DNA metabarcoding allows the analysis of insect communities faster and more efficiently than ever before. However, metabarcoding can be conducted through several approaches, and the consistency of results across methods has rarely been studied. We compare the results obtained by DNA metabarcoding of the same communities using two different markers – COI and 16S – and three different sampling methods: (a) homogenized Malaise trap samples (homogenate), (b) preservative ethanol from the same samples, and (c) soil samples. Our results indicate that COI and 16S offer partly complementary information on Malaise trap samples, with each marker detecting a significant number of species not detected by the other. Different sampling methods offer highly divergent estimates of community composition. The community recovered from preservative ethanol of Malaise trap samples is significantly different from that recovered from homogenate. Small and weakly sclerotized insects tend to be overrepresented in ethanol while strong and large taxa are overrepresented in homogenate. For soil samples, highly degenerate COI primers pick up large amounts of nontarget DNA and only 16S provides adequate analyses of insect diversity. However, even with 16S, very little overlap in molecular operational taxonomic unit (MOTU) content was found between the trap and the soil samples. Our results demonstrate that none of the tested sampling approaches is satisfactory on its own. For instance, DNA extraction from preservative ethanol is not a valid replacement for destructive bulk extraction but a complement. In future metabarcoding studies, both should ideally be used together to achieve comprehensive representation of the target community.

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the "barcoding region" of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein-coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon-based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.

Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95–100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, or heads) that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90% made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.

1!Insect metabarcoding has been mainly based on PCR amplification of short fragments 2! within the 'barcoding region' of the gene COI. However, because of the variability of 3! this gene, it has been difficult to design good universal PCR primers. Most primers 4! used today are associated with gaps in the taxonomic coverage or amplification biases 5! that make the results less reliable and impede the detection of species that are present 6! in the sample. We identify new primers for insect metabarcoding using computational 7! approaches (ECOPRIMERS and DEGEPRIME) applied to the most comprehensive 8! reference databases of mitochondrial genomes of Hexapoda assembled to date. New 9! primers are evaluated in silico against previously published primers in terms of 10! taxonomic coverage and resolution of the corresponding amplicons. For the latter 11! criterion, we propose a new index, exclusive taxonomic resolution, which is a more 12! biologically meaningful measure than the standard index used today. Our results show 13! that the best markers are found in the ribosomal RNA genes (12S and 16S); they 14! resolve about 90% of the genetically distinct species in the reference database. Some 15! markers in protein-coding genes provide similar performance but only at much higher 16! levels of primer degeneracy. Combining two of the best individual markers improves 17! the effective taxonomic resolution with up to 10%. The resolution is strongly 18! dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S 19! primers detect 12% of Collembola. Our results indicate that amplicon-based 20! metabarcoding of insect samples can be improved by choosing other primers than 21! those commonly used today.

1.Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well. Highly concentrated ethanol (95-100 %), preferred as a DNA preservative for molecular biology, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies of the correlation between ethanol concentration and specimen preservation are lacking.2. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After the treatments, we counted the number of appendages (legs, wings, antennae or heads) that each specimen had lost. 3.We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90 % made the insects more brittle and resulted in more shriveling, but insects with more robust or sclerotized exoskeletons actually retained more of their appendages at high concentrations. Surprisingly, neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the loss of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. 4.While higher ethanol concentrations might positively affect long-term DNA preservation, there is a clear trade-off between collecting and preserving insects for morphological examination and genetic analysis, since the optimal ethanol concentration for the latter is detrimental for the former and vice versa. The trade-off needs to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of the samples.

A phylogenetic analysis of Polycladida based on two partial mitochondrial genes (cox1 and 16S) is provided. The analysis includes 30 polyclad terminals that represent species from the two taxa which traditionally divide the groups Cotylea and Acotylea. Our phylogenetic analyses produced a well-supported hypothesis that confirms the monophyly of Polycladida, as well as Acotylea and Cotylea. Within Acotylea, there are two lineages not highly supported: on one hand, Leptoplanoidea (excluding Hoploplana elisabelloi) and one Stylochoidea member (Pseudostylochus intermedius) (classification sensu Faubel, 1983Faubel, , 1984, and on the other hand, Stylochoidea members together with Discocelis tigrina and H. elisabelloi. The genera Stylochus and Imogine are not monophyletic. Within Cotylea, Pseudocerotidae and Euryleptidae are monophyletic, though not highly supported, while Prosthiostomidae is not. Euryleptoidea is paraphyletic. The genera Pseudobiceros and Pseudoceros are monophyletic and highly supported. Our results suggest that, within Acotylea, the prostatoid organs of Discocelis may have been derived from a prostatic vesicle. The genus Hoploplana could be included in Stylochoidea. Within Cotylea, the common ancestor of Euryleptidae and Pseudocerotidae might have been an aposematic animal with tentacles.

DNA metabarcoding can accelerate research on insect diversity, as it is cheap and fast compared to manual sorting and identification. Most metabarcoding protocols require homogenisation of the sample, preventing further work on the specimens. Mild digestion of the tissue by incubation in a lysis buffer has been proposed as an alternative, and, although some mild lysis protocols have already been presented, they have so far not been evaluated against each other. Here, we analyse how two mild lysis buffers (one more aggressive, one gentler in terms of tissue degradation), two different incubation times, and two DNA purification methods (a manual precipitation and an automated protocol) affect the accuracy of retrieving the true composition of mock communities using two mitochondrial markers (COI and 16S). We found that protocol-specific variation in concentration and purity of the DNA extracts produced had little effect on the recovery of species. However, the two lysis treatments differed in quantification of species abundances. Digestion in the gentler buffer and for a shorter time yielded better representation of original sample composition. Digestion in a more aggressive buffer or longer incubation time yielded lower alpha diversity values and increased differences between metabarcoding results and the true species-abundance distribution. We conclude that the details of non-destructive protocols can have a significant effect on metabarcoding performance. A short and mild lysis treatment appears the best choice for recovering the true composition of the sample. This not only improves accuracy, but also comes with a faster processing time than the other treatments.

Massively parallel DNA sequencing opens up opportunities for bridging multiple temporal and spatial dimensions in biodiversity research, thanks to its efficiency to recover millions of nucleotide polymorphisms. Here, we identify the current status, discuss the main challenges, and look into future perspectives on biodiversity genomics focusing on insects, which arguably constitute the most diverse and ecologically important group among all animals. We suggest 10 simple rules that provide a succinct step-by-step guide and best-practices to anyone interested in biodiversity research through the study of insect genomics. To this end, we review relevant literature on biodiversity and evolutionary research in the field of entomology. Our compilation is targeted at researchers and students who may not yet be specialists in entomology or molecular biology. We foresee that the genomic revolution and its application to the study of non-model insect lineages will represent a major leap to our understanding of insect diversity.