ichthyoplankton is often impractical and many studies identify only fish larvae. Additionally, its accuracy shows great variation between taxonomists and laboratories according to their experience and specialty. DNA barcoding emerged as an alternative to provide assertive identification of fish eggs and larvae, but it becomes too expensive and laborious when the study demands the processing of huge amounts of organisms. DNA metabarcoding can overcome these limitations as a rapid, cost-effective, broad and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. Here, we present the identification of a sample containing 68 fish eggs and another containing 293 fish larvae from a single site in the São Francisco River Basin, Eastern Brazil, through DNA metabarcoding. We used a low-cost saline DNA extraction followed by PCR amplification with three primer sets targeting the 12S rRNA gene: MiFish (~170bp), Teleo_1 (~60bp), and NeoFish (~190bp). The latter was recently developed by our research group specifically for the identification of Neotropical fishes. All the amplified samples were sequenced in a single multiplexed Illumina MiniSeq run. We performed the filtering steps and assigned Amplicon Sequence Variants (ASVs) using a DADA2/Phyloseq based pipeline and a custom 12S reference sequence database including 101 species and 70 genera from the Jequitinhonha and São Francisco basins. The species Cyphocharax gilbert, Leporinus taeniatus, Megaleporinus elongatus, Prochilodus argenteus, P. costatus and Psalidodon fasciatus were detected by all three primer sets in the larva pool, while Pterygoplichthys etentaculatus was detected solely by NeoFish (Fig. 1). Within the egg pool, all three markers detected the species Characidium zebra, Curimatella lepidura, M. elongatus, Pimelodus fur and P. costatus, but Brycon orthotaenia was detected only by NeoFish, P. maculatus only by Teleo, and P. pohli by MiFish and Teleo (Fig. 1). The consistency in species detection among all three markers underpins the credibility of this method to accurately describe the sample composition. Considering that most of species were exclusive to the larvae or egg pool, our experiment highlights the importance of including the identification of fish eggs in reproduction studies, as it can provide additional information about which species are spawning in an area. Furthermore, the application of DNA metabarcoding to the study of ichthyoplankton can help decision makers create more informed guidelines for conservation of economically and ecologically important fish species.

scholarly journals New Perspective on Uncultured Bacterial Phylogenetic Division OP11

2004 ◽  
Vol 70 (2) ◽  
pp. 845-849 ◽  
Author(s):  
J. Kirk Harris ◽  
Scott T. Kelley ◽  
Norman R. Pace
Keyword(s):  
Environmental Samples ◽  
Rrna Gene ◽  
Molecular Signatures ◽  
Diverse Group ◽  
Environmental Distribution ◽  
Molecular Analyses ◽  
Partial Sequencing ◽  
Candidate Division ◽  
New Perspective ◽  
Pcr Techniques

ABSTRACT Organisms belonging to the OP11 candidate phylogenetic division of Bacteria have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.

scholarly journals Molecular taxonomy of polymicrobial diseases ? finding novel bacteria not previously considered to be associated with oral diseases

2005 ◽  
Vol 26 (3) ◽  
pp. 117
Author(s):  
Mangala Nadkarni ◽  
Roy Byun ◽  
Kim-Ly Chhour
Keyword(s):  
Recombinant Dna ◽  
Molecular Taxonomy ◽  
Rrna Gene ◽  
The Novel ◽  
Oral Diseases ◽  
Recombinant Dna Technology ◽  
Uncultured Bacteria ◽  
Novel Bacteria ◽  
Taxonomic Methods ◽  
New Perspective

Recombinant DNA technology and molecular biology have brought a revolution to the limitations imposed by traditional taxonomic methods of bacterial identification based solely on cultivation. Following on from the novel research of Carl Woese, molecular taxonomy is generally based on variations in the sequence of the 16S rRNA gene and has brought a new perspective to our understanding of the diversity of uncultured bacteria in a multitude of microbial habitats. Analysis of the polymicrobial oral diseases is no exception.

scholarly journals Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

2018 ◽  
Author(s):  
Aimee L van der Reis ◽  
Olivier Laroche ◽  
Andrew G Jeffs ◽  
Shane D Lavery
Keyword(s):  
New Zealand ◽  
Preliminary Analysis ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Gut Contents ◽  
Rrna Gene ◽  
Diverse Range ◽  
Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding outcomes indicated that the ethanol samples produced better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

scholarly journals Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

2018 ◽  
Author(s):  
Felix Heeger ◽  
Elizabeth C. Bourne ◽  
Christiane Baschien ◽  
Andrey Yurkov ◽  
Boyke Bunk ◽  
...  
Keyword(s):  
De Novo ◽  
Error Rates ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Sequencing Error ◽  
Rrna Gene ◽  
Dna Metabarcoding ◽  
Long Read ◽  
Reference Sequences ◽  
Ribosomal Rrna

ABSTRACTDNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well-established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels 15 comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By 20 allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 25 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.

scholarly journals New Insights Into Nematode DNA-metabarcoding as Revealed by the Characterization of Artificial and Spiked Nematode Communities

Diversity ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 52 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nancy de Sutter ◽  
Nicole Viaene ◽  
Annelies Haegeman
Keyword(s):  
Species Richness ◽  
18S Rrna ◽  
Nematode Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Soil Nematode ◽  
Mock Community ◽  
Nematode Communities ◽  
Dna Metabarcoding ◽  
Soil Nematode Communities

Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or ‘molecular’ species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

scholarly journals A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0245936
Author(s):  
Takuhei Shiozaki ◽  
Fumihiro Itoh ◽  
Yuu Hirose ◽  
Jonaotaro Onodera ◽  
Akira Kuwata ◽  
...  
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Formalin Fixation ◽  
Plankton Sample ◽  
Rrna Gene ◽  
Dna Metabarcoding ◽  
Plankton Diversity ◽  
The Impact ◽  
Formalin Fixed

Plankton samples have been routinely collected and preserved in formalin in many laboratories and museums for more than 100 years. Recently, attention has turned to use DNA information from formalin-fixed samples to examine changes in plankton diversity over time. However, no molecular ecological studies have evaluated the impact of formalin fixation on the genetic composition of the plankton community structure. Here, we developed a method for extracting DNA from archived formalin-preserved plankton samples to determine their community structure by a DNA metabarcoding approach. We found that a lysis solution consisting of borate-NaOH buffer (pH 11) with SDS and proteinase K effectively cleaved the cross-link formed by formalin fixation. DNA was extracted from samples preserved for decades in formalin, and the diatom community of the extracted DNA was in good agreement with the microscopy analysis. Furthermore, we stored a plankton sample for 1.5 years and demonstrated that 18S rRNA gene community structures did not change significantly from non-formalin-fixed, time-zero samples. These results indicate that our method can be used to describe the original community structure of plankton archived in formalin for years. Our approach will be useful for examining the long-term variation of plankton diversity by metabarcoding analysis of 18S rRNA gene community structure.

scholarly journals Bacterial, phytoplankton, and viral dynamics of meromictic Lake Cadagno offer insights into the Proterozoic ocean microbial loop

2021 ◽  
Author(s):  
Jaspreet Singh Saini ◽  
Christel Hassler ◽  
Rachel N Cable ◽  
Marion Fourquez ◽  
Francesco Danza ◽  
...  
Keyword(s):  
Meromictic Lake ◽  
Close Relative ◽  
Rrna Gene ◽  
Viral Dynamics ◽  
Comprehensive Understanding ◽  
Sulfur Bacteria ◽  
Eukaryotic Phytoplankton ◽  
Simultaneous Study ◽  
New Perspective ◽  
Secondary Productivity

Meromictic Lake Cadagno, with its permanent stratification and persistent microbial bloom within the anoxic chemocline, serves as an ancient ocean analogue. Although the lake has been studied for over 25 years, the absence of simultaneous study of the bacteria, phytoplankton, and viruses, together with primary and secondary productivity, has hindered a comprehensive understanding of its microbial food web. This study evaluated the identities, abundances, and productivity of microbes in the context of nutrient biogeochemical cycling across the stratified depths of Lake Cadagno. Photosynthetic pigments and chloroplast 16S rRNA gene phylogenies suggested high abundances of eukaryotic phytoplankton, primarily Chlorophyta, through the water column. Of these, a close relative of Ankyra judayi, a high-alpine adapted chlorophyte, peaked with oxygen in the mixolimnion, while Closteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. Chromatium, a genus of anoxygenic phototrophic sulfur bacteria, dominated the chemocline along with Lentimicrobium, a genus of known fermenters whose abundance was newly reported in Lake Cadagno. Secondary production peaked in the chemocline, suggesting anoxygenic primary producers depended on heterotrophic nutrient remineralization. Virus-to-microbe ratios (VMR) peaked in the zone of high phytoplankton abundances, yet were at a minimum at the peak of Chromatium, dynamic trends that suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical dynamics of Lake Cadagno, this study provides a new perspective on the biological and geochemical connections that comprised the food webs of the Proterozoic ocean.

scholarly journals Comparative study of benthic diatom community using 18S rDNA metabarcoding and light microscopy in the Bosphorus, Turkey

2021 ◽  
Vol 4 ◽  
Author(s):  
Aydın Kaleli ◽  
E. Gozde Ozbayram ◽  
Reyhan Akçaalan
Keyword(s):  
Light Microscopy ◽  
18S Rdna ◽  
18S Rrna Gene ◽  
Aquatic Systems ◽  
Rrna Gene ◽  
Sampling Effort ◽  
Diatom Community ◽  
Soil Dna ◽  
Dna Metabarcoding ◽  
Sequencing Platforms

Diatoms are one of the major algal groups having an important role in the aquatic systems in which they constitute the main primary production with dinoflagellates in the coastal regions, and can contribute to photosynthesis at great rates. There had been intense effort to reveal diatom community with light microscopy (LM) and scanning electron microscopy (SEM), on the other hand, DNA barcoding techniques had been an interest to understand the microbiome in the last decades. Barcoding provided rapid response on the targeted organisms and introduced many new species. Recently, monocultural molecular studies showed that species could be described with accurate and reliable results. DNA metabarcoding approaches yielded to determine the whole organisms with available DNA data in the sample and major advances on next-generation sequencing platforms enable to widen the application of metabarcoding approach to various environmental samples. The aim of this study was to perform a comparative assessment of the diatom community structures in Bosphorus of Istanbul, Turkey by LM and 18S rDNA metabarcoding. Diatom samples were collected by brushing from the submerged stones of 10 cm2 of area taken from the coast of Beykoz on the Asian part of the Bosphorus, Istanbul and processed for LM and metabarcoding in June 2020. To concentrate the sample, the scrapes of the biofilm filtered from a 0.22-micron filter, and eDNA was extracted from that filter paper using MN NucleoSpin Soil DNA isolation kit (Macherey-Nagel, Germany) following the manufacturer’s protocol and diatom community profile was analyzed by targeting the V4 region of the 18S rRNA gene using Illumina® MiSeq™. DNA metabarcoding results revealed two classes Bacillariophyceae (91%) and Mediophyceae (9%) (Fig. 1). While small-celled Hyalosira delicatula Kützing was the most abundant taxa with 40% abundance and it was followed by Licmophora spp. (33%). 28% of Licmophora sequences could not be assigned to any species and remained as unidentified, Licmophora gracilis (Ehrenberg) Grunow (4%), and L. flabellata (0.9%) detected at lower abundances. Besides, one of the common genera of diatoms, Navicula spp., showed a relative abundance of 4% and another common genus Nitzschia spp. were represented only by N. commutata Grunow and composed 0.5% of the community. While 18S rDNA metabarcoding revealed 11 genera, LM investigation identified 17 genera belonging to 21 species. 4 genera were common in both techniques and Licmophora flabellata was detected in both LM and metabarcoding methods. Interestingly, small celled taxa which could be easily overlooked in LM was detected with 18S metabarcoding. The results presented a promising number of genera which could be detected by both methods (Fig. 2). DNA metabarcoding of diatoms is a new area of research in the coastal waters and there are few studies performed so far and this is the first study relying on the rDNA metabarcoding of diatoms in the aquatic systems in Turkey. The comparison of the taxa using microscopy methods and metabarcoding techniques indicated some significant differences in the diatom composition. However, the results here with 18 species with metabarcoding vice-versa 21 taxa with microscopy methods confirms poor biodiversity in the Bosphorus. However, this study based on one sampling effort in one station for the comparison of the two methods, we had LM results from more stations in different seasons supporting the lower biodiversity. eDNA data is scarce from the coastal areas and our results comprise a promising number of genera. Results of this study could provide data for further research, which high number of diatoms could be determined with eDNA metabarcoding.

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