Assessment of Surface Sterilisation Approaches for the Removal of Pollen DNA from Philaenus spumarius.

Dietary analysis of herbivorous insects relies on successfully eliminating surface contamination. If this cannot be performed reliably, then it will not be possible to differentiate between plants that the insect is feeding on and plants the insect has been in contact with, either directly or via pollen. Methods in the literature often use bleach and alcohol washes to remove contamination. We perform a controlled metabarcoding baseline study on a herbivorous, xylem-feeding insect, the Meadow Spittlebug (Philaenus spumarius), using Oxford Nanopore Technologies (ONT) sequencing, and identify possible contamination that persists after washes. Despite the reported success of methods in the literature, we find that contamination is still present, leading to possible false-positive results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove, and conduct a further three experiments with the goal of removing pollen from the surface of Philaenus spumarius. This study investigates the effectiveness of robust bleach/Tween/alcohol washes, sterile gut excision (including combined with Distel application), and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination and still detect signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut.

Molecular Identification of Asian Hornet Vespa velutina nigrithorax Prey from Larval Gut Contents: A Promising Method to Study the Diet of an Invasive Pest.

The Asian hornet, Vespa velutina nigrithorax (Hymenoptera: Vespidae), is an invasive hornet that was accidentally introduced into Europe in 2004. It mainly preys on other invertebrates and arthropod species, and often targets honey bee (Apis mellifera) colonies. The introduction of these hornets may damage indigenous fauna and apiculture. Knowledge of V. velutina prey preference and the species composition of their diet is relatively limited. In this study, we assessed methodologies for the molecular identification of prey using dissected larvae from destroyed nests. Ten larval samples were taken from five nests in areas where the hornets had not yet established: two from the Channel Islands and three in the mainland UK. DNA was extracted from the gut contents and sequenced and analysed by metabarcoding with Oxford Nanopore Technologies' Flongle and MinION devices. Numerous taxa were detected in each larval sample with the species composition varying by individual and by nest. Between 15 and 26 species were found per nest, with wasps (Vespula spp.), spiders, honey bees and blow flies being the most abundant taxa. These results demonstrate that metabarcoding larval gut contents can be used to study the Asian hornet diet and give a first snapshot of the prey items captured by V. v. nigrithorax in the UK. This method could be used for future large-scale testing of the gut contents of hornet nests, in order to provide a greater insight into the foraging behaviour of this predator across Europe and elsewhere.

Managing incursions of Vespa velutina nigrithorax in the UK: an emerging threat to apiculture.

Vespa velutina nigrithorax is an invasive species of hornet accidentally introduced into Europe in 2004. It feeds on invertebrates, including honey bees, and represents a threat to European apiculture. In 2016, the first nest of this hornet was detected and destroyed on mainland UK. A further 8 nests were discovered between 2016 and 2019. Nest dissection was performed on all nests together with microsatellite analyses of different life stages found in the nests to address the reproductive output and success of nests found in the UK. None of the nests had produced the next generation of queens. Follow-up monitoring in those regions detected no new nests in the following years. Diploid males were found in many UK nests, while microsatellite analysis showed that nests had low genetic diversity and the majority of queens had mated with one or two males. All UK nests derived from the European zone of secondary colonisation, rather than from the native range of the species. None of the nests discovered so far have been direct offspring of another UK nest. The evidence suggests that these nests were separate incursions from a continental population rather than belonging to a single established UK population of this pest.

Development and validation of real-time PCR methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.

Parasitic gastroenteritis, a disease caused by parasitic nematodes, is of major concern to the sheep industry and threatens sustainability. Traditional methods for diagnosis of the type and level of infection in a sheep flock require laborious laboratory extraction, culture and microscopic examination of eggs or larvae from faecal samples. Advances in molecular technology offer the potential for more efficient and reliable methods. This study aimed to develop and test a real-time PCR method for routine diagnosis of infection by Teladorsagia circumcincta and Haemonchus contortus in sheep. Primer/probe sets were designed around the ribosomal internal transcribed spacer 2 (ITS2) region as sequence data was available from other studies and so tests used published primer/probe sets, as well as those designed at the Food and Environment Research Agency (Fera). Different primer/probe combinations were tested for specificity against DNA extracted from T. circumcincta larvae or H. contortus DNA. All sets were tested for cross reactivity against four other closely related species, using real-time PCR technology. Reactions were optimised with the best primer/probe combination for each species and then tested for sensitivity against samples containing different T. circumcincta or H. contortus DNA concentrations. Faecal samples were collected from sheep infected with T. circumcincta or H. contortus alone and the eggs harvested, counted and DNA extracted. Serial dilutions were prepared to give a range of concentrations between approximately 3000 and 50 eggs per sample and real-time PCR reactions were carried out for each and mean cycle time (Ct) values were calculated. These Ct values were plotted against the sample egg concentration to produce a standard curve. Regression analysis was carried out using the generated data. Eggs were then harvested from faecal samples collected in the field from sheep carrying natural mixed infections, DNA extracted and Ct values recorded as before. Results were compared to the standard curve data in order to calculate the approximate number of T. circumcincta and H. contortus eggs in each field sample. These values were compared to the number of eggs determined using the traditional laboratory methods and Pearson product-moment correlation coefficients calculated. Results showed a strong correlation between numbers of eggs determined using the traditional and novel molecular methods suggesting that the developed molecular tools adequately predict egg numbers in this range. Further development and validation work should allow practical use of the method. This assay has significant advantages over the traditional methods currently used routinely at Fera and other parasitology laboratories and these are discussed.

Genetic evidence that culling increases badger movement: implications for the spread of bovine tuberculosis.

The Eurasian badger (Meles meles) has been implicated in the transmission of bovine tuberculosis (TB, caused by Mycobacterium bovis) to cattle. However, evidence suggests that attempts to reduce the spread of TB among cattle in Britain by culling badgers have mixed effects. A large-scale field experiment (the randomized badger culling trial, RBCT) showed that widespread proactive badger culling reduced the incidence of TB in cattle within culled areas but that TB incidence increased in adjoining areas. Additionally, localized reactive badger culling increased the incidence of TB in cattle. It has been suggested that culling-induced perturbation of badger social structure may increase individual movements and elevate the risk of disease transmission between badgers and cattle. Field studies support this hypothesis, by demonstrating increases in badger group ranges and the prevalence of TB infection in badgers following culling. However, more evidence on the effect of culling on badger movements is needed in order to predict the epidemiological consequences of this control strategy. Here, analysis of the genetic signatures of badger populations in the RBCT revealed increased dispersal following culling. While standard tests provided evidence for greater dispersal after culling, a novel method indicated that this was due to medium- and long-distance dispersal, in addition to previously reported increases in home-range size. Our results also indicated that, on average, badgers infected with M. bovis moved significantly farther than did uninfected badgers. A disease control strategy that included culling would need to take account of the potentially negative epidemiological consequences of increased badger dispersal.