PT AU TI SO LA DT DE ID AB NR TC Z9 U1 U2 PD PY VL IS PN SU SI MA BP EP AR DI PG UT PM Order Platform Platform2 Approach J Mallott, EK; Malhi, RS; Garber, PA Brief Communication: High-Throughput Sequencing of Fecal DNA to Identify Insects Consumed by Wild Weddell's Saddleback Tamarins (Saguinus weddelli, Cebidae, Primates) in Bolivia AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY English Article pyrosequencing; metagenomics; faunivory; primates; feeding ecology PREY DNA; MITOCHONDRIAL-DNA; ARTHROPOD PREY; FUSCICOLLIS; DIET; MYSTAX; FECES; MONKEYS; CHIMPANZEES; PREDATION The genus Saguinus represents a successful radiation of over 20 species of small-bodied New World monkeys. Studies of the tamarin diet indicate that insects and small vertebrates account for approximate to 16-45% of total feeding and foraging time, and represent an important source of lipids, protein, and metabolizable energy. Although tamarins are reported to commonly consume large-bodied insects such as grasshoppers and walking sticks (Orthoptera), little is known concerning the degree to which smaller or less easily identifiable arthropod prey comprises an important component of their diet. To better understand tamarin arthropod feeding behavior, fecal samples from 20 wild Bolivian saddleback tamarins (members of five groups) were collected over a 3 week period in June 2012, and analyzed for the presence of arthropod DNA. DNA was extracted using a Qiagen stool extraction kit, and universal insect primers were created and used to amplify a approximate to 280 bp section of the COI mitochondrial gene. Amplicons were sequenced on the Roche 454 sequencing platform using high-throughput sequencing techniques. An analysis of these samples indicated the presence of 43 taxa of arthropods including 10 orders, 15 families, and 12 identified genera. Many of these taxa had not been previously identified in the tamarin diet. These results highlight molecular analysis of fecal DNA as an important research tool for identifying anthropod feeding patterns in primates, and reveal broad diversity in the taxa, foraging microhabitats, and size of arthropods consumed by tamarin monkeys. Am J Phys Anthropol 156:474-481, 2015. (c) 2014 Wiley Periodicals, Inc. 61 11 11 2 33 MAR 2015 156 3 474 481 10.1002/ajpa.22654 8 WOS:000350277800013 25369770 Feaces monkey 454 PCR Barcoding J Wood, JR; Holdaway, RJ; Orwin, KH; Morse, C; Bonner, KI; Davis, C; Bolstridge, N; Dickie, IA No single driver of biodiversity: divergent responses of multiple taxa across land use types ECOSPHERE English Article animals; bacteria; biodiversity; community structure; DNA metabarcoding; fungi; intensification; land use; plants; soil AGRICULTURAL INTENSIFICATION; SPECIES RICHNESS; MICROBIAL COMMUNITIES; CONSISTENT RESPONSES; GLOBAL BIODIVERSITY; FUNGAL COMMUNITIES; PLANT DIVERSITY; SOIL-PH; CONSERVATION; ABUNDANCE Understanding the responses of biodiversity to different land use regimes is critical for managing biodiversity in the face of future land use change. However, there is still significant uncertainty around how consistent the responses of different taxonomic groups to land use change are. Here, we use a combination of high-throughput environmental DNA sequencing and traditional field-based survey methods to examine how patterns of richness and community composition correlate among four domains/kingdoms (bacteria, fungi, plants, and metazoans) and the four most-abundant animal taxonomic groups (arachnids, Collembola, insects, and nematodes) across five different land use types (natural forest, planted forest, unimproved grassland, improved grassland, and vineyards). Richness for each taxonomic group varied between land use types, yet different taxa showed inconsistent responses to land use, and their richness was rarely correlated. This contrasted with community composition of taxonomic groups, for which there was relatively good discrimination of land use types and there was strong correlation between group responses. We found little evidence for consistent drivers of taxonomic richness, yet identified several significant drivers of community composition that were shared across many groups. Drivers of composition were not the same as the drivers of diversity, suggesting diversity and composition are independently controlled. While land use intensification has been viewed as having generally negative effects on biodiversity, our results provide evidence that different taxa respond divergently across different land uses. Further, our study demonstrates the power of high-throughput sequencing of environmental DNA as a tool for addressing broad ecological patterns relating to landscape biodiversity. 61 0 0 14 27 NOV 2017 8 11 e01997 10.1002/ecs2.1997 17 WOS:000417330000013 Soil/litter 454 PCR Barcoding J Malacrino, A; Schena, L; Campolo, O; Laudani, F; Mosca, S; Giunti, G; Strano, CP; Palmeri, V A Metabarcoding Survey on the Fungal Microbiota Associated to the Olive Fruit Fly MICROBIAL ECOLOGY English Article 454 Pyrosequencing; High-throughput sequencing; Bactrocera oleae; Cladosporium; Colletotrichum; Pseudocercospora PARASITOID PSYTTALIA-CONCOLOR; BIOLOGICAL-CONTROL; BACTROCERA-OLEAE; ABUNDANCE DISTRIBUTIONS; MOLECULAR ANALYSIS; HIGH-THROUGHPUT; ANTHRACNOSE; COMMUNITIES; TREE; DIVERSITY The occurrence of interaction between insects and fungi is interesting from an ecological point of view, particularly when these interactions involve insect pests and plant pathogens within an agroecosystem. In this study, we aimed to perform an accurate analysis on the fungal microbiota associated to Bactrocera oleae (Rossi) through a metabarcoding approach based on 454 pyrosequencing. From this analysis, we retrieved 43,549 reads that clustered into 128 operational taxonomic units (OTUs), of which 29 resulted in the "core" associate fungi of B. oleae. This fungal communitywasmainly represented by sooty mould fungi, such as Cladosporium spp., Alternaria spp. and Aureobasidium spp., by plant pathogens like Colletotrichum spp. and Pseudocercospora spp., along with several other less abundant taxa whose ecology is unclear in most of the cases. Our findings lead to new insights into the microbial ecology of this specific ecological niche, enabling the understanding of a complex network of interactions within the olive agroecosystem. 48 13 13 2 24 APR 2017 73 3 677 684 10.1007/s00248-016-0864-z 8 WOS:000397940000016 27687872 Diptera 454 PCR Barcoding Fungal J Kelley, ST; Dobler, S Comparative analysis of microbial diversity in Longitarsus flea beetles (Coleoptera: Chrysomelidae) GENETICA English Article 16S rRNA; Bacteria; Biodiversity; Herbivory; Metagenomics; Microbial ecology; Phylogeny CULTURE-INDEPENDENT METHODS; FLY CERATITIS-CAPITATA; BACTERIAL DIVERSITY; BARK BEETLES; HOST PLANTS; HERBIVOROUS INSECTS; SERRATIA-MARCESCENS; GUT MICROBES; EVOLUTION; COMMUNITY Herbivorous beetles comprise a significant fraction of eukaryotic biodiversity and their plant-feeding adaptations make them notorious agricultural pests. Despite more than a century of research on their ecology and evolution, we know little about the diversity and function of their symbiotic microbial communities. Recent culture-independent molecular studies have shown that insects possess diverse gut microbial communities that appear critical for their survival. In this study, we combined culture-independent methods and high-throughput sequencing strategies to perform a comparative analysis of Longitarsus flea-beetles microbial community diversity (MCD). This genus of beetle herbivores contains host plant specialists and generalists that feed on a diverse array of toxic plants. Using a deep-sequencing approach, we characterized the MCD of eleven Longitarsus species across the genus, several of which represented independent shifts to the same host plant families. Database comparisons found that Longitarsus-associated microbes came from two habitat types: insect guts and the soil rhizosphere. Statistical clustering of the Longitarsus microbial communities found little correlation with the beetle phylogeny, and uncovered discrepancies between bacterial communities extracted directly from beetles and those from frass. A Principal Coordinates Analysis also found some correspondence between beetle MCD and host plant family. Collectively, our data suggest that environmental factors play a dominant role in shaping Longitarsus MCD and that the root-feeding beetle larvae of these insects are inoculated by soil rhizosphere microbes. Future studies will investigate MCD of select Longitarsus species across their geographic ranges and explore the connection between the soil rhizosphere and the beetle MCD. 67 15 15 1 32 MAY 2011 139 5 541 550 10.1007/s10709-010-9498-0 10 WOS:000290320400002 20844936 Coleoptera Microbiota 454 PCR Barcoding J Pedro, PM; Piper, R; Neto, PB; Cullen, L; Dropa, M; Lorencao, R; Matte, MH; Rech, TC; Rufato, MO; Silva, M; Turati, DT Metabarcoding Analyses Enable Differentiation of Both Interspecific Assemblages and Intraspecific Divergence in Habitats With Differing Management Practices ENVIRONMENTAL ENTOMOLOGY English Article next-generation sequencing; metabarcoding; Beta diversity; insect dispersal BIODIVERSITY ASSESSMENT; COFFEE PLANTATIONS; DNA BARCODES; ARTHROPODS; DIVERSITY; METAGENOMICS; PARADIGM; SEARCH; PEST Spatial and temporal collections provide important data on the distribution and dispersal of species. Regional-scale monitoring invariably involves hundreds of thousands of samples, the identification of which is costly in both time and money. In this respect, metabarcoding is increasingly seen as a viable alternative to traditional morphological identification, as it eliminates the taxonomic bottleneck previously impeding such work. Here, we assess whether terrestrial arthropods collected from 12 pitfall traps in two farms of a coffee (Coffea arabica L.) growing region of Sao Paulo State, Brazil could differentiate the two locations. We sequenced a portion of the cytochrome oxidase 1 region from minimally processed pools of samples and assessed inter- and intraspecific parameters across the two locations. Our sequencing was sufficient to circumscribe the overall diversity, which was characterized by few dominant taxa, principally small Coleoptera species and Collembola. Thirty-four operational taxonomic units were detected and of these, eight were present in significantly different quantities between the two farms. Analysis of community-wide Beta diversity grouped collections based on farm provenance. Moreover, haplotype-based analyses for a species of Xyleborus beetle showed that there is significant population genetic structuring between the two farms, suggesting limited dispersal. We conclude that metabarcoding can provide important management input and, considering the rapidly declining cost of sequencing, suggest that large-scale monitoring is now feasible and can identify both the taxa present as well as contribute information about genetic diversity of focal species. 45 1 1 2 7 DEC 2017 46 6 1381 1389 10.1093/ee/nvx166 9 WOS:000417734400025 29069398 Trap pitfall 454 PCR Barcoding J Thomsen, PF; Kielgast, J; Iversen, LL; Wiuf, C; Rasmussen, M; Gilbert, MTP; Orlando, L; Willerslev, E Monitoring endangered freshwater biodiversity using environmental DNA MOLECULAR ECOLOGY English Article biological diversity; molecular detection; pyrosequencing; threatened species; wildlife conservation CONSERVATION; PERSISTENCE; PERMAFROST; STABILITY; SEDIMENTS; SAMPLES; ICE Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animalsrepresenting amphibians, fish, mammals, insects and crustaceanscan be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups. 39 318 333 22 492 JUN 2012 21 11 2565 2573 10.1111/j.1365-294X.2011.05418.x 9 WOS:000304389500004 22151771 Freshwater communities 454 PCR Barcoding J Yu, DW; Ji, YQ; Emerson, BC; Wang, XY; Ye, CX; Yang, CY; Ding, ZL Biodiversity soup: metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring METHODS IN ECOLOGY AND EVOLUTION English Article 454 Genome Sequencer FLX System; DNA barcoding; high-throughput sequencing; metagenetics; metagenomics; OTU picking; phylogenetic diversity 16S RIBOSOMAL-RNA; ENVIRONMENTAL DNA; DIVERSITY; SEQUENCES; COMMUNITIES; PITFALLS; ACCURATE; PRIMERS; PROGRAM; BARCODE 1. Traditional biodiversity assessment is costly in time, money and taxonomic expertise. Moreover, data are frequently collected in ways (e.g. visual bird lists) that are unsuitable for auditing by neutral parties, which is necessary for dispute resolution. 2. We present protocols for the extraction of ecological, taxonomic and phylogenetic information from bulk samples of arthropods. The protocols combine mass trapping of arthropods, mass-PCR amplification of the COI barcode gene, pyrosequencing and bioinformatic analysis, which together we call metabarcoding. 3. We construct seven communities of arthropods (mostly insects) and show that it is possible to recover a substantial proportion of the original taxonomic information. We further demonstrate, for the first time, that metabarcoding allows for the precise estimation of pairwise community dissimilarity (beta diversity) and within-community phylogenetic diversity (alpha diversity), despite the inevitable loss of taxonomic information inherent to metabarcoding. 4. Alpha and beta diversity metrics are the raw materials of ecology and the environmental sciences, facilitating assessment of the state of the environment with a broad and efficient measure of biodiversity. 57 202 217 19 285 AUG 2012 3 4 613 623 10.1111/j.2041-210X.2012.00198.x 11 WOS:000306961700001 Arthropod communities 454 PCR Barcoding J Russell, JA; Weldon, S; Smith, AH; Kim, KL; Hu, Y; Lukasik, P; Doll, S; Anastopoulos, I; Novin, M; Oliver, KM Uncovering symbiont-driven genetic diversity across North American pea aphids MOLECULAR ECOLOGY English Article biodiversity; co-infection; endosymbiont; strain diversity; Wolbachia FACULTATIVE BACTERIAL ENDOSYMBIONTS; HOST-PLANT SPECIALIZATION; ACYRTHOSIPHON-PISUM; SECONDARY SYMBIONTS; NATURAL-POPULATIONS; HORIZONTAL TRANSFER; HIGH-THROUGHPUT; DEFENSIVE SYMBIONTS; SEQUENCE ALIGNMENT; BEMISIA-TABACI Heritable genetic variation is required for evolution, and while typically encoded within nuclear and organellar genomes, several groups of invertebrates harbour heritable microbes serving as additional sources of genetic variation. Hailing from the symbiont-rich insect order Hemiptera, pea aphids (Acyrthosiphon pisum) possess several heritable symbionts with roles in host plant utilization, thermotolerance and protection against natural enemies. As pea aphids vary in the numbers and types of harboured symbionts, these bacteria provide heritable and functionally important variation within field populations. In this study, we quantified the cytoplasmically inherited genetic variation contributed by symbionts within North American pea aphids. Through the use of Denaturing Gradient Gel Electrophoresis (DGGE) and 454 amplicon pyrosequencing of 16S rRNA genes, we explored the diversity of bacteria harboured by pea aphids from five populations, spanning three locations and three host plants. We also characterized strain variation by analysing 16S rRNA, housekeeping and symbiont-associated bacteriophage genes. Our results identified eight species of facultative symbionts, which often varied in frequency between locations and host plants. We detected 28 cytoplasmic genotypes across 318 surveyed aphids, considering only the various combinations of secondary symbiont species infecting single hosts. Yet the detection of multiple Regiella insecticola, Hamiltonella defensa and Rickettsia strains, and diverse bacteriophage genotypes from H.defensa, suggest even greater diversity. Combined, these findings reveal that heritable bacteria contribute substantially to genetic variation in A.pisum. Given the costs and benefits of these symbionts, it is likely that fluctuating selective forces play a role in the maintenance of this diversity. 76 93 97 2 187 APR 2013 22 7 2045 2059 10.1111/mec.12211 15 WOS:000316575800020 23379399 Hemiptera 454 PCR Barcoding Simbionts J Boucias, DG; Cai, YP; Sun, YJ; Lietze, VU; Sen, R; Raychoudhury, R; Scharf, ME The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition MOLECULAR ECOLOGY English Article 16S rRNA gene; 454 pyrosequencing; bTEFAP; cellulose; Microbial symbionts BACTERIAL COMMUNITY STRUCTURE; FEEDING HIGHER TERMITE; RNA SEQUENCE DATA; PHYLOGENETIC DIVERSITY; SYMBIOTIC METHANOGENS; COPTOTERMES-FORMOSANUS; PHYSIOLOGICAL ECOLOGY; FLAGELLATED PROTISTS; SUBTERRANEAN TERMITE; SPECIES RICHNESS Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling prokaryotes and eukaryotes. In the R.flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture- and cloning-dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high-throughput 454 pyrosequencing of 16S V5-V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R.flavipes and to examine compositional changes in response to lignin-rich and lignin-poor cellulose diets after a 7-day feeding period. Of the 475000 high-quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R.flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species-level phylotypes. However, diet-dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony-level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition. 93 62 65 3 113 APR 2013 22 7 1836 1853 10.1111/mec.12230 18 WOS:000316575800007 23379767 Isoptera 454 PCR Barcoding Microbiota J Zhang, K; Lin, SL; Ji, YQ; Yang, CX; Wang, XY; Yang, CY; Wang, HS; Jiang, HS; Harrison, RD; Yu, DW Plant diversity accurately predicts insect diversity in two tropical landscapes MOLECULAR ECOLOGY English Article Arthropoda; biodiversity; biomonitoring; host specificity; insect-plant interactions; surrogate species SPECIES RICHNESS ESTIMATORS; BIODIVERSITY ASSESSMENT; HERBIVOROUS INSECTS; HOST-SPECIFICITY; MAXIMUM-LIKELIHOOD; BETA DIVERSITY; FOREST; PERFORMANCE; SEQUENCES; DNA Plant diversity surely determines arthropod diversity, but only moderate correlations between arthropod and plant species richness had been observed until Basset et al. (Science, 338, 2012 and 1481) finally undertook an unprecedentedly comprehensive sampling of a tropical forest and demonstrated that plant species richness could indeed accurately predict arthropod species richness. We now require a high-throughput pipeline to operationalize this result so that we can (i) test competing explanations for tropical arthropod megadiversity, (ii) improve estimates of global eukaryotic species diversity, and (iii) use plant and arthropod communities as efficient proxies for each other, thus improving the efficiency of conservation planning and of detecting forest degradation and recovery. We therefore applied metabarcoding to Malaise-trap samples across two tropical landscapes in China. We demonstrate that plant species richness can accurately predict arthropod (mostly insect) species richness and that plant and insect community compositions are highly correlated, even in landscapes that are large, heterogeneous and anthropogenically modified. Finally, we review how metabarcoding makes feasible highly replicated tests of the major competing explanations for tropical megadiversity. 64 6 7 5 70 SEP 2016 25 17 4407 4419 10.1111/mec.13770 13 WOS:000383343800021 27474399 Trap malaise 454 PCR Barcoding J Belda, E; Pedrola, L; Pereto, J; Martinez-Blanch, JF; Montagud, A; Navarro, E; Urchueguia, J; Ramon, D; Moya, A; Porcar, M Microbial Diversity in the Midguts of Field and Lab-Reared Populations of the European Corn Borer Ostrinia nubilalis PLOS ONE English Article CELLULOLYTIC BACTERIUM; GLYCOSIDE HYDROLASES; FUNCTIONAL-ANALYSIS; MULTIPLE ALIGNMENT; COMMUNITY; GUT; METAGENOMICS; RECONSTRUCTION; LEPIDOPTERA; ENVIRONMENT Background: Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications. Methodological/Principal Findings: A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories. Conclusions/Significance: Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria. 44 26 27 1 25 Jun-30 2011 6 6 e21751 10.1371/journal.pone.0021751 14 WOS:000292291800039 21738787 Lepidoptera 454 Skimming Microbiota J McCarthy, CB; Santini, MS; Pimenta, PFP; Diambra, LA First Comparative Transcriptomic Analysis of Wild Adult Male and Female Lutzomyia longipalpis, Vector of Visceral Leishmaniasis PLOS ONE English Article CYTOCHROME-C-OXIDASE; PROTEIN-COUPLED RECEPTORS; PERMETHRIN INDUCES OVEREXPRESSION; MOSQUITO ANOPHELES-GAMBIAE; DROSOPHILA ACTIN GENES; AEDES-AEGYPTI; PERITROPHIC MATRIX; SAND FLIES; CUTANEOUS LEISHMANIASIS; INSECTICIDE RESISTANCE Leishmaniasis is a vector-borne disease with a complex epidemiology and ecology. Visceral leishmaniasis (VL) is its most severe clinical form as it results in death if not treated. In Latin America VL is caused by the protist parasite Leishmania infantum (syn. chagasi) and transmitted by Lutzomyia longipalpis. This phlebotomine sand fly is only found in the New World, from Mexico to Argentina. However, due to deforestation, migration and urbanisation, among others, VL in Latin America is undergoing an evident geographic expansion as well as dramatic changes in its transmission patterns. In this context, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases. Insect vector transcriptomic analyses enable the identification of molecules involved in the insect's biology and vector-parasite interaction. Previous studies on laboratory reared Lu. longipalpis have provided a descriptive repertoire of gene expression in the whole insect, midgut, salivary gland and male reproductive organs. Nevertheless, the study of wild specimens would contribute a unique insight into the development of novel bioinsecticides. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. In this study, total RNA was extracted from the sand flies, submitted to sequence independent amplification and high-throughput pyrosequencing. This is the first time an unbiased and comprehensive transcriptomic approach has been used to analyse an infectious disease vector in its natural environment. Transcripts identified in the sand flies showed characteristic profiles which correlated with the environment of origin and with taxa previously identified in these same specimens. Among these, various genes represented putative targets for vector control via RNA interference (RNAi). 147 6 7 0 18 Mar-12 2013 8 3 e58645 10.1371/journal.pone.0058645 20 WOS:000316252500036 23554910 Diptera 454 Transcriptome J Berlanga, M; Llorens, C; Comas, J; Guerrero, R Gut Bacterial Community of the Xylophagous Cockroaches Cryptocercus punctulatus and Parasphaeria boleiriana PLOS ONE English Article WOOD-FEEDING COCKROACHES; H-2 PLUS CO2; TERMITE-GUT; PERIPLANETA-AMERICANA; MICROBIAL COMMUNITY; ATLANTIC FOREST; DIVERSITY; DICTYOPTERA; PHYLOGENY; PROTISTS Cryptocercus punctulatus and Parasphaeria boleiriana are two distantly related xylophagous and subsocial cockroaches. Cryptocercus is related to termites. Xylophagous cockroaches and termites are excellent model organisms for studying the symbiotic relationship between the insect and their microbiota. In this study, high-throughput 454 pyrosequencing of 16S rRNA was used to investigate the diversity of metagenomic gut communities of C. punctulatus and P. boleiriana, and thereby to identify possible shifts in symbiont allegiances during cockroaches evolution. Our results revealed that the hindgut prokaryotic communities of both xylophagous cockroaches are dominated by members of four Bacteria phyla: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Other identified phyla were Spirochaetes, Planctomycetes, candidatus Saccharibacteria (formerly TM7), and Acidobacteria, each of which represented 1-2% of the total population detected. Community similarity based on phylogenetic relatedness by unweighted UniFrac analyses indicated that the composition of the bacterial community in the two species was significantly different (P < 0.05). Phylogenetic analysis based on the characterized clusters of Bacteroidetes, Spirochaetes, and Deltaproteobacteria showed that many OTUs present in both cockroach species clustered with sequences previously described in termites and other cockroaches, but not with those from other animals or environments. These results suggest that, during their evolution, those cockroaches conserved several bacterial communities from the microbiota of a common ancestor. The ecological stability of those microbial communities may imply the important functional role for the survival of the host of providing nutrients in appropriate quantities and balance. 66 3 3 4 31 Apr-07 2016 11 4 e0152400 10.1371/journal.pone.0152400 16 WOS:000373608000019 27054320 Blattodea 454 PCR Barcoding Microbiota J Minard, G; Tran, FH; Dubost, A; Van, TV; Mavingui, P; Moro, CV Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY English Article high-throughput sequencing; 16S rDNA; bacterial diversity; Wolbachia; field-caught mosquitoes CHIKUNGUNYA VIRUS; GUT MICROBIOTA; CYTOPLASMIC INCOMPATIBILITY; WOLBACHIA; DIVERSITY; SYMBIONTS; VECTOR; POPULATIONS; MADAGASCAR; INFECTION The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 x 10(0.01) and 5.47 x 10(0.1) for wAlbA and between 2.03 x 10(0.1) and 1.4 x 10(1) for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area. 51 26 27 0 16 MAY 2014 4 59 10.3389/fcimb.2014.00059 9 WOS:000338986800006 24860790 Diptera 454 PCR Barcoding Microbiota J Gibson, J; Shokralla, S; Porter, TM; King, I; van Konynenburg, S; Janzen, DH; Hallwachs, W; Hajibabaei, M Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA English Article cytochrome c oxidase subunit I; Costa Rica; insect; Malaise trap; NGS GUT CONTENTS; BIODIVERSITY; DIVERSITY; BARCODE; AMPLIFICATION; REGION; PCR; SEQUENCES; RECOVERY; BACTERIA Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works. 54 87 89 8 175 Jun-03 2014 111 22 8007 8012 10.1073/pnas.1406468111 6 WOS:000336687900043 24808136 Monitoring 454; Illumina MiSeq PCR Barcoding J Martinson, EO; Wheeler, D; Wright, J; Mrinalini; Siebert, AL; Werren, JH Nasonia vitripennis venom causes targeted gene expression changes in its fly host MOLECULAR ECOLOGY English Article enhancer of split; extended phenotype; parasitoid wasps; venom WASP PIMPLA-HYPOCHONDRIACA; CULTURED INSECT CELLS; ECTOPARASITIC WASP; FLESH FLY; WALKER HYMENOPTERA; SARCOPHAGA-BULLATA; DROSOPHILA; PTEROMALIDAE; METABOLISM; EVOLUTION Parasitoid wasps are diverse and ecologically important insects that use venom to modify their host's metabolism for the benefit of the parasitoid's offspring. Thus, the effects of venom can be considered an extended phenotype' of the wasp. The model parasitoid wasp Nasonia vitripennis has approximately 100 venom proteins, 23 of which do not have sequence similarity to known proteins. Envenomation by N.vitripennis has previously been shown to induce developmental arrest, selective apoptosis and alterations in lipid metabolism in flesh fly hosts. However, the full effects of Nasonia venom are still largely unknown. In this study, we used high throughput RNA sequencing (RNA-Seq) to characterize global changes in Sarcophaga bullata (Diptera) gene expression in response to envenomation by N.vitripennis. Surprisingly, we show that Nasonia venom targets a small subset of S.bullata loci, with similar to 2% genes being differentially expressed in response to envenomation. Strong upregulation of enhancer of split complex genes provides a potential molecular mechanism that could explain the observed neural cell death and developmental arrest in envenomated hosts. Significant increases in antimicrobial peptides and their corresponding regulatory genes provide evidence that venom could be selectively activating certain immune responses of the hosts. Further, we found differential expression of genes in several metabolic pathways, including glycolysis and gluconeogenesis that may be responsible for the decrease in pyruvate levels found in envenomated hosts. The targeting of Nasonia venom effects to a specific and limited set of genes provides insight into the interaction between the ectoparasitoid wasp and its host. 48 25 26 1 31 DEC 2014 23 23 5918 5930 10.1111/mec.12967 13 WOS:000345630700020 25319487 Diptera 454; Illumina HiSeq Transcriptome J Sadd, BM; Barribeau, SM; Bloch, G; de Graaf, DC; Dearden, P; Elsik, CG; Gadau, J; Grimmelikhuijzen, CJP; Hasselmann, M; Lozier, JD; Robertson, HM; Smagghe, G; Stolle, E; Van Vaerenbergh, M; Waterhouse, RM; Bornberg-Bauer, E; Klasberg, S; Bennett, AK; Caamara, F; Guigo, R; Hoff, K; Mariotti, M; Munoz-Torres, M; Murphy, T; Santesmasses, D; Amdam, GV; Beckers, M; Beye, M; Biewer, M; Bitondi, MMG; Blaxter, ML; Bourke, AFG; Brown, MJF; Buechel, SD; Cameron, R; Cappelle, K; Carolan, JC; Christiaens, O; Ciborowski, KL; Clarke, DF; Colgan, TJ; Collins, DH; Cridge, AG; Dalmay, T; Dreier, S; du Plessis, L; Duncan, E; Erler, S; Evans, J; Falcon, T; Flores, K; Freitas, FCP; Fuchikawa, T; Gempe, T; Hartfelder, K; Hauser, F; Helbing, S; Humann, FC; Irvine, F; Jermiin, LS; Johnson, CE; Johnson, RM; Jones, AK; Kadowaki, T; Kidner, JH; Koch, V; Kohler, A; Kraus, FB; Lattorff, HMG; Leask, M; Lockett, GA; Mallon, EB; Antonio, DSM; Marxer, M; Meeus, I; Moritz, RFA; Nair, A; Napflin, K; Nissen, I; Niu, J; Nunes, FMF; Oakeshott, JG; Osborne, A; Otte, M; Pinheiro, DG; Rossie, N; Rueppell, O; Santos, CG; Schmid-Hempel, R; Schmitt, BD; Schulte, C; Simoes, ZLP; Soares, MPM; Swevers, L; Winnebeck, EC; Wolschin, F; Yu, N; Zdobnov, EM; Aqrawi, PK; Blankenburg, KP; Coyle, M; Francisco, L; Hernandez, AG; Holder, M; Hudson, ME; Jackson, L; Jayaseelan, J; Joshi, V; Kovar, C; Lee, SL; Mata, R; Mathew, T; Newsham, IF; Ngo, R; Okwuonu, G; Pham, C; Pu, LL; Saada, N; Santibanez, J; Simmons, D; Thornton, R; Venkat, A; Walden, KKO; Wu, YQ; Debyser, G; Devreese, B; Asher, C; Blommaert, J; Chipman, AD; Chittka, L; Fouks, B; Liu, J; O'Neill, MP; Sumner, S; Puiu, D; Qu, J; Salzberg, SL; Scherer, SE; Muzny, DM; Richards, S; Robinson, GE; Gibbs, RA; Schmid-Hempel, P; Worley, KC The genomes of two key bumblebee species with primitive eusocial organization GENOME BIOLOGY English Article BEE APIS-MELLIFERA; SEX-DETERMINATION PATHWAY; MULTIPLE SEQUENCE ALIGNMENT; BOMBUS-TERRESTRIS LINNAEUS; MALE COURTSHIP BEHAVIOR; HONEY-BEE; DROSOPHILA-MELANOGASTER; PHYLOGENETIC ANALYSES; DNA METHYLATION; HIGH-THROUGHPUT Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation. 213 103 106 15 200 Apr-24 2015 16 76 10.1186/s13059-015-0623-3 31 WOS:000353676700001 25908251 Hymenoptera 454; Illumina HiSeq WGS J Blanckenhorn, WU; Rohner, PT; Bernasconi, MV; Haugstetter, J; Buser, A Is qualitative and quantitative metabarcoding of dung fauna biodiversity feasible? ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY English Article Biodiversity assessment; Barcode; Species detection; Survival; Taxonomic resolution MITOCHONDRIAL-DNA SEQUENCES; INTERNATIONAL RING TEST; CHAIN-REACTION PRIMERS; VETERINARY PARASITICIDES; SCATHOPHAGIDAE DIPTERA; MOLECULAR SYSTEMATICS; IVERMECTIN RESIDUES; SPECIES-DIVERSITY; SEPSID FLIES; CATTLE DUNG In biodiversity assessments, especially of small-bodied organisms for which taxonomic expertise is lacking, identification by genetic barcoding may be a cost-effective and efficient alternative to traditional identification of species by morphology, ecology, and behavior. The authors tested the feasibility and accuracy of such an approach using dung insects of practical relevance in ecotoxicological assessments of veterinary pharmaceutical residues in the environment. They produced 8 known mixtures that varied in absolute and relative composition of small-bodied and large-bodied species to see whether mitochondrial cytochrome c oxidase subunit 1 barcoding picks up all species qualitatively and quantitatively. As demonstrated before in other contexts, such metabarcoding of large numbers of dung insect specimens is principally possible using next-generation sequencing. The authors recovered most species in a sample (low type I error), at minimum permitting analysis of species richness. They obtained even quantitative responses reflecting the body size of the species, although the number of specimens was not well detected. The latter is problematic when calculating diversity indices. Nevertheless, the method yielded too many closely related false positives (type II error), thus generally overestimating species diversity and richness. These errors can be reduced by refining methods and data filtering, although this requires bioinformatics expertise often unavailable where such research is carried out. Identification by barcoding foremost hinges on a good reference database, which does not yet exist for dung organisms but would be worth developing for practical applications. Environ Toxicol Chem 2016;35:1970-1977. (c) 2015 SETAC 59 7 7 1 32 AUG 2016 35 8 1970 1977 10.1002/etc.3275 8 WOS:000380057100011 26450644 Diptera; Coleoptera Illumina MiSeq PCR Barcoding J Zhang, F; Sun, XX; Zhang, XC; Zhang, S; Lu, J; Xia, YM; Huang, YH; Wang, XJ The interactions between gut microbiota and entomopathogenic fungi: a potential approach for biological control of Blattella germanica (L.) PEST MANAGEMENT SCIENCE English Article Blattella germanica; anti-entomopathogenic fungi assay; interactions; gut microbiota; biological control FREE DESERT LOCUSTS; METARHIZIUM-ANISOPLIAE; BEAUVERIA-BASSIANA; SCHISTOCERCA-GREGARIA; DELPHACODES-KUSCHELI; INTESTINAL FLORA; GENOMIC ANALYSIS; DALBULUS-MAIDIS; BACTERIA; COCKROACH BACKGROUNDMetarhizium anisopliae and Beauveria bassiana mainly infect insects through the cuticle; gut infection occasionally occurs. Micro-organisms existing in the gut may play a crucial role in the evolution and ecology of host defenses against fungal pathogens. To evaluate whether the gut bacteria participate in antifungal activity, and to determine their role in host protection, the interactions between gut bacteria and M. anisopliae and the diversity of gut microbiota in cockroaches were studied. RESULTSAn oral feeding test showed that the mortality of conventional cockroaches was significantly lower than that of germ-free cockroaches; both gut homogenates and aqueous fecal extracts showed antifungal activity, but the samples from germ-free cockroaches did not. Twenty-two bacterial strains with antifungal activity and siderophore-producing ability were isolated from the gut and feces of cockroaches. Using high-throughput sequencing techniques, a total of 23 different phyla and 212 genera were detected. The composition of the microbiota of the hindgut was vastly different from those of the foregut and midgut; higher diversity and abundance of Bacteroides and Pseudomonas were found in the hindgut. CONCLUSIONThe gut microbiota of German cockroaches may play a critical role in protecting cockroaches from fungal invasion and colonization. Removing certain bacteria from the B. germanica microbiota may facilitate microbial control using fungal pathogens. (c) 2017 Society of Chemical Industry 60 1 1 19 47 FEB 2018 74 2 438 447 10.1002/ps.4726 10 WOS:000419406700022 28888066 Blattodea Illumina MiSeq PCR Barcoding Microbiota J Fromont, C; Riegler, M; Cook, JM Relative Abundance and Strain Diversity in the Bacterial Endosymbiont Community of a Sap-Feeding Insect Across Its Native and Introduced Geographic Range MICROBIAL ECOLOGY English Article Endosymbiont; Geography; Carsonella; Wolbachia; Psyllid BAYESIAN PHYLOGENETIC INFERENCE; INFECTION DENSITY; WOLBACHIA DENSITY; ACYRTHOSIPHON-PISUM; RHAGOLETIS-CERASI; HOST GENOTYPE; SEQUENCE DATA; PEA APHID; 16S RDNA; POPULATIONS Most insects are associated with bacterial symbionts. The bacterial diversity and community composition within hosts may play an important role in shaping insect population biology, ecology and evolution. We focussed on the bacterial microbiome of the Australian fig homotomid Mycopsylla fici (Hemiptera: Psylloidea), which can cause defoliation of its only host tree, Ficus macrophylla. This sap-feeding insect is native to mainland Australia and Lord Howe Island (LHI) but also occurs where its host has been planted, notably in New Zealand. By using a high-throughput 16S rDNA amplicon sequencing approach, we compared the bacterial diversity and community composition in individual adult males of four host populations, Sydney, Brisbane, LHI and Auckland. We also compared males, females and nymphs of the Sydney population. The microbiome of M. fici was simple and consisted mostly of the following three maternally inherited endosymbiont species: the primary endosymbiont Carsonella, a secondary (S-) endosymbiont and Wolbachia. However, the relative abundance of their sequence reads varied between host populations, except for similarities between Sydney and Auckland. In addition, insects from Sydney and Auckland had identical bacterial strains supporting the hypothesis that Sydney is the source population for Auckland. In contrast, mainland and LHI populations harboured the same S-endosymbiont, co-diverged Carsonella but different Wolbachia strains. Besides detecting endosymbiont-specific patterns of either co-evolution or horizontal acquisition, our study highlights that relative abundance of maternally inherited endosymbionts should also be taken into account when studying bacterial communities across host populations, as variations in bacterial density may impact host biology and ecology. 85 2 2 8 39 OCT 2017 74 3 722 734 10.1007/s00248-017-0971-5 13 WOS:000408969500019 28386769 Hemiptera Illumina MiSeq PCR Barcoding Microbiota J Receveur, JP; Pechal, JL; Benbow, ME; Donato, G; Rainey, T; Wallace, JR Changes in Larval Mosquito Microbiota Reveal Non-target Effects of Insecticide Treatments in Hurricane-Created Habitats MICROBIAL ECOLOGY English Article Microbial ecology; Microbe; Higher organism interactions; Community genomics; Non-target effects AEDES-AEGYPTI; BACTERIAL COMMUNITIES; VECTOR COMPETENCE; METHOPRENE; DIVERSITY; ECOSYSTEM; PATTERNS; DECOMPOSITION; REPRODUCTION; ISRAELENSIS Ephemeral aquatic habitats and their associated microbial communities (microbiomes) play important roles in the growth and development of numerous aquatic insects, including mosquitoes (Diptera). Biological control agents, such as Bacillus thuringiensis israelensis (Biz) or insect growth regulators (e.g., methoprene), are commonly used to control mosquitoes in these habitats. However, it is unknown how commonly used control compounds affect the mosquito internal microbiome and potentially alter their life history traits. The objectives of this study were threefold: characterize the internal microbiota of Aedes larvae (Culicidae) in ephemeral forested mosquito habitat using high-throughput amplicon based sequencing, assess how mosquito control treatments affect the internal microbial communities of larval mosquitoes, and determine if changes to the microbiome resulted from direct or indirect treatment effects. The larval microbiome varied in community composition and diversity with development stage and treatment, suggesting potential effects of control compounds on insect microbial ecology. While microbial community differences due to Bti treatment were a result of indirect effects on larval development, methoprene had significant impacts on bacterial and algal taxa that could not be explained by indirect treatment effects. These results provide new information on the interactions between pesticide treatments and insect microbial communities. 64 0 0 7 9 OCT 2018 76 3 719 728 10.1007/s00248-018-1175-3 10 WOS:000443567600014 29549385 Diptera Illumina MiSeq PCR Barcoding Microbiota J Briem, F; Zeisler, C; Guenay, Y; Staudacher, K; Vogt, H; Traugott, M Identifying plant DNA in the sponging-feeding insect pest Drosophila suzukii JOURNAL OF PEST SCIENCE English Article Chloroplast DNA; Diet metabarcoding; Feeding experiment; Molecular gut content analysis; Spotted Wing Drosophila; High-throughput sequencing; NGS SPOTTED WING DROSOPHILA; GUT-CONTENT-ANALYSIS; DIPTERA DROSOPHILIDAE; PREY DNA; PREDATORS; HOST; PCR; IDENTIFICATION; PREFERENCES; MATSUMURA Drosophila suzukii (Diptera: Drosophilidae) is a highly polyphagous invasive pest threatening fruit production in the Americas and Europe. The current knowledge of its host plants is mainly based on oviposition and larval development in fruits, while little is known on the diet of the adult flies. This information is important for developing effective control strategies. Here, we examine DNA-based techniques to determine food plants of D. suzukii. Adult flies were fed with raspberries (Rubus idaeus) and allowed to digest up to 72 h after feeding. Raspberry DNA was detected by diagnostic PCR for up to 48 h post-feeding with a significant negative effect of time on DNA detection success but no significant differences between male and female flies in detection probabilities. As D. suzukii walks on plants, its body surface can get contaminated with DNA. With a bleaching experiment, we succeeded to remove contaminating external plant DNA, while the DNA in the gut content stayed unaffected. Finally, field-collected flies were subjected to a next-generation sequencing approach, demonstrating that plant DNA from different host plants can be efficiently detected in both bleached and non-bleached specimens. In order to safeguard against erroneous host plant detections, we recommend bleaching flies before they are subjected to DNA extraction. The current findings encourage the use of DNA-based gut content analysis in D. suzukii to obtain a better understanding of its feeding ecology which is a prerequisite for developing successful control strategies. 53 0 0 15 15 JUN 2018 91 3 985 994 10.1007/s10340-018-0963-3 10 WOS:000433026600005 Diptera Illumina MiSeq PCR Barcoding J Galliot, JN; Brunel, D; Berard, A; Chauveau, A; Blanchetete, A; Lanore, L; Farruggia, A Investigating a flower-insect forager network in a mountain grassland community using pollen DNA barcoding JOURNAL OF INSECT CONSERVATION English Article Flower-foraging insects; Semi-natural grasslands; Plant-pollinator interactions; DNA barcoding; Pollen loads CROP POLLINATION; AGRICULTURAL INTENSIFICATION; BEES; FLIES; BIODIVERSITY; DIVERSITY; PLANTS; SPECIALIZATION; IDENTIFICATION; LANDSCAPES Faced with the decline of pollinators, it is relevant to strengthen our understanding of the whole plant-pollinator web in semi-natural grasslands that serve as refuges for pollinator populations. The aim of this study was to explore the diversity of flower-foraging insects involved in pollen transfer in mountain semi-natural grasslands. Insects actively collecting pollen and/or nectar were caught in spring in six mountain semi-natural grasslands displaying a floristic richness gradient. Individual determinations of insects were made at the finest possible taxonomic scale and pollen loads were removed from the insect body. Using next-generation DNA sequencing, pollens were identified through the ribosomal DNA cistron using the ITS2 database and the ITS plant rDNA cistron sequences from Genbank. A total of 236 flower-foraging insects were collected. Diptera represented 82% of the total catches distantly followed by Hymenoptera (15%) and Apoidea (bees) (11%). Visual observations revealed that Diptera foraged on 16 of the 21 flower species visited by insects. DNA metabarcoding showed that 82% (191) of all of the collected insects were carrying pollen and 44% (104) were carrying two genera of plants or more. Our results demonstrate that Diptera are potential key-pollinators in mountain semi-natural grasslands that cannot be overlooked by the scientific community. However difficulties of taxonomic determination due to severe shortage of experts for Diptera have to be urgently overcome. Further studies on the link between pollen transfer and actual pollination in a global change context are also required. Moreover, our results support the idea that DNA metabarcoding provides accurate information about the plants-insects networks but it also pointed out sensitive issues, especially the necessity to build reliable national barcode databases. 66 1 1 11 32 DEC 2017 21 05-Jun 827 837 10.1007/s10841-017-0022-z 11 WOS:000417608400008 Pollen collectors Illumina MiSeq PCR Barcoding J Wang, QQ; Tang, GH Genomic and phylogenetic analysis of the complete mitochondrial DNA sequence of walnut leaf pest Paleosepharia posticata (Coleoptera: Chrysomeloidea) JOURNAL OF ASIA-PACIFIC ENTOMOLOGY English Article Coleoptera; Paleosepharia posticata; Mitochondrial genome; Phylogenetic analysis GENE ORGANIZATION; ORTHOPTERA; EVOLUTION; HEMIPTERA; SELECTION; SOFTWARE; PROGRAM; INSECTA; MODEL Mitochondrial DNA (mitogenome) has been widely used in species identification and comparison among closely related species and is valuable for phylogenetic studies. However, considering the large amount and high biodiversity of coleopteran insects, only about 100 complete or nearly complete mitogenome sequences of coleopterans were found in GenBank, largely limiting the research of biological evolution among coleopteran insects. In this study, the complete mitochondrial sequence of Paleosepharia posticata, an important walnut leaf pest, was determined by the next-generation sequencing technology and verified using Sanger sequencing method. The mitogenome of P. posticata was 15,729 bp with an A + T content of 79.8%, and was very similar with other reported coleopteran insects in genome complement, gene order and nucleotide composition. All of the protein-coding genes started with the typical ATN initiation codons except for nad1 (TTG). The ancestral gene order with trnI-trnQ-trnM located between the A + T-rich region and nad2 and the formerly identified conserved elements of coleopteran mitogenomes, such as the motif 'TAGTA' between trnS(UCN) and nad2, the overlap 'ATGATAA' between atp8 and atp6 genes and poly-T stretch in the A + T-rich region, were also present in the P. posticata mitogenome. A special 'ATGATAA' between nad41 and nad4, which was 'ATGTTAA' in many insect mitogenomes, was observed in the mitogenome of P. posticata and coleopteran species used in this study. Phylogenetic trees inferred from the amino acid and nucleotide sequence data for the 13 protein-coding genes strongly supported the monopoly of Elateriformia, the paraphyly of Staphyliniformia, and the sister relationship of Scarabaeoidea and Staphylinoidea. 62 2 3 6 12 SEP 2017 20 3 840 853 10.1016/j.aspcn.2017.05.010 14 WOS:000419749200015 Coleoptera Illumina HiSeq Skimming J Haran, J; Delvare, G; Vayssieres, JF; Benoit, L; Cruaud, P; Rasplus, JY; Cruaud, A Increasing the utility of barcode databases through high-throughput sequencing of amplicons from dried museum specimens, an example on parasitic hymenoptera (Braconidae) BIOLOGICAL CONTROL English Article Braconidae; Biological control; Gut content; Dry insect collections; Sub-Saharan Africa SPECIES COMPLEX HYMENOPTERA; BIOLOGICAL-CONTROL; DIPTERA TEPHRITIDAE; WASPS HYMENOPTERA; DNA; COLLECTIONS; TAXONOMY; POPULATION; CERATITIS; PLATFORM Parasitic hymenoptera are natural enemies of a wide range of crop pests. However, the complex taxonomy of this group and the scarcity of taxonomic expertise make difficult the accurate identification of species, which limits their use in biological control programs. In this study, we explored the potential of a two-step PCR approach combined with Illumina sequencing to recover mitochondrial protein coding genes (COI and Cytb) from dry specimens from a reference collection, whose DNA was extracted non-destructively. We studied 18 species of afrotropical Braconidae (Microgastrinae, Opiinae and Braconinae) collected 10 to 35 years ago. Depending on the target region, sequences were obtained from 61 to 90% of the specimens. The full barcode was reconstructed in ca 90% of the species. A customized analysis pipeline allowed us to manage the high rate of contamination and to identify co-amplified sequences of hosts of parasitoids. As a result, in a single analysis, we were able to 1) generate new barcodes for poorly known species, 2) obtain a preliminary overview of intra specific variability, 3) generate a few data on host-parasitoid associations based on remains of hosts in adult wasps. This cost-effective approach should allow to improve the quality of reference molecular databases by capitalizing on museum specimens identified by expert taxonomists, in order to set up more efficient biological control programs. 56 0 0 12 12 JUL 2018 122 93 100 10.1016/j.biocontrol.2018.04.001 8 WOS:000432518000012 Hymenoptera Illumina MiSeq PCR Barcoding J Jacobsen, RM; Kauserud, H; Sverdrup-Thygeson, A; Bjorbwkmo, MM; Birkemoe, T Wood-inhabiting insects can function as targeted vectors for decomposer fungi FUNGAL ECOLOGY English Article Animal-mediated dispersal; Dead wood; Decomposition; DNA barcoding; High-throughput sequencing; Insect-vectored dispersal; Polypores; Saproxylic insects; Scanning electron microscopy; Wood-decay fungi BASIDIOCARP-FEEDING BEETLES; LONG-DISTANCE DISPERSAL; SEED DISPERSAL; FOREST FRAGMENTATION; COMMUNITY COMPOSITION; POPULATION-STRUCTURE; FOMITOPSIS-PINICOLA; SPORE DISPERSAL; CLIMATE-CHANGE; NORWAY SPRUCE Most wood-inhabiting fungi are assumed to be dispersed primarily by wind, with the exception of a few species involved in mutualistic relationships with insects. In this study we tested whether several species of wood-inhabiting insects can function as dispersal vectors for non-mutualistic fungi, which would indicate that wood-inhabiting fungi can benefit from targeted animal-mediated dispersal. We sampled wood-inhabiting beetles (Coleoptera) from freshly felled wood experimentally added to forests and used DNA metabarcoding to investigate the fungal DNA carried by these insects. Staphylinid beetles rarely contained fungal DNA, while Endomychus coccineus, Glischrochilus hortensis and Glischrochilus quadripunctatus frequently carried fungal DNA with a composition specific to the insect taxon. A large proportion of the obtained fungal sequences (34%) represented decomposer fungi, including well-known wood-decay fungi such as Fomitopsis pinicola, Fames fomentarius, Trichaptum abietinum and Trametes versicolor. Scanning electron microscopy further showed that some of the fungal material was carried as spores or yeast cells on the insect exoskeletons. Our results suggest that insect vectored dispersal is of broader importance to wood-inhabiting fungi than previously assumed. (C) 2017 The Authors. Published by Elsevier Ltd. 109 4 4 3 21 OCT 2017 29 76 84 10.1016/j.funeco.2017.06.006 9 WOS:000411305400010 Coleoptera Illumina HiSeq PCR Barcoding J Osimani, A; Milanovic, V; Garofalo, C; Cardinali, F; Roncolini, A; Sabbatini, R; De Filippis, F; Ercolini, D; Gabucci, C; Petruzzelli, A; Tonucci, F; Clementi, F; Aquilanti, L Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY English Article Novel foods; Spore-forming bacteria; Risk analysis; Food safety; Edible insects LARVAE TENEBRIO-MOLITOR; TRANSFERABLE ANTIBIOTIC RESISTANCES; LOCUSTA-MIGRATORIA-MIGRATORIOIDES; LACTIC-ACID BACTERIA; COXIELLA-BURNETII; ESCHERICHIA-COLI; FATTY-ACIDS; STENOTROPHOMONAS-MALTOPHILIA; ALPHITOBIUS-DIAPERINUS; INDEPENDENT METHODS The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. 84 2 2 26 31 Jul-02 2018 276 54 62 10.1016/j.ijfoodmicro.2018.04.013 9 WOS:000432510900008 29665523 Edible insects Illumina MiSeq PCR Barcoding J Dupuis, JR; Brunet, BMT; Bird, HM; Lumley, LM; Fagua, G; Boyle, B; Levesque, R; Cusson, M; Powell, JA; Sperling, FAH Genome-wide SNPs resolve phylogenetic relationships in the North American spruce budworm (Choristoneura fumiferana) species complex MOLECULAR PHYLOGENETICS AND EVOLUTION English Article Phylogenomics; Systematics; Genotyping-by-sequencing; RAD-seq; Population genetics; Species relationships MAXIMUM-LIKELIHOOD PHYLOGENIES; MULTILOCUS GENOTYPE DATA; MITOCHONDRIAL-DNA; GENE TREES; LEPIDOPTERA-TORTRICIDAE; EVOLUTIONARY HISTORY; POPULATION-STRUCTURE; RAD MARKERS; DE-NOVO; INFERENCE High throughput sequencing technologies have revolutionized the potential to reconcile incongruence between gene and species trees, and numerous approaches have been developed to take advantage of these advances. Genotyping-by-sequencing is becoming a regular tool for gathering phylogenetic data, yet comprehensive evaluations of phylogenetic methods using these data are sparse. Here we use multiple phylogenetic and population genetic methods for genotyping-by-sequencing data to assess species relationships in a group of forest insect pests, the spruce budworm (Choristoneura fumiferana) species complex. With few exceptions, all methods agree on the same relationships, most notably placing C. pinus as basal to the remainder of the group, rather than C fumiferana as previously suggested. We found strong support for the monophyly of C pinus, C fumiferana, and C retiniana, but more ambiguous relationships and signatures of introgression in a Glade of western lineages, including C carnana, C lambertiana, C occidentalis occidentalis, C. occidentalis biennis, and C orae. This represents the most taxonomically comprehensive genomic treatment of the spruce budworm species group, which is further supported by the broad agreement among multiple methodologies. (C) 2017 Elsevier Inc. All rights reserved. 109 6 6 4 51 JUN 2017 111 158 168 10.1016/j.ympev.2017.04.001 11 WOS:000400715100013 28390910 Lepidoptera Illumina HiSeq GBS J Brandon-Mong, GJ; Gan, HM; Sing, KW; Lee, PS; Lim, PE; Wilson, JJ DNA metabarcoding of insects and allies: an evaluation of primers and pipelines BULLETIN OF ENTOMOLOGICAL RESEARCH English Article Arthropoda; biodiversity; COI; high-throughput sequencing; Illumina MiSeq; Malaise trap BIODIVERSITY ASSESSMENT; ANNEALING TEMPERATURE; PCR; GENERATION; DIVERSITY; BARCODE; SAMPLES; RECOMBINATION; COMMUNITIES; ARTHROPODS Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80-90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity. 77 35 36 7 90 DEC 2015 105 6 717 727 10.1017/S0007485315000681 11 WOS:000365166000008 26344799 Trap malaise Illumina MiSeq PCR Barcoding J Tay, WT; Elfekih, S; Polaszek, A; Court, LN; Evans, GA; Gordon, KHJ; De Barro, PJ Novel molecular approach to define pest species status and tritrophic interactions from historical Bemisia specimens SCIENTIFIC REPORTS English Article TABACI HEMIPTERA ALEYRODIDAE; MITOCHONDRIAL GENOMES; HYMENOPTERA; APHELINIDAE; COMPLEX; PARASITOIDS; POPULATIONS; WHITEFLIES; EVOLUTION; SEQUENCE Museum specimens represent valuable genomic resources for understanding host-endosymbiont/parasitoid evolutionary relationships, resolving species complexes and nomenclatural problems. However, museum collections suffer DNA degradation, making them challenging for molecular-based studies. Here, the mitogenomes of a single 1912 Sri Lankan Bemisia emiliae cotype puparium, and of a 1942 Japanese Bemisia puparium are characterised using a Next-Generation Sequencing approach. Whiteflies are small sap-sucking insects including B. tabaci pest species complex. Bemisia emiliae's draft mitogenome showed a high degree of homology with published B. tabaci mitogenomes, and exhibited 98-100% partial mitochondrial DNA Cytochrome Oxidase I (mtCOI) gene identity with the B. tabaci species known as Asia II-7. The partial mtCOI gene of the Japanese specimen shared 99% sequence identity with the Bemisia 'JpL' genetic group. Metagenomic analysis identified bacterial sequences in both Bemisia specimens, while hymenopteran sequences were also identified in the Japanese Bemisia puparium, including complete mtCOI and rRNA genes, and various partial mtDNA genes. At 88-90% mtCOI sequence identity to Aphelinidae wasps, we concluded that the 1942 Bemisia nymph was parasitized by an Eretmocerus parasitoid wasp. Our approach enables the characterisation of genomes and associated metagenomic communities of museum specimens using 1.5 ng gDNA, and to infer historical tritrophic relationships in Bemisia whiteflies. 66 4 4 0 9 Mar-27 2017 7 429 10.1038/s41598-017-00528-7 13 WOS:000397391000005 28348369 Hemiptera Illumina MiSeq Skimming J Zhao, Y; Zhang, S; Luo, JY; Wang, CY; Lv, LM; Cui, JJ Bacterial communities of the cotton aphid Aphis gossypii associated with Bt cotton in northern China SCIENTIFIC REPORTS English Article SAP-FEEDING INSECTS; FACULTATIVE SYMBIONTS; ACYRTHOSIPHON-PISUM; SECONDARY SYMBIONTS; HORIZONTAL TRANSFER; HIGH-THROUGHPUT; HOST GENOTYPE; PEA APHIDS; ENDOSYMBIONT; DIVERSITY Aphids are infected with a wide variety of endosymbionts that can confer ecologically relevant traits. However, the bacterial communities of most aphid species are still poorly characterized. This study investigated the bacterial diversity of the cotton aphid Aphis gossypii associated with Bt cotton in northern China by targeting the V4 region of the 16S rDNA using the Illumina MiSeq platform. Our sequencing data revealed that bacterial communities of A. gossypii were generally dominated by the primary symbiont Buchnera, together with the facultative symbionts Arsenophonus and Hamiltonella. To our knowledge, this is the first report documenting the facultative symbiont Hamiltonella in A. gossypii. Moreover, the bacterial community structure was similar within aphids from the same province, but distinct among those from different provinces. The taxonomic diversity of the bacterial community is greater in Hebei Province compared with in samples from Henan and Shandong Provinces. The selection pressure exerted by the different geographical locations could explain the differences found among the various provinces. These findings broaden our understanding of the interactions among aphids, endosymbionts and their environments, and provide clues to develop potential biocontrol techniques against this cotton aphid. 57 2 2 3 32 Apr-15 2016 6 22958 10.1038/srep22958 8 WOS:000374161000001 27079679 Hemiptera Illumina MiSeq PCR Barcoding Microbiota J Lee, CC; Wang, J; Matsuura, K; Yang, CCS The complete mitochondrial genome of yellow crazy ant, Anoplolepis gracilipes (Hymenoptera: Formicidae) MITOCHONDRIAL DNA PART B-RESOURCES English Article Yellow crazy ant; mitochondrial genome; invasive ant ISLAND The yellow crazy ant Anoplolepis gracilipes is an invasive species that threatens biodiversity in introduced ecosystems. We sequenced the A. gracilipes mitogenome using next-generation sequencing methods. The circular mitogenome of A. gracilipes was 16,943 bp included 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single large non-coding region of 893 bp. The base composition was AT-biased (72%). Three genomic rearrangements compared to ancestral insects were found. Phylogenetic analysis based on the concatenated nucleotide sequences of the 13 protein-coding genes supports A. gracilipes belonging to the Formicinae subfamily. We announce the A. gracilipes mitogenome as a DNA reference for further population genetic, phylogenetic, and evolutionary analyses. 17 0 0 1 1 MAY 2018 3 2 622 623 10.1080/23802359.2018.1467739 2 WOS:000445298500067 Hymenoptera Illumina HiSeq Skimming J Weatherbee, CR; Pechal, JL; Benbow, ME The Dynamic Maggot Mass Microbiome ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA English Article Calliphoridae; decomposition; necrobiome; epinecrotic; community BACTERIAL COMMUNITY SUCCESSION; CARRION DECOMPOSITION; POSTMORTEM INTERVAL; POTENTIAL USE; BLOW FLIES; XANTHOMONADACEAE; ECOLOGY; FLY Necrophagous insect studies have shown that decomposing vertebrate remains are an important ephemeral resource within an ecosystem. However, the microbes (e.g., bacteria and archaea) that were a part of the once living organism and the exogenous taxa that colonize this postmortem resource remain largely underexplored. Also, it is not well understood how these two kingdoms interact to recycle decaying biomass, an important mechanistic question for ecosystem function ecology. To better understand microbial community dynamics throughout decomposition, we used swine carcasses (N = 6) as models for mammalian postmortem decomposition to characterize epinecrotic microbial communities from: the abdominal skin of replicate carcasses; the internal microbiome of individual necrophagous dipteran larvae (maggots); and the microbiome of dipteran larval masses that had colonized the carcasses. Sampling occurred every 12 h for the duration of the decomposition process. We characterized these microbial communities over time using high-throughput 16S amplicon sequencing. The relative abundance of microbial taxa changed over decomposition as well as across sampling locations, suggesting significant interactions between the environment, microbes, and insect larvae. Maggot masses were represented by multiple blow fly species in each mass: Phormia regina (Meigen), Lucilia coeruleiviridis (Macquart), and Cochliomyia macellaria (F.). Relative abundance of these species within the mass also changed as decomposition progressed, suggesting the presence of certain Calliphoridae species within a mass may be associated with temporal shifts of the microbial communities. These results provide new insight into the community ecology of carrion decomposition by providing new data on interactions of microbes and dipteran larvae over time. 42 3 3 3 18 JAN 2017 110 1 45 53 10.1093/aesa/saw088 9 WOS:000397091700006 Diptera Illumina MiSeq PCR Barcoding Microbiota J Mata, VA; Amorim, F; Corley, MFV; McCracken, GF; Rebelo, H; Beja, P Female dietary bias towards large migratory moths in the European free-tailed bat (Tadarida teniotis) BIOLOGY LETTERS English Article resource partitioning; bat diet; gender segregation; Tadarida teniotis; metabarcoding; COI EPTESICUS-FUSCUS; FORAGING BEHAVIOR; INSECTS; SEGREGATION; SEX In bats, sexual segregation has been described in relation to differential use of roosting and foraging habitats. It is possible that variation may also exist between genders in the use of different prey types. However, until recently this idea was difficult to test owing to poorly resolved taxonomy of dietary studies. Here, we use high-throughput sequencing to describe gender-related variation in diet composition of the European free-tailed bat (Tadarida teniotis), while controlling for effects of age and season. We analysed guano pellets collected from 143 individuals mist-netted from April to October 2012 and 2013, in northeast Portugal. Moths (Lepidoptera; mainly Noctuidae and Geometridae) were by far the most frequently recorded prey, occurring in nearly all samples and accounting for 96 out of 115 prey taxa. There were significant dietary differences between males and females, irrespective of age and season. Compared to males, females tended to consume larger moths and more moths of migratory behaviour (e.g. Autographa gamma). Our study provides the first example of gender-related dietary variation in bats, illustrating the value of novel molecular tools for revealing intraspecific variation in food resource use in bats and other insectivores. 24 12 12 3 36 Mar-01 2016 12 3 20150988 10.1098/rsbl.2015.0988 5 WOS:000373934700005 27009885 Feaces bat Illumina MiSeq PCR Barcoding J Poelchau, MF; Reynolds, JA; Elsik, CG; Denlinger, DL; Armbruster, PA Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES English Article Aedes albopictus; RNA-Seq; diapause preparation; embryonic development; molecular physiology; invasive species CELL NUCLEAR ANTIGEN; LARGE GENE LISTS; INSECT DIAPAUSE; SARCOPHAGA-CRASSIPALPIS; DROSOPHILA-MELANOGASTER; EMBRYONIC-DEVELOPMENT; FLESH FLY; EXPRESSION; CULICIDAE; DIPTERA Seasonal environments present fundamental physiological challenges to a wide range of insects. Many temperate insects surmount the exigencies of winter by undergoing photoperiodic diapause, in which photoperiod provides a token cue that initiates an alternative developmental programme leading to dormancy. Pre-diapause is a crucial preparatory phase of this process, preceding developmental arrest. However, the regulatory and physiological mechanisms of diapause preparation are largely unknown. Using high-throughput gene expression profiling in the Asian tiger mosquito, Aedes albopictus, we reveal major shifts in endocrine signalling, cell proliferation, metabolism, energy production and cellular structure across pre-diapause development. While some hallmarks of diapause, such as insulin signalling and stress response, were not important at the transcriptional level, two genes, Pepck and PCNA, appear to show diapause-induced transcriptional changes across insect taxa. These processes demonstrate physiological commonalities between Ae. albopictus pre-diapause and diapause strategies across insects, and support the idea of a genetic 'toolkit' for diapause. Observations of gene expression trends from a comparative developmental perspective suggest that individual physiological processes are delayed against a background of a fixed morphological ontogeny. Our results demonstrate how deep sequencing can provide new insights into elusive molecular bases of complex ecological adaptations. 61 47 50 2 51 May-22 2013 280 1759 20130143 10.1098/rspb.2013.0143 9 WOS:000317482100011 23516243 Diptera Illumina HiSeq Transcriptome J Pechal, JL; Benbow, ME Microbial ecology of the salmon necrobiome: evidence salmon carrion decomposition influences aquatic and terrestrial insect microbiomes ENVIRONMENTAL MICROBIOLOGY English Article TROUT ONCORHYNCHUS-MYKISS; FLIES DIPTERA; MAGGOT EXCRETIONS/SECRETIONS; STAPHYLOCOCCUS-AUREUS; INTERACTIVE TREE; RAIN-FOREST; BLOW FLIES; FOOD WEBS; DIVERSITY; BACTERIAL Carrion decomposition is driven by complex relationships that affect necrobiome community (i.e. all organisms and their genes associated with a dead animal) interactions, such as insect species arrival time to carrion and microbial succession. Little is understood about how microbial communities interact with invertebrates at the aquatic-terrestrial habitat interface. The first objective of the study was to characterize internal microbial communities using high-throughput sequencing of 16S rRNA gene amplicons for aquatic insects (three mayfly species) in streams with salmon carcasses compared with those in streams without salmon carcasses. The second objective was to assess the epinecrotic microbial communities of decomposing salmon carcasses (Oncorhynchus keta) compared with those of terrestrial necrophagous insects (Calliphora terraenovae larvae and adults) associated with the carcasses. There was a significant difference in the internal microbiomes of mayflies collected in salmon carcass-bearing streams and in non-carcass streams, while the developmental stage of blow flies was the governing factor in structuring necrophagous insect internal microbiota. Furthermore, the necrophagous internal microbiome was influenced by the resource on which the larvae developed, and changes in the adult microbiome varied temporally. Overall, these carrion subsidy-driven networks respond to resource pulses with bottom-up effects on consumer microbial structure, as revealed by shifting communities over space and time. 77 10 10 0 21 MAY 2016 18 5 1511 1522 10.1111/1462-2920.13187 12 WOS:000375481200019 26690563 Ephemeroptera Illumina MiSeq PCR Barcoding Microbiota J Liu, SL; Wang, X; Xie, L; Tan, MH; Li, ZY; Su, X; Zhang, H; Misof, B; Kjer, KM; Tang, M; Niehuis, O; Jiang, H; Zhou, X Mitochondrial capture enriches mito-DNA 100 fold, enabling PCR-free mitogenomics biodiversity analysis MOLECULAR ECOLOGY RESOURCES English Article biodiversity; gene capture; microarray; mitochondrial genome DE-NOVO ASSEMBLER; GENOMES; PHYLOGENY; BARCODE; EVOLUTION; SAMPLES; ARRAY Biodiversity analyses based on next-generation sequencing (NGS) platforms have developed by leaps and bounds in recent years. A PCR-free strategy, which can alleviate taxonomic bias, was considered as a promising approach to delivering reliable species compositions of targeted environments. The major impediment of such a method is the lack of appropriate mitochondrial DNA enrichment ways. Because mitochondrial genomes (mitogenomes) make up only a small proportion of total DNA, PCR-free methods will inevitably result in a huge excess of data (>99%). Furthermore, the massive volume of sequence data is highly demanding on computing resources. Here, we present a mitogenome enrichment pipeline via a gene capture chip that was designed by virtue of the mitogenome sequences of the 1000 Insect Transcriptome Evolution project (1KITE, ). A mock sample containing 49 species was used to evaluate the efficiency of the mitogenome capture method. We demonstrate that the proportion of mitochondrial DNA can be increased by approximately 100-fold (from the original 0.47% to 42.52%). Variation in phylogenetic distances of target taxa to the probe set could in principle result in bias in abundance. However, the frequencies of input taxa were largely maintained after capture (R-2=0.81). We suggest that our mitogenome capture approach coupled with PCR-free shotgun sequencing could provide ecological researchers an efficient NGS method to deliver reliable biodiversity assessment. 45 24 26 1 42 MAR 2016 16 2 470 479 10.1111/1755-0998.12472 10 WOS:000369137000010 26425990 DNA soup Illumina HiSeq Transcriptome Chip (Capture) J Reiskind, MOB; Coyle, K; Daniels, HV; Labadie, P; Reiskind, MH; Roberts, NB; Roberts, RB; Schaff, J; Vargo, EL Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa MOLECULAR ECOLOGY RESOURCES English Article double-digest RAD sequencing; high-throughput sequencing; reduced representation sequencing; restriction enzyme digests POPULATION GENOMICS; ADAPTIVE RADIATION; HIGH-THROUGHPUT; READ ALIGNMENT; CICHLID FISHES; MARKERS; DIVERGENCE; PHYLOGENY; EVOLUTION; DISCOVERY The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies. 40 5 5 6 36 NOV 2016 16 6 1303 1314 10.1111/1755-0998.12527 12 WOS:000385941500003 27739656 Several Orders Illumina HiSeq RAD-seq J Carew, ME; Metzeling, L; St Clair, R; Hoffmann, AA Detecting invertebrate species in archived collections using next-generation sequencing MOLECULAR ECOLOGY RESOURCES English Article aquatic; DNA barcoding; insect; invertebrates; metagenomic DNA BARCODES; WATER-QUALITY; RAPID ASSESSMENT; FAMILY-LEVEL; MACROINVERTEBRATES; AUSTRALIA; IDENTIFICATION; BIOASSESSMENT; AMPLIFICATION; SPECIMENS Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next-generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill 'gaps' in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material. 64 3 3 4 10 SEP 2017 17 5 915 930 10.1111/1755-0998.12644 16 WOS:000415921600008 27987268 Monitoring Illumina MiSeq PCR Barcoding J Sproul, JS; Maddison, DR Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA MOLECULAR ECOLOGY RESOURCES English Article high-throughput DNA sequencing; insects; museum specimens; natural history collections; sample preparation; type specimens MUSEUM SPECIMENS; ANCIENT DNA; GENOMICS; RHODOPHYTA; PHYLOGENY; BEMBIDION; CAPTURE; MODELS; FUTURE; SHIFTS Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens. 37 4 4 6 14 NOV 2017 17 6 1183 1201 10.1111/1755-0998.12660 19 WOS:000415921900032 28199781 Coleoptera Illumina HiSeq Skimming J Batovska, J; Lynch, SE; Cogan, NOI; Brown, K; Darbro, JM; Kho, EA; Blacket, MJ Effective mosquito and arbovirus surveillance using metabarcoding MOLECULAR ECOLOGY RESOURCES English Article bulk sample; Culicidae; cytochrome c oxidase subunit I; DNA barcoding; pooled samples; virus ROSS-RIVER-VIRUS; DNA; IDENTIFICATION; BIODIVERSITY; ARTHROPODS; ABUNDANCE; BARCODES; VECTORS; CAPTURE; SAMPLES Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next-generation sequencing (NGS) is a high-throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed 'metabarcoding.' The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269-bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67-bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches. 52 0 0 8 10 JAN 2018 18 1 32 40 10.1111/1755-0998.12682 9 WOS:000424112000004 28417591 Diptera Illumina MiSeq PCR Barcoding J Birer, C; Tysklind, N; Zinger, L; Duplais, C Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: A case study with ant cuticular bacteria MOLECULAR ECOLOGY RESOURCES English Article 16S rRNA; bacterial communities; cuticular microbiome; insect cuticle; metabarcoding SKIN MICROBIOTA; COMMUNITIES; HYMENOPTERA; FORMICIDAE; STRATEGIES; DIVERSITY; EVOLUTION; SYMBIOSIS High-throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behaviour of their hosts. However, our understanding of host-cuticular microbiota feedbacks remains hampered by the difficulties of working with low bacterial DNA quantities as with individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (similar to 15mm) and Pseudomyrmex penetrator (similar to 5mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial community characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in terms of bacterial diversity and composition for A.cephalotes (similar to 15mm). In contrast, greater amounts of artefactual sequences and contaminations, as well as noticeable differences in bacterial community characteristics were observed between extraction methods for P.penetrator (similar to 5mm). We also found that large (similar to 15mm) and small (similar to 5mm) A.cephalotes individuals harbour different bacterial communities. Our benchmark suggests that cuticular microbiota of single individual insects can be reliably retrieved provided that blank controls, appropriate data cleaning, and individual body size and functional role within insect society are considered in the experiment. 48 1 1 11 30 NOV 2017 17 6 e34 e45 10.1111/1755-0998.12688 12 WOS:000415921900004 28477337 Formicidae Cuticle Illumina MiSeq PCR Barcoding J Galan, M; Pons, JB; Tournayre, O; Pierre, E; Leuchtmann, M; Pontier, D; Charbonnel, N Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis MOLECULAR ECOLOGY RESOURCES English Article Arthropoda; Chiroptera; environmental DNA (eDNA); false positives; high-throughput sequencing; predator-prey interactions DESMAN GALEMYS-PYRENAICUS; ENVIRONMENTAL DNA; PYRENEAN DESMAN; BIODIVERSITY; EXTRACTION; IDENTIFY; PLATFORM; BARCODE; FOOD Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two-step PCR protocol enabling the "all at once" taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis-assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost-effective screening tool for addressing evolutionary ecological issues or developing "chirosurveillance" and conservation strategies. 55 1 1 38 38 MAY 2018 18 3 474 489 10.1111/1755-0998.12749 16 WOS:000432662400008 29288544 Feaces bat Illumina MiSeq PCR Barcoding J Wang, WY; Srivathsan, A; Foo, M; Yamane, SK; Meier, R Sorting specimen-rich invertebrate samples with cost-effective NGS barcodes: Validating a reverse workflow for specimen processing MOLECULAR ECOLOGY RESOURCES English Article community ecology; DNA barcoding; insects; invertebrates; systematics DNA BARCODES; BIOLOGICAL IDENTIFICATIONS; BIODIVERSITY ASSESSMENT; DIRECT PCR; TAXONOMY; ANTS; DELIMITATION; ORGANIZATION; PSEUDOGENES; EXTRACTION Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313bp of COI; laboratory cost <$0.50 per specimen) through next-generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88-90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information. 64 6 7 18 18 MAY 2018 18 3 490 501 10.1111/1755-0998.12751 12 WOS:000432662400009 29314756 Hymenoptera Illumina MiSeq PCR Barcoding J Dupuis, JR; Bremer, FT; Kauwe, A; San Jose, M; Leblanc, L; Rubinoff, D; Geib, SM HiMAP: Robust phylogenomics from highly multiplexed amplicon sequencing MOLECULAR ECOLOGY RESOURCES English Article Bactrocera; high-throughput sequencing; phylogenetics; systematics; Tephritidae SPECIES TREE ESTIMATION; MAXIMUM-LIKELIHOOD PHYLOGENIES; ANCHORED HYBRID ENRICHMENT; FLIES DIPTERA TEPHRITIDAE; ULTRACONSERVED ELEMENTS; FRUIT-FLIES; TARGET ENRICHMENT; GENETIC-MARKERS; ORTHOLOG GROUPS; TRANSCRIPTOME High-throughput sequencing has fundamentally changed how molecular phylogenetic data sets are assembled, and phylogenomic data sets commonly contain 50- to 100-fold more loci than those generated using traditional Sanger sequencing-based approaches. Here, we demonstrate a new approach for building phylogenomic data sets using single-tube, highly multiplexed amplicon sequencing, which we name HiMAP (highly multiplexed amplicon-based phylogenomics) and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and postsequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously and requires minimal hands-on time at the bench (<1/2day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically important agricultural insect pests. The resulting filtered data set (>150,000-bp concatenated alignment, 20% missing character sites across all individuals and amplicons) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting data set provides into relationships of this diverse insect group. 127 1 1 10 13 SEP 2018 18 5 1000 1019 10.1111/1755-0998.12783 20 WOS:000441753000008 29633537 Diptera Illumina MiSeq PCR Multiplex J Liu, SL; Li, YY; Lu, JL; Su, X; Tang, M; Zhang, R; Zhou, LL; Zhou, CR; Yang, Q; Ji, YQ; Yu, DW; Zhou, X SOAPBarcode: revealing arthropod biodiversity through assembly of Illumina shotgun sequences of PCR amplicons METHODS IN ECOLOGY AND EVOLUTION English Article high-throughput sequencing; metabarcoding; next-generation-sequencing; operational taxonomic units; phylogenetic diversity; species richness; standard barcode ENVIRONMENTAL DNA; GENERATION; ALIGNMENT; BARCODE Metabarcoding of mixed arthropod samples for biodiversity assessment has mostly been carried out on the 454 GS FLX sequencer (Roche, Branford, Connecticut, USA), due to its ability to produce long reads (400bp) that are believed to allow higher taxonomic resolution. The Illumina sequencing platforms, with their much higher throughputs, could potentially reduce sequencing costs and improve sequence quality, but the associated shorter read length (typically <150bp) has deterred their usage in next-generation-sequencing (NGS)-based analyses of eukaryotic biodiversity, which often utilize standard barcode markers (e.g. COI, rbcL, matK, ITS) that are hundreds of nucleotides long. We present a new Illumina-based pipeline to recover full-length COI barcodes from mixed arthropod samples. Our new assembly program, SOAPBarcode, a variant of the genome assembly program SOAPdenovo, uses paired-end reads of the standard COI barcode region as anchors to extract the correct pathways (sequences) out of otherwise chaotic de Bruijn graphs', which are caused by the presence of large numbers of COI homologs of high sequence similarity. Two bulk insect samples of known species composition have been analysed in a recently published 454 metabarcoding study (Yu etal. 2012) and are re-analysed by our analysis pipeline. Compared to the results of Roche 454 (c.400-bp reads), our pipeline recovered full-length COI barcodes (658bp) and 17-31% more species-level operational taxonomic units (OTUs) from bulk insect samples, with fewer untraceable (novel) OTUs. On the other hand, our PCR-based pipeline also revealed higher rates of contamination across samples, due to the Illumina's increased sequencing depth. On balance, the assembled full-length barcodes and increased OTU recovery rates resulted in more resolved taxonomic assignments and more accurate beta diversity estimation. The HiSeq 2000 and the SOAPBarcode pipeline together can achieve more accurate biodiversity assessment at a much reduced sequencing cost in metabarcoding analyses. However, greater precaution is needed to prevent cross-sample contamination during field preparation and laboratory operation because of greater ability to detect non-target DNA amplicons present in low-copy numbers. 28 23 25 2 77 DEC 2013 4 12 1142 1150 10.1111/2041-210X.12120 9 WOS:000327997900005 Trap malaise Illumina HiSeq PCR Barcoding J Bittleston, LS; Baker, CCM; Strominger, LB; Pringle, A; Pierce, NE Metabarcoding as a tool for investigating arthropod diversity in Nepenthes pitcher plants AUSTRAL ECOLOGY English Article 18S amplicon sequencing; carnivorous plant; insect; microcosm; network BIODIVERSITY ASSESSMENT; SARRACENIA-PURPUREA; HIGH-THROUGHPUT; COPY NUMBER; COMMUNITY; DNA; AMPLIFICATION; EVOLUTION; ALIGNMENT; RESOURCE The biodiversity of tropical forests consists primarily of small organisms that are difficult to detect and characterize. Next-generation sequencing (NGS) methods can facilitate analyses of these arthropod and microbial communities, leading to a better understanding of existing diversity and factors influencing community assembly. The pitchers of carnivorous pitcher plants often house surprisingly discrete communities and provide ideal systems for analysis using an NGS approach. The plants digest insects in order to access essential nutrients while growing in poor soils; however, the pitchers are also home to communities of living organisms, called inquilines. Certain arthropods appear to have coevolved with their pitcher plant hosts and are not found in other environments. We used Illumina amplicon sequencing of 18S rDNA to characterize the eukaryotes in three species of Nepenthes (Nepenthaceae) pitcher plants - N.gracilis, N.rafflesiana and N.ampullaria - in each of three different parks in Singapore. The data reveal an unexpected diversity of eukaryotes, significant differences in community diversity among host species, variation in host specificity of inquilines and the presence of gregarine parasites. Counts of whole inquiline arthropods from the first collection year were roughly correlated with scaled 18S sequence abundances, indicating that amplicon sequencing is an effective means of gauging community structure. We barcoded a subset of the dipteran larvae using COI primers, and the resulting phylogenetic tree is mostly congruent with that found using the 18S locus, with the exception of one of five morphospecies. For many 18S and COI sequences, the best BLASTn matches showed low sequence identity, illustrating the need for better databases of Southeast Asian dipterans. Finally, networks of core arthropods and their host species were used to investigate degree of host specificity across multiple hosts, and this revealed significant specialization of certain arthropod fauna. 50 4 4 5 56 APR 2016 41 2 120 132 10.1111/aec.12271 13 WOS:000374301600002 Nepenthes trap Illumina MiSeq PCR Barcoding J Swift, JF; Lance, RF; Guan, X; Britzke, ER; Lindsay, DL; Edwards, CE Multifaceted DNA metabarcoding: Validation of a noninvasive, next-generation approach to studying bat populations EVOLUTIONARY APPLICATIONS English Article bats; DNA metabarcoding; next-generation DNA sequencing; noninvasive sampling; population assessment WHITE-NOSE SYNDROME; ELUSIVE ANIMALS; DIET; IDENTIFICATION; PRIMERS; PHYLOGEOGRAPHY; MICROBIOTA; TAXONOMY; ACCURACY; WILDLIFE As multiple species of bats are currently experiencing dramatic declines in populations due to white-nose syndrome (WNS) and other factors, conservation managers have an urgent need for data on the ecology and overall status of populations of once-common bat species. Standard approaches to obtain data on bat populations often involve capture and handling, requiring extensive expertise and unavoidably resulting in stress to the bats. New methods to rapidly obtain critical data are needed that minimize both the stress on bats and the spread of WNS. Guano provides a noninvasive source of DNA that includes information from the bat, but also dietary items, parasites, and pathogens. DNA metabarcoding is a high-throughput, DNA-based identification technique to assess the biodiversity of environmental or fecal samples. We investigated the use of multifaceted DNA metabarcoding (MDM), a technique combining next-generation DNA sequencing (NGS), DNA barcodes, and bioinformatic analysis, to simultaneously collect data on multiple parameters of interest (bat species composition, individual genotype, sex ratios, diet, parasites, and presence of WNS) from fecal samples using a single NGS run. We tested the accuracy of each MDM assay using samples in which these parameters were previously determined using conventional approaches. We found that assays for bat species identification, insect diet, parasite diversity, and genotype were both sensitive and accurate, the assay to detect WNS was highly sensitive but requires careful sample processing steps to ensure the reliability of results, while assays for nectivorous diet and sex showed lower sensitivity. MDM was able to quantify multiple data classes from fecal samples simultaneously, and results were consistent whether we included assays for a single data class or multiple data classes. Overall, MDM is a useful approach that employs noninvasive sampling and a customizable suite of assays to gain important and largely accurate information on bat ecology and population dynamics. 72 1 1 29 29 AUG 2018 11 7 SI 1120 1138 10.1111/eva.12644 19 WOS:000439505200007 30026801 Feaces bat Illumina MiSeq PCR Barcoding J Nakadai, R; Kawakita, A Patterns of temporal and enemy niche use by a community of leaf cone moths (Caloptilia) coexisting on maples (Acer) as revealed by metabarcoding MOLECULAR ECOLOGY English Article closely related species; herbivorous insect; metabarcoding; parasitoid; phenology; species coexistence PLANT-FEEDING INSECTS; SYMPATRIC HOST RACES; REPRODUCTIVE ISOLATION; HERBIVORE ASSEMBLAGES; AREA RELATIONSHIPS; SPECIES-AREA; GUT CONTENTS; DIVERSITY; DNA; DIVERSIFICATION The diversity of herbivorous insects is often considered a function of host plant diversity. However, recent research has uncovered many examples of closely related herbivores using the same host plant(s), suggesting that partitioning of host plants is not the only mechanism generating diversity. Herbivores sharing hosts may utilize different parts of the same plant, but such resource partitioning is often not apparent; hence, the factors that allow closely related herbivores to coexist are still largely undetermined. We examined whether partitioning of phenology or natural enemies may explain the coexistence of leaf cone moths (Caloptilia; Gracillariidae) associated with maples (Acer; Sapindaceae). Larval activity of 10 sympatric Caloptilia species found on nine maple species was monitored every 2-3 weeks for a total of 13 sampling events, and an exhaustive search for internal parasitoid wasps was conducted using high-throughput sequencing. Blocking primers were used to facilitate the detection of wasp larvae inside moth tissue. We found considerable phenological overlap among Caloptilia species, with two clear peaks in July and September-October. Coexisting Caloptilia species also had largely overlapping parasitoid communities; a total of 13 chalcid and ichneumon wasp species attacked Caloptilia in a nonspecific fashion at an overall parasitism rate of 46.4%. Although coexistence may be facilitated by factors not accounted for in this study, it appears that niche partitioning is not necessary for closely related herbivores to stably coexist on shared hosts. Co-occurrence without resource partitioning may provide an additional axis along which herbivorous insects attain increased species richness. 61 2 2 4 18 JUN 2017 26 12 3309 3319 10.1111/mec.14105 11 WOS:000402836700018 28316099 Lepidoptera Illumina MiSeq PCR Barcoding J Kocher, A; de Thoisy, B; Catzeflis, F; Valiere, S; Banuls, AL; Murienne, J iDNA screening: Disease vectors as vertebrate samplers MOLECULAR ECOLOGY English Article bloodmeal; dipteran; feeding preference; insect; mosquito; sand fly PANAMANIAN PHLEBOTOMINE SANDFLIES; NATURAL HOST PREFERENCES; HIGH-THROUGHPUT; BLOOD MEALS; SOUTH-AMERICA; FRENCH-GUIANA; RAIN-FOREST; SAND FLIES; IDENTIFICATION; BIODIVERSITY In the current context of global change and human-induced biodiversity decline, there is an urgent need for developing sampling approaches able to accurately describe the state of biodiversity. Traditional surveys of vertebrate fauna involve time-consuming and skill-demanding field methods. Recently, the use of DNA derived from invertebrate parasites (leeches and blowflies) was suggested as a new tool for vertebrate diversity assessment. Bloodmeal analyses of arthropod disease vectors have long been performed to describe their feeding behaviour, for epidemiological purposes. On the other hand, this existing expertise has not yet been applied to investigate vertebrate fauna per se. Here, we evaluate the usefulness of hematophagous dipterans as vertebrate samplers. Blood-fed sand flies and mosquitoes were collected in Amazonian forest sites and analysed using high-throughput sequencing of short mitochondrial markers. Bloodmeal identifications highlighted contrasting ecological features and feeding behaviour among dipteran species, which allowed unveiling arboreal and terrestrial mammals of various body size, as well as birds, lizards and amphibians. Additionally, lower vertebrate diversity was found in sites undergoing higher levels of human-induced perturbation. These results suggest that, in addition to providing precious information on disease vector host use, dipteran bloodmeal analyses may represent a useful tool in the study of vertebrate communities. Although further effort is required to validate the approach and consider its application to large-scale studies, this first work opens up promising perspectives for biodiversity monitoring and eco-epidemiology. 66 3 3 6 23 NOV 2017 26 22 6478 6486 10.1111/mec.14362 9 WOS:000417241800020 28926155 Blood-feeding Diptera Illumina MiSeq PCR Barcoding J Yeo, D; Puniamoorthy, J; Ngiam, RWJ; Meier, R Towards holomorphology in entomology: rapid and cost-effective adult-larva matching using NGS barcodes SYSTEMATIC ENTOMOLOGY English Article SPECIES DELIMITATION; MOLECULAR-IDENTIFICATION; ANISOPTERA AESHNIDAE; ODONATA AESHNIDAE; NATURAL-HISTORY; DNA-SEQUENCES; INSTAR LARVA; TAXONOMY; DIPTERA; ASSOCIATION In many taxa the morphology of females and immatures is poorly known because species descriptions and identification tools have a male bias. The root causes are problems with matching life-history stages and genders belonging to the same species. Such matching is time-consuming when conventional methods are used (e.g. rearing) and expensive when the stages are matched with DNA barcodes. Unfortunately, the lack of associations is not a trivial problem because it renders a large part of the phenome of insects unexplored, although larvae and females are useful sources of characters for descriptive and phylogenetic purposes. In addition, many collectors intentionally avoid females and immature stages, which skews survey results, interferes with collecting life-history information, and makes it less likely that rare species are discovered. These problems even exist for well-studied taxa like Odonata, where obtaining adult-larva matches relies largely on rearing. Here we demonstrate how the matching problem can be addressed with cost-effective tagged amplicon sequencing of a 313-bp segment of cox1 with next-generation sequencing (NGS) ('NGS barcoding'). We illustrate the value of this approach based on Singapore's odonate fauna which is of a similar size as the European fauna (Singapore, 122 extant species; Europe, 138 recorded species). We match the larvae and adults of 59 species by first creating a barcode database for 338 identified adult specimens representing 83 species. We then sequence 1178 larvae from a wide range of sources. We successfully barcode 1123 specimens, which leads to adult-larva matches for 59 species based on our own barcodes (55) and online barcode databases (4). With these additions, 84 of the 131 species recorded in Singapore have now been associated with a species name. Most common species are now matched (83%), and good progress has been made for vulnerable/near-threatened (55%), endangered (53%), and critically endangered species (38%). We used nondestructive DNA extraction methods in order to be able to use high-resolution imaging of matched larvae to establish a publicly available digital reference collection for odonates which is incorporated into 'Biodiversity of Singapore' (https://singapore.biodiversity.online/). We suggest that the methods described here are suitable for many insect taxa because NGS barcoding allows for fast and low-cost matching of well-studied life-history stages with neglected semaphoronts (eggs, larvae, females). We estimate that the specimen-specific amplicons in this study (c. 1500 specimens) can now be obtained within eight working days and that the laboratory and sequencing cost is c. US$600 (