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.