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Our study aimed to develop an optimised laboratory protocol ensuring the preservation of morphological structures and extraction of high-quality DNA sequences from Psychodidae (Insecta, Diptera) specimens. With 310 analysed specimens, we investigated the impact of distinct laboratory treatments by employing two shaking categories (constant and interrupted) with five different incubation periods (16, 12, 8, 4 and 2 hours) during the DNA extraction process. Notably, 80.65% of the specimens exhibited morphological changes during DNA extraction. Our results indicated no statistical difference between constant and interrupted shaking for the total of morphological structures lost. However, within each shaking category, the loss of structures was influenced significantly by the incubation period. Prolonged incubation correlated with increased structural losses, whereas shorter incubation periods caused minor alterations in structures lost. In addition, our results showed a significant difference between constant and interrupted shaking treatments for DNA concentration. Likewise, the incubation period showed differences within each shaking category. Successful COI sequencing was achieved in 89.6% of specimens, with negligible differences in DNA fragment lengths across treatments. Our findings underscore the importance of an optimised protocol and its potential in systematic research involving nematoceran dipteran specimens by balancing morphological integrity and DNA extraction efficiency., Competing Interests: No conflict of interest to declare Disclaimer: This article is (co-)authored by any of the Editors-in-Chief, Managing Editors or their deputies in this journal., (Santiago Jaume-Schinkel, Björn Müller, Sergio Avila-Calero, Sandra Kukowka, Vera Rduch, Ximo Mengual.)

Copepods are important ecologically and represent a large amount of aquatic biomass in both freshwater and marine systems. Despite this, the taxonomy of copepods and other meiofauna is not well understood, hampered by tiny sizes, cryptic taxa, intraspecific polymorphisms and total specimen destruction where DNA methods are employed. In this article we highlight these issues and propose a more up-to-date approach for dealing with them. Namely, we recommend non-destructive DNA extraction methods, coupled with high-throughput sequencing (HTS). Whilst DNA yields may be low, they should still be sufficient for HTS library preparation and DNA sequencing. At the same time morphological specimens can be preserved and the crucial link between morphology and DNA sequence is maintained. This is critical for an integrative taxonomy and a fuller understanding of biodiversity patterns as well as evolutionary processes in meiofauna. [ABSTRACT FROM AUTHOR]

We examined the utility of a non-destructive DNA extraction method for identification of Bradysia odoriphaga Yang and Zhang, 1985 (Diptera: Sciaridae) based on PCR using species-specific primers and DNA barcoding targeting the mitochondrial cytochrome c oxidase subunit I gene. Using species-specific primers, PCR success rates for adults and pupae were more than 90%, while those for larvae and eggs were 51% and 13%, respectively. The success rates of PCR for DNA barcode region were slightly higher than those of PCR using species-specific primers. In all sequenced samples, the 658 bp of sequences in the DNA barcode region were identical to the sequences from B. odoriphaga that we previously obtained using a destructive DNA extraction method. The adults were successfully identified based on morphological characters after non-destructive DNA extraction. The non-destructive DNA extraction method examined in this study is useful for the identification of adults and pupae of B. odoriphaga collected in the fields. [ABSTRACT FROM AUTHOR]

Plant bio-protection and biosecurity programs worldwide use trap-based surveillance for the early detection of agricultural pests and pathogens to contain their incursions and spread. This task is reliant on effective preservation in insect traps, which is required to maintain specimen quality for extended periods under variable environmental conditions. Furthermore, with traditional morphological examinations now increasingly paired with modern molecular diagnostic techniques, insect traps are required to preserve both the specimens’ morphology and the DNA of insects and vectored bacterial pathogens. Here, we used psyllids (Hemiptera) and their hosted bacteria as a model to test the preservative ability of propylene glycol (PG): a non-flammable, easily transportable preservative agent that could be used in pitfall, suction or malaise traps. We tested preservation using various PG concentrations, at different temperatures and for multiple time periods, paired with non-destructive DNA extraction methods, which allow isolation of both insect and arbobacterial DNA while retaining a morphological voucher of the insect host specimens. PG concentrations between 40% and 100% performed best for both insect and bacterial DNA preservation up to 28 days. Ultimately, given the viscous nature of PG at high concentrations, we recommend using it at a concentration between 40% and 60% to enable insects to sink into the solution, thus enhancing DNA preservation.

Spider mites (Acari: Tetranychidae) include serious agricultural pests and some species have spread globally as invasive species.. For this reason, rapid and simple identification of spider mite species is necessary for agricultural field and plant quarantine inspection. DNA sequence-based molecular techniques can rapidly identify spider mites. However, extracting DNA from minute invertebrates is difficult, expensive and time consuming. Here, we describe a polymerase chain reaction (PCR) technique in which the whole body of a spider mite adult is non-destructively soaked in PCR solution. The mite is then removed intact and can be used as a voucher specimen, leaving the PCR solution as the template and avoiding the need for a DNA extraction kit. For this study, we used six common spider mite species from four genera [ Tetranychus urticae Koch (red form), Tetranychus kanzawai Kishida, Tetranychus parakanzawai Ehara, Oligonychus gotohi Ehara, Eotetranychus smithi Pritchard & Baker and Panonychus citri (McGregor)]. A portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with a universal primer pair from all individuals examined and sequenced. This method shortened the time for molecular identification from 9 to 5 h and eliminated the cost of commercial kits for DNA extraction [ca. 600 yen (~5.3 USD) per sample]. [ABSTRACT FROM AUTHOR]

Although it is important to identify species of pest and natural enemy insects in agriculture, morphological identification alone is often difficult. Because a combination of morphological identification and DNA barcoding is very important for identifying indiscriminable species, there is a need to develop non-destructive DNA extraction methods. Here, we compared three general methods of DNA extraction-the Chelex method, PrepMan Ultra Reagent, and DNeasy Blood and Tissue Kit-in the search for a rapid, simple, and non-destructive method of DNA extraction from pest and natural enemy specimens. Amplification of PCR production was achieved with the Chelex method and the DNeasy Blood and Tissue Kit. However, not all specimens tested by the PrepMan Ultra Reagent method yielded amplification products. Observation of the morphological features of dried Encarsia formosa specimens revealed that all heads and leg segments remained intact after DNA extraction by each of the three methods. Almost all samples treated with the PrepMan Ultra Reagent method had large, discernible morphological changes, in particular, of the antennae and forewings, which are important features for identifying minute wasps. In contrast, the antennae and forewings of specimens on which we used the Chelex method suffered little damage. Use of the DNeasy Blood and Tissue Kit method did not alter the forewing features, but strong wrinkles appeared in the antennae. We discuss the DNA extraction techniques from the perspective of these results. [ABSTRACT FROM AUTHOR]

Molecular analyses of small-sized copepods (≤1 mm) generally involve the complete destruction of the specimens. Consequently, incongruences between the molecular and morphological results cannot be investigated since no specimen vouchers remain. The present study provides a modified column-based DNA extraction method to retain the exoskeleton of the specimen and thus, to enable molecular and morphological analysis of the same specimens. The method has been tested on ethanol preserved specimens of nine pelagic copepod genera. [ABSTRACT FROM PUBLISHER]

The soybean aphid ( Aphis glycines Matsumura) is an economically significant pest in North America, causing extensive damage to soybean crops through direct feeding damage and disease transmission. If unchecked, this pest could cause billions of dollars of damage to soybean crops. Identification of the soybean aphid can be difficult due to its small size, complex life cycle and morphological plasticity. Generally, an expert is required to identify a specimen. Additionally, identification of some life stages, such as eggs, is impossible. DNA barcoding has been successfully used to differentiate aphid species, including A. glycines, based on sequencing of a standardized gene region. Although this method represents an important step towards accurate identification, samples must still be sent to specialized facilities for analysis. Using existing DNA barcode sequences in the publically accessible Barcode of Life Data System ( BOLD; ), species-specific differences were identified and used to develop a real-time PCR assay to identify soybean aphids. This assay can be run on portable systems for rapid, accurate and simple identification in the field. The use of a non-destructive DNA extraction protocol allows the original insect to be vouchered and therefore available for further study if necessary. This work represents an important step in soybean aphid management. [ABSTRACT FROM AUTHOR]

WallaceaphytisPolaszek and Fusu gen. nov. (type speciesWallaceaphytis kikiaeAyshford and Polaszek sp. nov.) is described from Danum Valley, Sabah, in Malaysian Borneo. Although known from just a single female individual, the genus is extremely unusual morphologically, being the only member of the large subfamily Aphelininae with four-segmented tarsi. The form of the fore wings and head are also unique in the subfamily, and its status as a new genus is confirmed by analysis of nuclear ribosomal DNA. DNA sequence analysis was undertaken by comparison with more than 60 aphelinid sequences from GenBank. The sequence for the standard DNA barcode region (cytochrome oxidase c subunit I; COI) is provided. The new genus is named in honour of Alfred Russel Wallace, co-discoverer of the theory of evolution by natural selection. The new genus and species are published on the exact date of the centenary of his death. http://www.zoobank.org/urn:lsid:zoobank.org:pub:700D2B2A-1586-4100-85D2-24844EFE3F90 [ABSTRACT FROM AUTHOR]

Here, we describe a simple method adapted for high-throughput protocols allowing voucher specimen recovery for Collembola and by extension for other soft-bodied small arthropods. A standard extraction protocol was tested to examine the effects of lysis duration (1, 2, 4, 12 h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1 h of lysis, while voucher condition was fine for up to 12 h. The lysis step substantially shortens the clearing process necessary for morphological examination. [ABSTRACT FROM AUTHOR]

Plant bio-protection and biosecurity programs worldwide use trap-based surveillance for the early detection of agricultural pests and pathogens to contain their incursions and spread. This task is reliant on effective preservation in insect traps, which is required to maintain specimen quality for extended periods under variable environmental conditions. Furthermore, with traditional morphological examinations now increasingly paired with modern molecular diagnostic techniques, insect traps are required to preserve both the specimens' morphology and the DNA of insects and vectored bacterial pathogens. Here, we used psyllids (Hemiptera) and their hosted bacteria as a model to test the preservative ability of propylene glycol (PG): a non-flammable, easily transportable preservative agent that could be used in pitfall, suction or malaise traps. We tested preservation using various PG concentrations, at different temperatures and for multiple time periods, paired with non-destructive DNA extraction methods, which allow isolation of both insect and arbobacterial DNA while retaining a morphological voucher of the insect host specimens. PG concentrations between 40% and 100% performed best for both insect and bacterial DNA preservation up to 28 days. Ultimately, given the viscous nature of PG at high concentrations, we recommend using it at a concentration between 40% and 60% to enable insects to sink into the solution, thus enhancing DNA preservation. [ABSTRACT FROM AUTHOR]

One of the major challenges in molecular analysis of arthropods, especially for natural enemies of insect pests, is the intact preservation of the specimens to be integrated into entomological collections. However, most of the DNA extraction protocols involve maceration of the tissue, avoiding the preservation of the original specimen. Two general methods were adapted into non-destructive DNA extraction protocols, DNeasy® Blood & Tissue Kit (A) and the CaCl 2 lysis buffer method (B), while the potential of the method with the alkaline lysis buffer (HotSHOT; C) was evaluated for the first time on insect specimens. These protocols were assessed for the recovery of DNA from Ceraeochrysa valida , Tamarixia radiata , and Hippodamia convergens . Photographical records showed that morphological features of the specimens were preserved after the DNA extraction process. COI fragments were successfully amplified with method A (100%), B (77%), and C (88%), respectively. We conclude that these non-destructive DNA extraction methods avoid the destruction of tissue and preserve the original insects and their morphological characteristics for future reference.

The rapid loss of biodiversity, coupled with difficulties in species identification, call for innovative approaches to assess biodiversity. Insects make up a substantial proportion of extant diversity and play fundamental roles in any given ecosystem. To complement morphological species identification, new techniques such as metabarcoding make it possible to quantify insect diversity and insect-ecosystem interactions through DNA sequencing. Here we examine the potential of bulk insect samples (i.e., containing many non-sorted specimens) to assess prokaryote and eukaryote biodiversity and to complement the taxonomic coverage of soil samples. We sampled 25 sites on three continents and in various ecosystems, collecting insects with SLAM traps (Brazil) and Malaise traps (South Africa and Sweden). We then compared our diversity estimates with the results obtained with biodiversity data from soil samples from the same localities. We found a largely different taxonomic composition between the soil and insect samples, testifying to the potential of bulk insect samples to complement soil samples. Finally, we found that non-destructive DNA extraction protocols, which preserve insect specimens for morphological studies, constitute a promising choice for cost-effective biodiversity assessments. We propose that the sampling and sequencing of insect samples should become a standard complement for biodiversity studies based on environmental DNA.