Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

Whole mitochondrial genome capture from faecal samples and museum‐preserved specimens

Population‐scale molecular studies of endangered and cryptic species are often limited by access to high‐quality samples. The use of noninvasively collected samples or museum‐preserved specimens reduces the pressure on modern populations by removing the need to capture and handle live animals. However, endogenous DNA content in such samples is low, making shotgun sequencing a financially prohibitive approach. Here, we apply a target enrichment method to retrieve mitochondrial genomes from 65 museum specimens and 56 noninvasively collected faecal samples of two endangered great ape species, Grauer's gorilla and the eastern chimpanzee. We show that the applied method is suitable for a wide range of sample types that differ in endogenous DNA content, increasing the proportion of target reads to over 300‐fold. By systematically evaluating biases introduced during target enrichment of pooled museum samples, we show that capture is less efficient for fragments shorter or longer than the baits, that the proportion of human contaminating reads increases postcapture although capture efficiency is lower for human compared to gorilla fragments with a gorilla‐generated bait, and that the rate of jumping PCR is considerable, but can be controlled for with a double‐barcoding approach. We succeed in capturing complete mitochondrial genomes from faecal samples, but observe reduced capture efficiency as sequence divergence increases between the bait and target species. As previously shown for museum specimens, we demonstrate here that mitochondrial genome capture from field‐collected faecal samples is a robust and reliable approach for population‐wide studies of nonmodel organisms.     

Construction of a bacterial artificial chromosome library of Endoclita excrescens as a tool for comparative gene mapping in Lepidoptera

Karyotype evolution in one of the most diverse and species‐rich group of insects, moths and butterflies (Lepidoptera), has interesting features that remain to be resolved. Recent studies showed that fluorescence in situ hybridization using bacterial artificial chromosome clones (BAC‐FISH) is an efficient cytogenetic method for identification and gene mapping of lepidopteran chromosomes. Using comparative mapping by BAC‐FISH, extensive synteny of genes was revealed between chromosomes of different lepidopteran species based on Bombyx mori genomic information. However, this comparative mapping has been done only in representatives of advanced groups of Lepidoptera. Here we constructed a BAC library of Endoclita excrescens, which belongs to the primitive lepidopteran family Hepialidae. High molecular weight DNA for the library construction was prepared from the pupae by using a rapid nuclear isolation method known in plants. The BAC clones of E. excrescens contain 66.6 kb inserts on average. The successful application of BAC‐FISH showed that the BAC library of E. excrescens is a useful tool for comparative gene mapping on chromosomes of this species.     

New approaches to species delimitation and population structure of anthozoans: Two case studies of octocorals using ultraconserved elements and exons

As coral populations decline worldwide in the face of ongoing environmental change, documenting their distribution, diversity and conservation status is now more imperative than ever. Accurate delimitation and identification of species is a critical first step. This task, however, is not trivial as morphological variation and slowly evolving molecular markers confound species identification. New approaches to species delimitation in corals are needed to overcome these challenges. Here, we test whether target enrichment of ultraconserved elements (UCEs) and exons can be used for delimiting species boundaries and population structure within species of corals by focusing on two octocoral genera, Alcyonium and Sinularia, as exemplary case studies. We designed an updated bait set (29,181 baits) to target‐capture 3,023 UCE and exon loci, recovering a mean of 1,910 ± 168 SD per sample with a mean length of 1,055 ± 208 bp. Similar numbers of loci were recovered from Sinularia (1,946 ± 227 SD) and Alcyonium (1,863 ± 177 SD). Species‐level phylogenies were highly supported for both genera. Clustering methods based on filtered single nucleotide polymorphisms delimited species and populations that are congruent with previous allozyme, DNA barcoding, reproductive and ecological data for Alcyonium, and offered further evidence of hybridization among species. For Sinularia, results were congruent with those obtained from a previous study using restriction site associated DNA sequencing. Both case studies demonstrate the utility of target‐enrichment of UCEs and exons to address a wide range of evolutionary and taxonomic questions across deep to shallow timescales in corals.     

Hydrogel baits with low‐dose thiamethoxam for sustainable Argentine ant management in commercial orchards

Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae), are a significant pest in various agricultural systems around the world, and are often associated with outbreaks of phloem‐feeding hemipteran insects. Previous research has evaluated a number of active ingredients and management approaches for controlling Argentine ant populations in agricultural systems, but various regulatory and economic issues have limited the development of effective management tools. Current chemical controls rely on residual sprays or toxic baits, each one posing unique disadvantages that limit their usefulness and efficacy. This study evaluated the potential of water‐storing crystals to effectively deliver liquid baits to Argentine ants. The efficacy of bait crystals containing 0.007% thiamethoxam was first evaluated in laboratory colonies. In addition, field studies were performed in a commercial plum orchard to determine the efficacy of the bait crystals. Protein marking was used within the orchard to examine the distribution of the bait in Argentine ant populations when delivered via water‐storing crystals. Results of laboratory tests showed that water‐storing crystals containing 0.007% thiamethoxam are highly attractive and effective against Argentine ants and require ca. 3–5 days to kill all castes and life stages. Results of the protein‐marking study demonstrated that the percentage of ants carrying protein‐labeled sugar water decreases sharply with increasing distance from the bait station. Bait movement was limited to within 17 m of the bait dispenser. Furthermore, bait efficacy tests in the field showed that Argentine ants can be effectively controlled using liquid thiamethoxam baits deployed via water‐storing crystals. The bait was highly effective and ant densities throughout the baited plots declined by 94 ± 2% within 14 days. The results of this study demonstrate that (1) thiamethoxam is highly effective for Argentine ant control in fruit orchards when used in low concentrations (0.007%), and (2) water‐storing crystals are an effective tool for delivering liquid baits to Argentine ants in agricultural settings.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

Interference between male‐targeted and female‐targeted lures of the Mediterranean fruit fly Ceratitis capitata (Dipt., Tephritidae) in Italy

The efficacy of male‐targeted and female‐targeted baits was compared when lures were presented together or singly in traps for capturing the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). For male‐targeted baits, either trimedlure or ceralure presented singly attracted large numbers of flies, supporting data from many previous reports. The present results are the first published data on the attractiveness of ceralure to a European population of C. capitata. The quaternary female bait consisting of ammonium carbonate, putrescine, trimethylamine and acetic acid was a potent attractant for female flies (and also showed some activity for males). Replacing acetic acid with ammonium acetate in the quaternary female bait did not influence activity. Traps with female‐targeted and male‐targeted baits together always showed a tendency of catching fewer flies than traps with only one type of bait. The decrease was significant in females, regardless of whether ceralure or trimedlure was the male‐targeted bait. In males, the tendency was the same for traps with trimedlure or ceralure alone, catching higher numbers than those with both male and female baits. Our present results suggest that both types of baits mutually decrease the numbers of the non‐target sex in the trap. In conclusion, it is advisable to use both male‐ and female‐targeted baits in separate and distant traps and not jointly in the same trap, lest the efficacy of detection or monitoring trials be compromised.     

Transcriptome‐based target‐enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia)

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.     

用于高通量测序的基因组靶序列捕获方法的建立

文章旨在建立一种基因组目标靶序列捕捉文库的方法,并结合第二代测序技术,以实现候选基因区段的深度测序。利用Agilent公司的eArray在线平台,对1250个基因的11824个外显子共2414977bp的基因组序列进行120个碱基长度的捕捉探针(钓饵)设计,并制备成SureSelect液相靶序列捕获试剂。选用2例人基因组DNA,超声打断后末端补平并磷酸化,连接SOLiD接头,回收150bp～200bp的DNA片段,与靶序列探针杂交捕获目标序列,油包水微乳滴PCR扩增后,磁珠分离富集,上SOLiD测序系统通过工作流程分析(WFA)进行文库质量的评价,或正式测序反应。结果显示对所包含的11147个基因外显子片段设计出并合成了46509个捕捉探针,制备成SureSelect试剂盒。探针可有效地捕捉并富集基因组DNA的目标靶片段,定量PCR显示富集效率可达29倍。WFA分析表明文库可以在SOLiD仪器进行正式测序。测序结果显示靶序列区域的测序数占有效总测序数的比例达到70%,覆盖率均在200×以上。结果表明本研究所建立的SureSelect基因组靶序列捕捉、富集建立测序文库的技术路线可行,可直接用于SOLiD测序仪的测序。     

Differential consumption of baits by pest blattid and blattellid cockroaches and resulting direct and secondary effects

Consumption was measured of three commercially available cockroach gel baits (0.01 and 0.05% fipronil and 0.6% indoxacarb) by two pest blattellid (German cockroach, Blattella germanica L., and brownbanded cockroach, Supella longipalpa Fabricius) and three pest blattid [oriental cockroach, Blatta orientalis L., American cockroach, Periplaneta americana L., and smokybrown cockroach, Periplaneta fuliginosa (Serville)] species (Dictyoptera), and direct and secondary effects were quantified. All three baits were greatly preferred for consumption over dog food; however, virtually all consumption (ca. 98%) by pest blattids was gel baits containing sugar feeding stimulants and water. Pest blattid greater preference for gel baits was probably due to their greater need for nutrients in baits due to their greater cuticular water permeability and higher metabolism than the pest blattellids. Brownbanded cockroaches had lowest percentage gel bait selection. Pest blattellids consumed greater amounts of bait per g body weight than pest blattids. Cockroaches consumed more active ingredient than needed to cause mortality; however, based on bait consumption, a 30‐g tube of gel bait potentially killed from 394 to 6 966 adult cockroaches, depending on species. Mortality for all cockroach species was faster for adults (≥3 days) than for nymphs (≥7 days); however, most brownbanded cockroaches exposed to indoxacarb survived despite consuming 1.5‐ to >3‐fold more than other baits, suggesting low enzyme production by brownbanded cockroaches and consequently lower conversion of indoxacarb into its toxic form. Besides direct mortality, German cockroaches died from indirect effects: exposure to debris from other cockroaches that had direct access to the gel baits or bait contact without ingestion. Although maximization of bait consumption is important, factors that enhance secondary mortality and contact toxicity should also be considered.     

Testing target‐specific bait delivery for controlling feral pigs in a tropical rainforest

Mitigation of feral pig (Sus scrofa) impacts in Australia’s Wet Tropics World Heritage Area has been impeded by the lack of a target‐specific method for delivering toxic baits in the region. This study evaluated methods to reduce bait‐take by susceptible nontarget species without inhibiting bait‐take by pigs, to enable more effective pig management. We predicted that dingoes would not select an unprocessed corn bait and that other potential nontarget bait consumers would be unable to access the same bait presented under a lightweight cover. Neither of these methods was expected to reduce bait selection or access by pigs. We tested these predictions by monitoring animal interactions with covered and uncovered corn baits, and covered corn and manufactured baits. Use of corn as a bait substrate effectively prevented bait‐take by dingoes. Covering baits substantially reduced bait‐take by other nontarget species and completely prevented nontarget bait‐take when uncovered feed was provided simultaneously. The corn bait preparation was highly acceptable and accessible to feral pigs. We conclude that the methods evaluated here could enable the consideration of poison baiting as a viable method for controlling feral pigs in the World Heritage Area, where it has previously been unavailable.     

Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades‐old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high‐throughput laboratory workflows. The strategy uses hemi‐nested, degenerate, M13‐tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard‐compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.     

Testing baits prepared from by‐product of the shrimp and snow crab industry in the pot fishery for Gadus morhua (Linnaeus, 1758) and Pollachius virens (Linnaeus, 1758)

In a study between October and November 2015 in northern Norway, bait prepared from the by‐product of the shrimp and snow crab industry was employed in the pot fishery to capture cod and saithe. Frozen natural herring was used as control bait. Pots with the three bait types were placed 1,000 m apart in order to eliminate the risk of interaction between the different baits. In total 11 sampling periods were conducted. There were no significant differences in catch rates between the different baits tested on cod and saithe. The manufactured bait tested in this study showed a continuous release of odours over a 14‐day period. Results show that the water content of the bait increases rapidly in the first phase of the immersion (until day 7), and the corresponding loss of bait mass (dry weight) suggest some physical disintegration (erosion) of the bait until the water content stabilizes. This study illustrates that it is possible to replace natural herring with hydrolysates from by‐products from shrimp or snow crab industry in the Norwegian pot fishery of cod and saithe.     

A chromosome‐scale genome assembly of Antheraea pernyi (Saturniidae,Lepidoptera)

Antheraea pernyi is a semi‐domesticated lepidopteran insect species valuable to the silk industry, human health, and ecological tourism. Owing to its economic influence and developmental properties, it serves as an ideal model for investigating divergence of the Bombycoidea super family. However, studies on the karyotype evolution and functional genomics of A. pernyi are limited by scarce genomic resource. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first high‐quality A. pernyi genome from a single male individual. The genome is 720.67 Mb long with 49 chromosomes and a 13.77‐Mb scaffold N50. Approximately 441.75 Mb, accounting for 60.74% of the genome, was identified as repeats. The genome comprises 21,431 protein‐coding genes, 85.22% of which were functionally annotated. Comparative genomics analysis suggested that A. pernyi diverged from its common ancestor with A. yamamai ~30.3 million years ago, and that chromosome fission contributed to the increased chromosome number. The genome assembled in this work will not only facilitate future research on A. pernyi and related species but also help to progress comparative genomics analyses in Lepidoptera.     

Simpler is better: fewer non‐target insects trapped with a four‐component chemical lure vs. a chemically more complex food‐type bait for Drosophila suzukii

Baits – fermented food products – are generally attractive to many types of insects, which makes it difficult to sort through non‐target insects to monitor a pest species of interest. We test the hypothesis that a chemically simpler and more defined attractant developed for a target insect is more specific and attracts fewer non‐target insects than a chemically more complex food‐type bait. A four‐component chemical lure isolated from a food bait and optimized for the spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), was compared to the original wine/vinegar bait to assess the relative responses of non‐target insects. In several field experiments in Washington State, USA, it was shown that numbers of pest muscid flies, cutworm and armyworm moths, and pest yellowjackets were reduced in traps baited with the chemical lure compared to the wine/vinegar bait. In other field experiments in the states of Washington, Oregon, and New York, numbers of non‐target drosophilid flies were also reduced in traps baited with the chemical lure relative to wine/vinegar bait. In Washington, numbers of Drosophila melanogaster Meigen and Drosophila obscura Fallen species groups and Drosophila immigrans Sturtevant were reduced in the chemical lure traps, whereas in New York, D. melanogaster and D. obscura species groups, D. immigrans, Drosophila putrida Sturtevant, Drosophila simulans Sturtevant, Drosophila tripunctata Loew, and Chymomyza spp. numbers were reduced. In Oregon, this same effect was observed with the D. melanogaster species group. Taken together, these results indicate that the four‐component SWD chemical lure will be more selective for SWD compared to fermentation baits, which should reduce time and cost involved in trapping in order to monitor SWD.     

Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses

Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.     

Universal target‐enrichment baits for anthozoan (Cnidaria) phylogenomics: New approaches to long‐standing problems

Anthozoans (e.g., corals, anemones) are an ecologically important and diverse group of marine metazoans that occur from shallow to deep waters worldwide. However, our understanding of the evolutionary relationships among the ~7,500 species within this class is hindered by the lack of phylogenetically informative markers that can be reliably sequenced across a diversity of taxa. We designed and tested 16,306 RNA baits to capture 720 ultraconserved element loci and 1,071 exon loci. Library preparation and target enrichment were performed on 33 taxa from all orders within the class Anthozoa. Following Illumina sequencing and Trinity assembly, we recovered 1,774 of 1,791 targeted loci. The mean number of loci recovered from each species was 638 ± 222, with more loci recovered from octocorals (783 ± 138 loci) than hexacorals (475 ± 187 loci). Parsimony informative sites ranged from 26 to 49% for alignments at differing hierarchical taxonomic levels (e.g., Anthozoa, Octocorallia, Hexacorallia). The per cent of variable sites within each of three genera (Acropora, Alcyonium, and Sinularia) for which multiple species were sequenced ranged from 4.7% to 30%. Maximum‐likelihood analyses recovered highly resolved trees with topologies matching those supported by other studies, including the monophyly of the order Scleractinia. Our results demonstrate the utility of this target‐enrichment approach to resolve phylogenetic relationships from relatively old to recent divergences. Redesigning the baits with improved affinities to capture loci within each subclass will provide a valuable toolset to address systematic questions, further our understanding of the timing of diversifications and help resolve long‐standing controversial relationships in the class Anthozoa.     

Target and non‐target effects of a spider venom toxin produced in transgenic cotton and tobacco plants

The peptide ω‐Hexatoxin‐Hv1a (Hvt) is one of the most studied spider toxins. Its insecticidal potential has been reported against species belonging to the arthropod orders Lepidoptera, Diptera and Orthoptera. The gene encoding Hvt has been transformed into cotton and tobacco to protect the plants from damage by lepidopteran pests. This study evaluated the expression of the ω‐HXTX‐Hv1a gene in transgenic plants, and the toxicity of plant‐expressed and purified Hvt on target lepidopteran insects and on several non‐target species. Transgenic Bollgard II cotton plants, which produce Cry1Ac and Cry2Ab2 and purified Cry2Ab2 protein were included in the study as comparators. LC₉₅ values of purified Hvt against Spodoptera littoralis and Heliothis virescens were 28.31 and 27.57 μg/ml of artificial diet, respectively. Larval mortality was 100% on Hvt‐transgenic tobacco plants but not on Hvt‐transgenic cotton, probably because of the significantly lower toxin expression level in the transgenic cotton line. Non‐target studies were conducted with larvae of the predators Chrysoperla carnea and Coccinella septempunctata, adults of the aphid parasitoid Aphidius colemani, and adult workers of the honey bee, Apis mellifera. Even at 40 μg/ml, Hvt did not adversely affect the four non‐target species. Purified Cry2Ab2 at 10 μg/ml also did not adversely affect any of the non‐target species. Our results show that Hvt might be useful for developing insecticidal plant varieties to control pest Lepidoptera.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.