Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding.

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding.

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at - 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

Environmental monitoring study of pesticide contamination in Denmark through honey bee colonies using APIStrip-based sampling.

Due to their extensive use in both agricultural and non-agricultural applications, pesticides are a major source of environmental contamination. Honey bee colonies are proven sentinels of these and other contaminants, as they come into contact with them during their foraging activities. However, active sampling strategies involve a negative impact on these organisms and, in most cases, the need of analyzing multiple heterogeneous matrices. Conversely, the APIStrip-based passive sampling is innocuous for the bees and allows for long-term monitorings using the same colony. The versatility of the sorbent Tenax, included in the APIStrip composition, ensures that comprehensive information regarding the contaminants inside the beehive will be obtained in one single matrix. In the present study, 180 APIStrips were placed in nine apiaries distributed in Denmark throughout a six-month sampling period (10 subsequent samplings, April to September 2020). Seventy-five pesticide residues were detected (out of a 428-pesticide scope), boscalid and azoxystrobin being the most frequently detected compounds. There were significant variations in the findings of the sampling sites in terms of number of detections, pesticide diversity and average concentration. A relative indicator of the potential risk of pesticide exposure for the honey bees was calculated for each sampling site. The evolution of pesticide detections over the sampling periods, as well as the individual tendencies of selected pesticides, is herein described. The findings of this large-scale monitoring were compared to the ones obtained in a previous Danish, APIStrip-based pilot monitoring program in 2019. Samples of honey and wax were also analyzed and compared to the APIStrip findings.

Repellent activity of desiccant dusts and conidia of the entomopathogenic fungus Beauveria bassiana when tested against poultry red mites (Dermanyssus gallinae) in laboratory experiments.

Desiccant dusts and entomopathogenic fungi have previously been found to hold potential against the poultry red mite, which is an important pest in egg production and notoriously difficult to control. Both control agents may cause repellence in other arthropods and potentially also influence control levels adversely when used against the poultry red mite. Five desiccant dust products with good efficacy against the poultry red mite Dermanyssus gallinae caused avoidance behavior in mites when tested in bioassays. The repellent activity was correlated with efficacy, which was found to depend on both dose and relative humidity (RH). However, one desiccant dust was significantly less repellent compared to other dusts with similar levels of efficacy. Further, dry conidia of the fungus Beauveria bassiana were also shown to be repellent to poultry red mites, both when applied on its own and when admixed with a low dose of the desiccant dust Diamol. The pick-up of desiccant dust particles and fungus conidia from treated surfaces by mites did not differ depending on RH, whereas the overall efficacy of the two control agents were significantly higher at 75 than at 85 % RH. In addition, the combined effect of the two substances was synergistic when tested in a bioassay where mites could choose whether to cross a treated surface. This is the first time a member of Acari has been shown to be repelled by desiccant dusts and by conidia of an entomopathogenic fungus.

Synergistic interaction between the fungus Beauveria bassiana and desiccant dusts applied against poultry red mites (Dermanyssus gallinae).

The poultry red mite, Dermanyssus gallinae, is a major pest in egg production, feeding on laying hens. Widely used non-chemical control methods include desiccant dusts, although their persistence under field conditions is often short. Entomopathogenic fungi may also hold potential for mite control, but these fungi often take several days to kill mites. Laboratory experiments were carried out to study the efficacy of 3 types of desiccant dusts, the fungus Beauveria bassiana and combinations of the two control agents against D. gallinae. There was significant synergistic interaction between each of the desiccant dusts and the fungus, with observed levels of mite mortality significantly higher than those expected for an additive effect (up to 38 % higher). Synergistic interaction between desiccant dust and fungus was found also when different application methods were used for the fungus and at different levels of relative humidity. Although increased levels of mortality were reached due to the synergistic interaction, the speed of lethal action was not influenced by combining the two components. The persistence of the control agents applied separately or in combination did not change over a period of 4 weeks. Overall, combinations of desiccant dusts and fungus conidia seem to hold considerable promise for future non-chemical control of poultry red mites.

Real-time measurement of volatile chemicals released by bed bugs during mating activities.

In recent years, bed bug (Hemiptera: Cimicidae) problems have increased dramatically in many parts of the world, leading to a renewed interest in their chemical ecology. Most studies of bed bug semiochemicals have been based on the collection of volatiles over a period of time followed by chemical analysis. Here we present for the first time, a combination of proton transfer reaction mass spectrometry and video analysis for real-time measurement of semiochemicals emitted by isolated groups of bed bugs during specific behavioural activities. The most distinct peaks in the proton transfer reaction mass spectrometry recordings were always observed close to the termination of mating attempts, corresponding to the defensive emissions that bed bugs have been suspected to exploit for prevention of unwanted copulations. The main components of these emissions were (E)-2-hexenal and (E)-2-octenal recorded in ratios between 1:3 and 3:1. In the current study, the quantity varied over 1000 fold for both of the compounds with up to 40 µg total release in a single emission. Males also emit defensive compounds due to homosexual copulation attempts by other males, and no significant differences were observed in the ratio or the amount of the two components released from males or females. In summary, this study has demonstrated that combining proton-transfer-reaction mass spectrometry with video analysis can provide detailed information about semiochemicals emitted during specific behavioural activities.

Resistance differences between chlorpyrifos and synthetic pyrethroids in Cimex lectularius population from Denmark.

Bed bug, Cimex lectularius L., populations were investigated for resistance against permethrin and chlorpyrifos in a topical application bioassay, after an initial establishment of a discriminating dose with a susceptible population. For both insecticides, ca. two times the lethal dose LD(99) was selected: 2,560 ng of permethrin and 200 ng of chlorpyrifos per bed bug, respectively. Bed bugs were collected from infested homes in Denmark at ten locations and bred in the laboratory. The frequency of permethrin-resistant individuals was high in Danish bed bug populations as susceptible individuals were only found in three of ten populations. In contrast, the frequency of chlorpyrifos-resistant individuals was low in Danish bed bug populations, but resistant individuals were found in five of ten populations. To test the significance of the observed resistance, we performed tarsal contact test with commercially available insecticides. The test indicated that both a permethrin and a deltamethrin product had very low efficacy against the field-collected bed bug populations. Despite the reduced sensitivity to synthetic pyrethroids, all populations tested in the tarsal test on the commercial product with micro-encapsulated chlorpyrifos resulted in close to 100% mortality.

Inert dusts and their effects on the poultry red mite (Dermanyssus gallinae).

The haematophagous poultry red mite (Dermanyssus gallinae) is the most important pest of egg laying hens in many parts of the world. Control has often relied on chemical pesticides, but inert dusts, which are thought to kill target hosts primarily by desiccation, have become one of the most commonly applied alternative control methods for poultry red mite in Europe. This development has occurred despite a lack of knowledge of the efficacy of the different types of inert dusts and how this is affected by environmental parameters, e.g. the high relative humidity found in poultry houses. In this laboratory study the efficacy of different commercial inert dust products against D. gallinae is compared. All tested compounds killed mites, but there was a clear ranking of efficacy (measured as weight loss after 24 h and as time until 50% mortality), particularly at 75% relative humidity (RH). At 85% RH the efficacy was significantly lower for all tested compounds (P < 0.001). Weight changes over time followed an exponential evaporation model until the mites started dying whereafter the rate of evaporation increased again and followed a slightly different exponential evaporation model. A tarsal test showed that 24 h exposure to surfaces treated with doses much lower than those recommended by the producers is sufficient to kill mites as fast as when they were dusted with massive doses. These data emphasise the need for thorough treatment of all surfaces in a poultry house in order to combat D. gallinae.

Prevalence and key figures for the poultry red mite Dermanyssus gallinae infections in poultry farm systems.

Recent surveys and sample collection have confirmed the endemicity of Dermanyssus gallinae in poultry farming worldwide. The reduction in number and efficacy of many acaricide products has accentuated the prevalence rates of this poultry ectoparasite observed more often in non intensive systems such as free-range, barns or backyards and more often in laying hens than in broiler birds. The lack of knowledge from producers and the utilisation of inadequate, ineffective or illegal chemicals in many countries have been responsible for the increase in infestation rates due to the spread of acaricide resistance. The costs for control methods and treatment are showing the tremendous economic impact of this ectoparasite on poultry meat and egg industries. This paper reviews the prevalence rates of this poultry pest in different countries and for different farming systems and the production parameters which could be linked to this pest proliferation.