The insect superfamily Psylloidea (Hemiptera) includes economically important biocontrol agents, pests and plant pathogen vectors, for which a rapid and accurate identification is fundamental for international biosecurity. Australasia is a hot spot for psyllid diversity, but previous species assessments in the region were largely based on morphology and host plant association. Morphological identification of psyllids remains challenging for a wide number of species and for juvenile insects, while a robust molecular framework for identification is not available. Consequently, knowledge of psyllid biology is compromised. Here, incorporating morphological evidence and host plant associations, insects collected from almost 600 primarily New Zealand locations were linked to 67 previously described species. By applying species delimitation methods including GYMC (General Mixed Yule-Coalescent method), PTP (Poisson Tree Processes), mPTP (multi-rate Poisson Tree Processes) and ABGD (Automatic Barcode Gap Discovery) to a dataset composed of 425 cytochrome oxidase I (COI) DNA barcode sequences, further cryptic diversity was revealed among the psyllid collection; more than 20 undescribed taxa are reported here for the first time, resulting in a total of 90 taxa across 21 genera and six families included in this study. Our improved understanding of psyllid diversity in New Zealand revealed new plant host-psyllid associations and geographical variation. The DNA barcode resource will enable future studies of psyllid ecology and more accurate, rapid identifications of psyllids that pose biosecurity threats to Australasia.
A checklist of extant species of Psylloidea in New Zealand is presented. The list is structured according to the latest taxonomic classification of families, subfamilies and genera. It includes 99 species, 71 of which are formally described and named, along with information on the host plants and the natural enemies as aspects that are either species-specific or assist in their recognition. An updated distribution of each species is given based on literature records and material held in the major New Zealand entomological collections and databases, including from very recent field surveys. A new record for New Zealand is Phellopsylla formicosa.
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.
Insects preserved as reference specimens are important in a wide range of fields, including health, pest management and forensics. The aim of the present study was to test a non‐destructive DNA extraction method on samples of soft‐bodied insects, fly larvae, which are otherwise hard to identify morphologically. This not only provides DNA enabling molecular identification but also retains morphological reference specimens for samples belonging to collections and museums that cannot be destroyed. In this work, fly larvae identified as belonging to the family Muscidae were non‐destructively processed. DNA barcoding molecular identification allowed most of these specimens to be assigned to species. Furthermore, the visibility of seven important larval morphological characters – anterior and posterior spiracles, mouth hooks, cephalopharyngeal skeleton, locomotory welts, segmentation and colour – was assessed pre‐ and post‐DNA extraction. It was found that the morphology generally did not deteriorate post‐DNA extraction but actually improved through increased visibility of internal features. Therefore, this non‐destructive DNA extraction method not only allowed COI barcode sequences to be obtained, but also enabled a better morphological identification of the fly larvae retaining physical reference voucher specimens and avoiding the need for dissections.
A fast and reliable method for obtaining a species-level identification is a fundamental requirement for a wide range of activities, from plant protection and invasive species management to biodiversity assessments and ecological studies. For insects, novel molecular techniques such as DNA metabarcoding have emerged as a rapid alternative to traditional morphological identification, reducing the dependence on limited taxonomic experts. Until recently, molecular techniques have required a destructive DNA extraction, precluding the possibility of preserving voucher specimens for future studies, or species descriptions. Here we paired insect metabarcoding with two recent non-destructive DNA extraction protocols, to obtain a rapid and high-throughput taxonomic identification of diverse insect taxa while retaining a physical voucher specimen. The aim of this work was to explore how non-destructive extraction protocols impact the semi-quantitative nature of metabarcoding, which alongside species presence/absence also provides a quantitative, but biased, representation of their relative abundances. By using a series of mock communities representing each stage of a typical metabarcoding workflow we were able to determine how different morphological (i.e., insect biomass and exoskeleton hardness) and molecular traits (i.e., primer mismatch and amplicon GC%), interact with different protocol steps to introduce quantitative bias into non-destructive metabarcoding results. We discuss the relevance of taxonomic bias to metabarcoding identification of insects and potential approaches to account for it.
The ‘Eugenia psyllid’ or ‘Lilly pilly psyllid’, widely recognized in Australia and in the USA as Trioza eugeniae Froggatt (Hemiptera: Triozidae), is not T. eugeniae, but rather T. adventicia Tuthill. In this study we assessed morphological comparisons of materials from throughout the native and introduced ranges and re-examined original descriptions of both taxa, together with Froggatt's type specimens of T. eugeniae. Furthermore, through DNA barcoding analyses, we confirmed the validity of both T. adventicia and T. eugeniae as separate species. We re-described both species to include additional characters not previously included and designated a lectotype for T. eugeniae. T. eugeniae has smaller fore wings that are slightly more elongate. These lack infuscation around veins R and R1, vein Rs is relatively longer, meeting the costa closer to the wing apex; with certain veins bearing long, fine divergent setae, a character not previously described. It has consistently three inner and one outer metatibial spurs. The male parameres appear narrowly pyriform with a weak dorsolateral lobe and weakly sclerotized apices. T. adventicia has larger fore wings that are slightly more ovate with dark infuscation around veins R and R1; vein Rs is relatively shorter, meeting the costa further from the wing apex, with veins lacking long, fine divergent setae. The usual configuration of two inner and one outer metatibial spurs, previously used to separate the two species, appears inconsistent. The male parameres appear a little more broadly pyriform with slightly more sclerotized apices. T. eugeniae refers to a distinct species which has a restricted distribution only in its native range in southern subcoastal New South Wales, Australia. T. adventicia refers to a separate species, with a natural distribution in eastern subcoastal Australia, but has been introduced widely in southern Australia, to New Zealand and the USA. This study elucidates a long history of misidentification of T. eugeniae in the nursery industry and in almost 30 years of literature on its biological control in the USA. Regardless, the biological control program, unknowingly, targeted the correct species of psyllid, T. adventicia, in its foreign exploration and importation of the appropriate parasitoid as a biocontrol agent in the USA. Despite being firmly entrenched in both the nursery trade and scientific literature, the name T. eugeniae is misapplied. While the acceptance of the valid name, T. adventicia, might be regarded as both problematic and protracted, this is the correct taxonomical attribution.
High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.
High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of
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