Lessons from genome skimming of arthropod‐preserving ethanol

Field‐collected specimens of invertebrates are regularly killed and preserved in ethanol, prior to DNA extraction from the specimens, while the ethanol fraction is usually discarded. However, DNA may be released from the specimens into the ethanol, which can potentially be exploited to study species diversity in the sample without the need for DNA extraction from tissue. We used shallow shotgun sequencing of the total DNA to characterize the preservative ethanol from two pools of insects (from a freshwater habitat and terrestrial habitat) to evaluate the efficiency of DNA transfer from the specimens to the ethanol. In parallel, the specimens themselves were subjected to bulk DNA extraction and shotgun sequencing, followed by assembly of mitochondrial genomes for 39 of 40 species in the two pools. Shotgun sequencing from the ethanol fraction and read‐matching to the mitogenomes detected ~40% of the arthropod species in the ethanol, confirming the transfer of DNA whose quantity was correlated to the biomass of specimens. The comparison of diversity profiles of microbiota in specimen and ethanol samples showed that ‘closed association’ (internal tissue) bacterial species tend to be more abundant in DNA extracted from the specimens, while ‘open association’ symbionts were enriched in the preservative fluid. The vomiting reflex of many insects also ensures that gut content is released into the ethanol, which provides easy access to DNA from prey items. Shotgun sequencing of DNA from preservative ethanol provides novel opportunities for characterizing the functional or ecological components of an ecosystem and their trophic interactions.

[1]  Tami D. Lieberman,et al.  Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes , 2015, bioRxiv.

[2]  Mehrdad Hajibabaei,et al.  Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics , 2014, Proceedings of the National Academy of Sciences.

[3]  M. Johnson,et al.  Circulating microRNAs in Sera Correlate with Soluble Biomarkers of Immune Activation but Do Not Predict Mortality in ART Treated Individuals with HIV-1 Infection: A Case Control Study , 2015, PloS one.

[4]  D. P. Paula,et al.  Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics , 2015, Molecular ecology resources.

[5]  Susan M. Huse,et al.  Microbial diversity in the deep sea and the underexplored “rare biosphere” , 2006, Proceedings of the National Academy of Sciences.

[6]  M. Thomas P. Gilbert,et al.  Screening mammal biodiversity using DNA from leeches , 2012, Current Biology.

[7]  T. Eickbush,et al.  Sequence variation within the rRNA gene loci of 12 Drosophila species. , 2007, Genome research.

[8]  A. Vogler,et al.  Bulk De Novo Mitogenome Assembly from Pooled Total DNA Elucidates the Phylogeny of Weevils (Coleoptera: Curculionoidea) , 2014, Molecular biology and evolution.

[9]  A. Suarez,et al.  The Value of Museum Collections for Research and Society , 2004 .

[10]  S. Koren,et al.  Assembly algorithms for next-generation sequencing data. , 2010, Genomics.

[11]  H. Dumont.  Aquatic Oligochaeta , 1987, Developments in Hydrobiology.

[12]  M. Källersjö,et al.  Molecular evidence for the non-monophyletic status of Naidinae (Annelida, Clitellata, Tubificidae). , 2006, Molecular phylogenetics and evolution.

[13]  T. Fukatsu,et al.  Phenotypic Effect of “Candidatus Rickettsiella viridis,” a Facultative Symbiont of the Pea Aphid (Acyrthosiphon pisum), and Its Interaction with a Coexisting Symbiont , 2013, Applied and Environmental Microbiology.

[14]  N. Moran,et al.  Evolutionary Relationships of Three New Species of Enterobacteriaceae Living as Symbionts of Aphids and Other Insects , 2005, Applied and Environmental Microbiology.

[15]  A. Douglas.  Multiorganismal insects: diversity and function of resident microorganisms. , 2015, Annual review of entomology.

[16]  Mark Fishbein,et al.  Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics. , 2012, American journal of botany.

[17]  Sarah R. Carrino‐Kyker,et al.  Temporal and Spatial Patterns of Eukaryotic and Bacterial Communities Found in Vernal Pools , 2008, Applied and Environmental Microbiology.

[18]  Alexander F. Auch,et al.  MEGAN analysis of metagenomic data. , 2007, Genome research.

[19]  L. Villa-Tanaca,et al.  Bacterial Community and Nitrogen Fixation in the Red Turpentine Beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae) , 2009, Microbial Ecology.

[20]  L. Orlando,et al.  Meta‐barcoding of ‘dirt’ DNA from soil reflects vertebrate biodiversity , 2012, Molecular ecology.

[21]  Yun-Ji Kim,et al.  Insect Gut Bacterial Diversity Determined by Environmental Habitat, Diet, Developmental Stage, and Phylogeny of Host , 2014, Applied and Environmental Microbiology.

[22]  P. Stadler,et al.  MITOS: improved de novo metazoan mitochondrial genome annotation. , 2013, Molecular phylogenetics and evolution.

[23]  M. Inbar,et al.  Rickettsia ‘In’ and ‘Out’: Two Different Localization Patterns of a Bacterial Symbiont in the Same Insect Species , 2011, PloS one.

[24]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[25]  M. Jackson,et al.  Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects. , 2009, Mycological research.

[26]  Mehrdad Hajibabaei,et al.  Assessing biodiversity of a freshwater benthic macroinvertebrate community through non-destructive environmental barcoding of DNA from preservative ethanol , 2012, BMC Ecology.

[27]  J. Werren,et al.  Wolbachia: master manipulators of invertebrate biology , 2008, Nature Reviews Microbiology.

[28]  A. Vogler,et al.  Phylogenetic community ecology of soil biodiversity using mitochondrial metagenomics , 2015, Molecular ecology.

[29]  Eugene W. Myers,et al.  A whole-genome assembly of Drosophila. , 2000, Science.

[30]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[31]  F. Grimont,et al.  The genus Serratia. , 1978, Annual review of microbiology.

[32]  A. Willems.  The family Comamonadaceae , 2014 .

[33]  G. Singer,et al.  Direct PCR amplification and sequencing of specimens' DNA from preservative ethanol. , 2010, BioTechniques.

[34]  L. Dijkshoorn,et al.  1 The Diversity of the Genus Acinetobacter , 2007 .

[35]  C. Moreau,et al.  A Veritable Menagerie of Heritable Bacteria from Ants, Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review , 2012, PloS one.

[36]  Guanliang Meng,et al.  High‐throughput monitoring of wild bee diversity and abundance via mitogenomics , 2015, Methods in ecology and evolution.

[37]  M. Schlaepfer,et al.  Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds. , 2014 .

[38]  T. Hashimoto,et al.  Ultrastructural and Phylogenetic Studies on Blastocystis Isolates from Cockroaches , 2007, The Journal of eukaryotic microbiology.

[39]  Douglas W. Yu,et al.  Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. , 2013, Ecology letters.

[40]  T. Fukatsu,et al.  Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte–embryo interface , 2012, Proceedings of the National Academy of Sciences.

[41]  A. Vogler,et al.  Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample , 2015, Molecular biology and evolution.

[42]  A. Vogler,et al.  Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages , 2015 .

[43]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[44]  Douglas W. Yu,et al.  Mitochondrial metagenomics: letting the genes out of the bottle , 2016, GigaScience.

[45]  A. Reid,et al.  The Rare Biosphere , 2011 .

[46]  N. Moran,et al.  Multiple introductions of the Spiroplasma bacterial endosymbiont into Drosophila , 2009, Molecular ecology.

[47]  C. Wiuf,et al.  Monitoring endangered freshwater biodiversity using environmental DNA. , 2012, Molecular ecology.

[48]  F. Rodríguez-Valera,et al.  The bacterial pan-genome:a new paradigm in microbiology. , 2010, International microbiology : the official journal of the Spanish Society for Microbiology.

[49]  A. Shostak.  Hymenolepis diminuta Infections In Tenebrionid Beetles As A Model System for Ecological Interactions Between Helminth Parasites and Terrestrial Intermediate Hosts: A Review and Meta-Analysis , 2014, The Journal of parasitology.

[50]  C. Braquart-Varnier,et al.  A host as an ecosystem: Wolbachia coping with environmental constraints. , 2014, Environmental microbiology.

[51]  R. Cordaux,et al.  Molecular Characterization and Evolution of Arthropod-Pathogenic Rickettsiella Bacteria , 2007, Applied and Environmental Microbiology.

[52]  F. Raymond,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ray Meta: scalable de novo metagenome assembly and profiling , 2012 .

[53]  J. Glass,et al.  The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade. , 2004, International journal of systematic and evolutionary microbiology.

[54]  Siu-Ming Yiu,et al.  IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth , 2012, Bioinform..

[55]  A. Steinbüchel,et al.  Aerobic Degradation of Mercaptosuccinate by the Gram-Negative Bacterium Variovorax paradoxus Strain B4 , 2010, Journal of bacteriology.

[56]  Mark L. Blaxter,et al.  Second-generation environmental sequencing unmasks marine metazoan biodiversity , 2010, Nature communications.

[57]  Douglas W. Yu,et al.  Environmental DNA for wildlife biology and biodiversity monitoring. , 2014, Trends in ecology & evolution.

[58]  A. Vogler,et al.  Metagenome Skimming of Insect Specimen Pools: Potential for Comparative Genomics , 2015, Genome biology and evolution.

[59]  W. L. Chadderton,et al.  “Sight‐unseen” detection of rare aquatic species using environmental DNA , 2011 .

[60]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..