BackgroundAdvancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field.FindingsWe demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding.ConclusionsOverall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Metaseiulus occidentalis is an eyeless phytoseiid predatory mite employed for the biological control of agricultural pests including spider mites. Despite appearances, these predator and prey mites are separated by some 400 Myr of evolution and radically different lifestyles. We present a 152-Mb draft assembly of the M. occidentalis genome: Larger than that of its favored prey, Tetranychus urticae, but considerably smaller than those of many other chelicerates, enabling an extremely contiguous and complete assembly to be built—the best arachnid to date. Aided by transcriptome data, genome annotation cataloged 18,338 protein-coding genes and identified large numbers of Helitron transposable elements. Comparisons with other arthropods revealed a particularly dynamic and turbulent genomic evolutionary history. Its genes exhibit elevated molecular evolution, with strikingly high numbers of intron gains and losses, in stark contrast to the deer tick Ixodes scapularis. Uniquely among examined arthropods, this predatory mite’s Hox genes are completely atomized, dispersed across the genome, and it encodes five copies of the normally single-copy RNA processing Dicer-2 gene. Examining gene families linked to characteristic biological traits of this tiny predator provides initial insights into processes of sex determination, development, immune defense, and how it detects, disables, and digests its prey. As the first reference genome for the Phytoseiidae, and for any species with the rare sex determination system of parahaploidy, the genome of the western orchard predatory mite improves genomic sampling of chelicerates and provides invaluable new resources for functional genomic analyses of this family of agriculturally important mites.

Background In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). Findings Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. Conclusions Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development.

BackgroundIn light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community, but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). ResultsUsing a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. ConclusionsSequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost effective, portable and universal approach for eukaryote DNA barcoding. Long rDNA amplicons scale up DNA barcoding by enabling the accurate recovery of taxonomic and phylogenetic diversity. However, bulk community analyses using long-read approaches may introduce biases and will require further exploration. MiniBarWe created a de-multiplexing software, called MiniBar. It allows customization of search parameters to account for the high read error rates and has built-in awareness of the dual barcode and primer pairs flanking the sequences. MiniBar takes as input a tab-delimited barcode file and a sequence file in either fasta or fastq format. The barcode file contains, at a minimum, sample name, forward barcode, forward primer, reverse barcode, and reverse primer for each of the samples potentially in the sequence file. The software searches for barcodes and for a primer, each permitting a user defined number of errors, an error being a mismatch or indel. Error count to determine a match can either be a percentage of each of their lengths or can be separately specified for barcode and primer as a maximum edit distance [49]. Output options permit saving each sample in its own file or all samples in a single file, with the sample names in the fasta or fastq headers. The found barcode primer pairs can be trimmed from the sequence or can remain in the sequence distinguished by case or color. MiniBar, written in Python 2.7, can also run in Python 3 and has the single dependency of the Edlib library module for edit distance measured approximate search [50]. MiniBar can be found at...

21In water, transparency seems an ideal concealment strategy, as testified by the variety of transparent 22 aquatic organisms. By contrast, transparency is nearly absent on land, with the exception of insect 23 wings, and knowledge is scarce about its functions and evolution, with fragmentary studies and no 24 comparative perspective. Lepidoptera (butterflies and moths) represent an outstanding group to 25 investigate transparency on land, as species typically harbour opaque wings covered with coloured 26 2 scales, a key multifunctional innovation. Yet, many Lepidoptera species have evolved partially or 27 fully transparent wings. At the interface between physics and biology, the present study investigates 28 transparency in 123 Lepidopteran species (from 31 families) for its structural basis, optical 29 properties and biological relevance in relation to thermoregulation and vision. Our results establish 30 that transparency has likely evolved multiple times independently. Efficiency at transmitting light 31 is largely determined by clearwing microstructure (scale shape, insertion, colouration, dimensions 32 and density) and macrostructure (clearwing area, species size or wing area). Microstructural traits -33 density, dimensions -are tightly linked in their evolution, with different constraints according to 34 scale shape, insertion, and colouration. Transparency appears highly relevant for vision, especially 35 for camouflage, with size-dependent and activity-rhythm dependent variations. Links between 36 transparency and latitude are consistent with an ecological relevance of transparency in 37 thermoregulation, and not so for protection against UV radiation. Altogether, our results shed new 38 light on the physical and ecological processes driving the evolution of transparency on land and 39 underline that transparency is a more complex than previously thought colouration strategy. 40 41

As biodiversity loss continues to accelerate, there is a critical need for education and biomonitoring across the globe. Portable technologies allow for in situ molecular biodiversity monitoring that has been historically out of reach for many researchers in habitat nations. In the realm of education, portable tools such as DNA sequencers facilitate in situ hands-on training in real-time sequencing and interpretation techniques. Here, we provide step-by-step protocols as a blueprint for a terrestrial conservation genetics field training program that uses low-cost, portable devices to conduct genomics-based training directly in biodiverse habitat countries.

Characterization and expression analyses are essential to gain insight into sex-determination pathways in members of the Acari. Little is known about sex determination at the molecular level in the western orchard predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Arachnida: Acari: Phytoseiidae), a parahaploid species. In this study, eight genes previously identified as putative homologs to genes involved in the sex-determination pathway in Drosophila melanogaster were evaluated for sex-specific alternative splicing and sex-biased expression using reverse-transcriptase PCR and quantitative real-time PCR techniques, respectively. The homologs evaluated in M. occidentalis included two doublesex-like genes (Moccdsx1 and Moccdsx2), transformer-2 (Mocctra-2), intersex (Moccix), two fruitless-like genes (MoccBTB1 and MoccBTB2), as well as two vitellogenin-like genes (Moccvg1 and Moccvg2). Single transcripts of equal size were detected in males and females for Moccdsx1, Moccdsx2, Mocctra-2, Moccix, and MoccBTB2, suggesting that their pre-mRNAs do not undergo alternative splicing in a sex-specific manner. Three genes, Moccdsx1, Moccdsx2 and MoccBTB2, displayed male-biased expression relative to females. One gene, Moccix, displayed female-biased expression relative to males. Two genes, Mocctra-2 and MoccBTB1, did not display detectable differences in transcript abundance in males and females. Expression of Moccvg1 and Moccvg2 were detected in females only, and transcript levels were up-regulated in mated females relative to unmated females. To our knowledge, this represents the first attempt to elucidate expression patterns of putative sex-determination genes in an acarine. This study is an initial step towards understanding the sex-determination pathway in the parahaploid M. occidentalis.