The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular ...biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5' fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage ≥10; CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples; whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming presorting process will yield approximately 30% more high score BINs compared to the non-sorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline.
Situated in the Eastern section of the European Alps, Austria encompasses a great diversity of different habitat types, ranging from alpine to lowland Pannonian ecosystems, and a correspondingly high ...level of species diversity, some of which has been addressed in various DNA barcoding projects. Here, we report a DNA barcode library of all the 476 species of Geometridae (Lepidoptera) that have been recorded in Austria. As far as possible, species were sampled from different Austrian regions in order to capture intraspecific genetic variation. In total, 2500 DNA barcode sequences, representing 438 species, were generated in this study. For complete coverage of Austrian geometrid species in the subsequent analyses, the dataset was supplemented with DNA barcodes from specimens of non-Austrian origin. Species delimitations by ASAP, BIN and bPTP methods yielded 465, 510 and 948 molecular operational taxonomic units, respectively. Congruency of BIN and ASAP partitions with morphospecies assignments was reasonably high (85% of morphospecies in unique partitions), whereas bPTP appeared to overestimate the number of taxonomic units. The study furthermore identified taxonomically relevant cases of morphospecies splitting and sharing in the molecular partitions. We conclude that DNA barcoding and sequence analysis revealed a high potential for accurate DNA-based identification of the Austrian Geometridae species. Additionally, the study provides an updated checklist of the geometrid moths of Austria.
The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides ...a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families.
This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species.
The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity.
We investigated the distribution and species circumscription of Nychiodes obscurariaVillers (1789) and Nychiodes ragusaria Millière (1884). The morphology of male and female genitalia of Calabrian ...populations (southern Italy) revealed them to belong to N. ragusaria while specimens from Basilicata, Molise and the rest of Italy turned out to be N. obscuraria. Although the DNA barcodes showed a divergence of 1.55% between typical N. obscuraria and N. ragusaria, 9 out of 28 Calabrian specimens of N. ragusaria shared the N. obscuraria haplotype, and sometimes both haplotypes were found at the same site. Genetic introgression appears as the most probable scenario to explain the observed pattern. A correct identification for the investigated species pair should be based on morphology instead of DNA barcode analysis because of the observed mitonuclear discordance at mountain sites. Further studies are needed to clarify the occurrence of this discordance at high elevations only.
This publication provides the first comprehensive DNA barcode data set for the Neuropterida of Central Europe, including 80 of the 102 species (78%) recorded from Bavaria (Germany) and three other ...species from nearby regions (Austria, France and the UK). Although the 286 specimens analyzed had a heterogeneous conservation history (60% dried; 30% in 80% EtOH; 10% fresh specimens in 95% EtOH), 237 (83%) generated a DNA barcode. Eleven species (13%) shared a BIN, but three of these taxa could be discriminated through barcodes. Four pairs of closely allied species shared barcodes including Chrysoperla pallida Henry et al., 2002 and C. lucasina Lacroix, 1912; Wesmaelius concinnus (Stephens, 1836) and W. quadrifasciatus (Reuter, 1894); Hemerobius handschini Tjeder, 1957 and H. nitidulus Fabricius, 1777; and H. atrifrons McLachlan, 1868 and H. contumax Tjeder, 1932. Further studies are needed to test the possible synonymy of these species pairs or to determine if other genetic markers permit their discrimination. Our data highlight five cases of potential cryptic diversity within Bavarian Neuropterida: Nineta flava (Scopoli, 1763), Sympherobius pygmaeus (Rambur, 1842), Sisyra nigra (Retzius, 1783), Semidalis aleyrodiformis (Stephens, 1836) and Coniopteryx pygmaea Enderlein, 1906 are each split into two or three BINs. The present DNA barcode library not only allows the identification of adult and larval stages, but also provides valuable information for alpha-taxonomy, and for ecological and evolutionary research.
The present study aimed to perform molecular identification of lepidopteran larvae from canopy fogging including gut-content analyses. A total of 130 lepidopteran larvae were selected from 37 fogging ...samples at the Panguana station, district Yuyapichis, province Puerto Inca, department Huánuco, Peru. Target trees were pre-identified and subsequently submitted to molecular confirmation of identity with three markers (rbcL, psbA and trnL-F). The COI gene of 119 lepidopteran larvae was successfully sequenced and found to belong to 92 species: Comparison of DNA barcodes with the reference database of adult moths resulted in 65 (55%) matches at species level, 32 (27%) at genus level, 19 (16%) at subfamily or family level, three just to order level. Three larvae could not be assigned to a family. For these larvae the fogged target tree now suggests a potential host-plant relationship. Molecular gut content analysis, based on High-Throughput-Sequencing was successfully tested for ten larvae corroborating feeding on the target plant in some cases but elucidating several other cases of potential 'alternative feeding'. We propose a larger-scale approach using this rapid and efficient method including molecular gut-content analyses for comprehensively testing the ratio of 'alternative feeders' and pitfalls caused by collateral fogging of larvae from neighboring trees.
Background: The geometrid moths of Europe are one of the best investigated insect groups in traditional taxonomy making them an ideal model group to test the accuracy of the Barcode Index Number ...(BIN) system of BOLD (Barcode of Life Datasystems), a method that supports automated, rapid species delineation and identification. Methodology/Principal Findings: This study provides a DNA barcode library for 219 of the 249 European geometrid moth species (88%) in five selected subfamilies. The data set includes COI sequences for 2130 specimens. Most species (93%) were found to possess diagnostic barcode sequences at the European level while only three species pairs (3%) were genetically indistinguishable in areas of sympatry. As a consequence, 97% of the European species we examined were unequivocally discriminated by barcodes within their natural areas of distribution. We found a 1:1 correspondence between BINs and traditionally recognized species for 67% of these species. Another 17% of the species (15 pairs, three triads) shared BINs, while specimens from the remaining species (18%) were divided among two or more BINs. Five of these species are mixtures, both sharing and splitting BINs. For 82% of the species with two or more BINs, the genetic splits involved allopatric populations, many of which have previously been hypothesized to represent distinct species or subspecies. Conclusions/Significance: This study confirms the effectiveness of DNA barcoding as a tool for species identification and illustrates the potential of the BIN system to characterize formal genetic units independently of an existing classification. This suggests the system can be used to efficiently assess the biodiversity of large, poorly known assemblages of organisms. For the moths examined in this study, cases of discordance between traditionally recognized species and BINs arose from several causes including overlooked species, synonymy, and cases where DNA barcodes revealed regional variation of uncertain taxonomic significance.
Habitat destruction and deterioration of habitat quality caused a severe decline of biodiversity, such as insect diversity. In this study, we analyze insect diversity and biomass across ...agro-environments. We collected flying insects with 20 malaise traps across a landscape mosaic consisting of organic (eight traps) and conventional (four traps) farmland, as well as across agricultural land that has been recently converted from conventional to organic farming (eight traps). Sampling was conducted over 2 years, in 2019 and 2020, with in total 340 sampling events. We measured the dry weight of the captured organisms and identified species diversity by analyzing Operational Taxonomic Units (OTUs) and Barcode Index Numbers (BINs) via metabarcoding. The results obtained show temporal dynamics. The number of OTUs were always higher than the number of BINs. OTUs and BINs were moderately to highly correlated, while the number of OTUs and BINs were only moderately positively correlated with dry biomass. OTUs and BINs as well as biomass were highest in the recently transformed farmland if compared with pure organic and conventional farmland sites, which showed no significant differences in respect of insect diversity. OTU and BIN numbers but not the OTU/BIN ratio significantly decreased with increasing distance from the nearest forest fringe. The numbers of OTUs, BINs and the OTU/BIN proportion, as well as OTU and BIN/biomass proportions varied strongly over seasons, irrespective of agricultural practice. Based on our findings, we suggest to combine data on insect species richness and biomass measured over a period of time, to derive a largely complete and meaningful assessment of biodiversity for a specific region.
The moth family Geometridae (inchworms or loopers), with approximately 23,000 described species, is the second most diverse family of the Lepidoptera. Apart from a few recent attempts based on ...morphology and molecular studies, the phylogeny of these moths has remained largely uninvestigated.
We performed a rigorous and extensive molecular analysis of eight genes to examine the geometrid affinities in a global context, including a search for its potential sister-taxa. Our maximum likelihood analyses included 164 taxa distributed worldwide, of which 150 belong to the Geometridae. The selected taxa represent all previously recognized subfamilies and nearly 90% of recognized tribes, and originate from all over world. We found the Geometridae to be monophyletic with the Sematuridae+Epicopeiidae clade potentially being its sister-taxon. We found all previously recognized subfamilies to be monophyletic, with a few taxa misplaced, except the Oenochrominae+Desmobathrinae complex that is a polyphyletic assemblage of taxa and the Orthostixinae, which was positioned within the Ennominae. The Sterrhinae and Larentiinae were found to be sister to the remaining taxa, followed by Archiearinae, the polyphyletic assemblage of Oenochrominae+Desmobathrinae moths, Geometrinae and Ennominae.
Our study provides the first comprehensive phylogeny of the Geometridae in a global context. Our results generally agree with the other, more restricted studies, suggesting that the general phylogenetic patterns of the Geometridae are now well-established. Generally the subfamilies, many tribes, and assemblages of tribes were well supported but their interrelationships were often weakly supported by our data. The Eumeleini were particularly difficult to place in the current system, and several tribes were found to be para- or polyphyletic.
The aim of this field trial was to evaluate the efficacy of attractive toxic sugar baits (ATSB) in Mali, where sustained malaria transmission occurs despite the use of long-lasting insecticidal nets ...(LLINs). ATSB bait stations were deployed in seven of 14 similar study villages, where LLINs were already in widespread use. The combined use of ATSB and LLINs was tested to see if it would substantially reduce parasite transmission by Anopheles gambiae sensu lato beyond use of LLINs alone.
A 2-day field experiment was conducted to determine the number of mosquitoes feeding on natural sugar versus those feeding on bait stations containing attractive sugar bait without toxin (ASB)-but with food dye. This was done each month in seven random villages from April to December 2016. In the following year, in seven treatment villages from May to December 2017, two ATSB bait stations containing the insecticide dinotefuran were placed on the outer walls of each building. Vector population density was evaluated monthly by CDC UV light traps, malaise traps, pyrethrum spray (PSCs) and human landing catches (HLCs). Female samples of the catch were tested for age by examination of the ovarioles in dissected ovaries and identification of Plasmodium falciparum sporozoite infection by ELISA. Entomological inoculation rates (EIR) were calculated, and reductions between treated and untreated villages were determined.
In the 2-day experiment with ASB each month, there was a lower number of male and female mosquitoes feeding on the natural sugar sources than on the ASB. ATSB deployment reduced CDC-UV trap female catches in September, when catches were highest, were by 57.4% compared to catches in control sites. Similarly, malaise trap catches showed a 44.3% reduction of females in August and PSC catches of females were reduced by 48.7% in September. Reductions of females in HLCs were lower by 19.8% indoors and 26.3% outdoors in September. The high reduction seen in the rainy season was similar for males and reductions in population density for both males and females were > 70% during the dry season. Reductions of females with ≥ 3 gonotrophic cycles were recorded every month amounting to 97.1% in October and 100.0% in December. Reductions in monthly EIRs ranged from 77.76 to 100.00% indoors and 84.95% to 100.00% outdoors. The number of sporozoite infected females from traps was reduced by 97.83% at treated villages compared to controls.
Attractive toxic sugar baits used against Anopheles mosquitoes in Mali drastically reduced the density of mosquitoes, the number of older females, the number of sporozoite infected females and the EIR demonstrating how ATSB significantly reduces malaria parasite transmission.
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