Advancing pathogen surveillance by nanopore sequencing and genotype characterization of Acheta domesticus densovirus in mass-reared house crickets.

Rapid and reliable detection of pathogens is crucial to complement the growing industry of mass-reared insects, in order to safeguard the insect colonies from outbreak of diseases, which may cause significant economic loss. Current diagnostic methods are mainly based on conventional PCR and microscopic examination, requiring prior knowledge of disease symptoms and are limited to identifying known pathogens. Here, we present a rapid nanopore-based metagenomics approach for detecting entomopathogens from the European house cricket (Acheta domesticus). In this study, the Acheta domesticus densovirus (AdDV) was detected from diseased individuals using solely Nanopore sequencing. Virus reads and genome assemblies were obtained within twenty-four hours after sequencing. Subsequently, due to the length of the Nanopore reads, it was possible to reconstruct significantly large parts or even the entire AdDV genome to conduct studies for genotype identification. Variant analysis indicated the presence of three AdDV genotypes within the same house cricket population, with association to the vital status of the diseased crickets. This contrast provided compelling evidence for the existence of non-lethal AdDV genotypes. These findings demonstrated nanopore-based metagenomics sequencing as a powerful addition to the diagnostic tool kit for routine pathogen surveillance and diagnosis in the insect rearing industry.

An insect's energy bar: the potential role of plant guttation on biological control.

Plant guttation is an exudation fluid composed of xylem and phloem sap secreted at the margins of leaves of many agricultural crops. Although plant guttation is a widespread phenomenon, its effect on natural enemies remains largely unexplored. A recent study showed that plant guttation can be a reliable nutrient-rich food source for natural enemies, affecting their communities in highbush blueberries. This review highlights the potential role of plant guttation as a food source for natural enemies, with a particular emphasis on its nutritional value, effects on insect communities, and potential use in conservation biological control. We also discuss possible negative implications and conclude with some open questions and future directions for research.

Confirmation of the Y215H mutation in the ß2 -octopamine receptor in Varroa destructor is associated with contemporary cases of amitraz resistance in the United States.

BACKGROUND: The parasitic mite, Varroa destructor (Anderson and Trueman), is a leading cause of honey bee colony losses around the world. Application of miticides such as amitraz are often the primary method of Varroa control in commercial beekeeping operations in the United States. It is likely that excessive and exclusive amitraz application has led to the development of amitraz resistance in Varroa. A mutation of tyrosine at amino acid position 215 to histidine (Y215H) in the ß2 -octopamine receptor was identified in putatively amitraz-resistant Varroa in the United States. This research investigated the presence of the Y215H mutation in quantitatively confirmed amitraz-resistant Varroa from the United States. RESULTS: There was a strong association of susceptible and resistant phenotypes with the corresponding susceptible and resistant genotypes respectively, and vice versa. The resistance bioassay may understate resistance levels because of the influence of environmental conditions on the outcome of the test, whereby Varroa with an amitraz-resistant genotype may appear with a susceptible phenotype. CONCLUSION: Confirmation of the Y215H mutation in the ß2 -octopamine receptor of amitraz-resistant Varroa encourages the development and validation of low-cost, high-throughput genotyping protocols to assess amitraz resistance. Resistance monitoring via genotyping will allow for large-scale passive monitoring to accurately determine the prevalence of amitraz resistance rather than directed sampling of apiaries with known resistance issues. Genotyping of Varroa for amitraz resistance early in the beekeeping season may predict late-season resistance at the colony level and provide beekeepers with enough time to develop an effective Varroa management strategy. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Transcriptomic profile of the predatory mite Amblyseius swirskii (Acari: Phytoseiidae) on different host plants.

Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) is a predatory mite, effective at controlling whiteflies and thrips in protected crops. However, on tomato its efficacy as a biocontrol agent is hindered, most probably by the plant trichomes and their exudates. Our aim was to characterize the response of A. swirskii to the tomato trichome exudates and identify three major detoxification gene sets in this species: cytochromes P450 (CYPs), glutathione S-transferases (GSTs) and carboxyl/cholinesterases (CCEs). Mites were exposed separately to tomato and pepper, a favourable host plant for A. swirskii, after which their transcriptional responses were analysed and compared. The de novo transcriptome assembly resulted in 71,336 unigenes with 66.1% of them annotated. Thirty-nine A. swirskii genes were differentially expressed after transfer on tomato leaves when compared to pepper leaves; some of the expressed genes were associated with the metabolism of tomato exudates. Our results illustrate that the detoxification gene sets CYPs, GSTs and CCEs are abundant in A. swirskii, but do not play a significant role when in contact with the tomato exudates.

Neonicotinoids from coated seeds toxic for honeydew-feeding biological control agents.

Seed coating ('seed treatment') is the leading delivery method of neonicotinoid insecticides in major crops such as soybean, wheat, cotton and maize. However, this prophylactic use of neonicotinoids is widely discussed from the standpoint of environmental costs. Growing soybean plants from neonicotinoid-coated seeds in field, we demonstrate that soybean aphids (Aphis glycines) survived the treatment, and excreted honeydew containing neonicotinoids. Biochemical analyses demonstrated that honeydew excreted by the soybean aphid contained substantial concentrations of neonicotinoids even one month after sowing of the crop. Consuming this honeydew reduced the longevity of two biological control agents of the soybean aphid, the predatory midge Aphidoletes aphidimyza and the parasitic wasp Aphelinus certus. These results have important environmental and economic implications because honeydew is the main carbohydrate source for many beneficial insects in agricultural landscapes.

Mutations associated with pyrethroid resistance in Varroa mite, a parasite of honey bees, are widespread across the United States.

BACKGROUND: Managed honey bees are key pollinators of many crops and play an essential role in the United States food production. For more than ten years, beekeepers in the United States have been reporting high rates of colony losses. One of the drivers of these losses is the parasitic mite Varroa destructor. Maintaining healthy honey bee colonies in the United States is dependent on a successful control of this mite. The pyrethroid tau-fluvalinate (Apistan®) was among the first synthetic varroacides registered in the United States. With over 20 years of use, mites resistant to Apistan® have emerged, and so it is unsurprising that treatment failures have been reported. Resistance to tau-fluvalinate in US mite populations is associated with point mutations at position 925 of the voltage-gated sodium channel. RESULTS: Here, we have generated a distribution map of pyrethroid resistance alleles in Varroa samples collected from US apiaries in 2016 and 2017, using a high throughput allelic discrimination assay based on TaqMan®. Our results evidence that knockdown resistance (kdr)-type mutations are widely distributed in Varroa populations across the country showing high variability among apiaries. We used these data to predict the phenotype of the mites in the case of treatments with pyrethroids. CONCLUSION: We highlight the relevance of monitoring the resistance in mite populations to achieve an efficient control of this pest. We also put forward the benefits of implementing this methodology to provide data for designing pest management programs aiming to control Varroa. © 2021 Society of Chemical Industry.

Large-Scale Monitoring of Resistance to Coumaphos, Amitraz, and Pyrethroids in Varroa destructor.

Varroa destructor is an ectoparasitic mite causing devastating damages to honey bee colonies around the world. Its impact is considered a major factor contributing to the significant seasonal losses of colonies recorded every year. Beekeepers usually rely on a reduced set of acaricides to manage the parasite, usually the pyrethroids tau-fluvalinate or flumethrin, the organophosphate coumaphos, and the formamidine amitraz. However, the evolution of resistance in the mite populations is leading to an unsustainable scenario with almost no alternatives to reach an adequate control of the mite. Here, we present the results from the first large-scale and extensive monitoring of the susceptibility to acaricides in the Comunitat Valenciana, one of the most prominent apicultural regions in Spain. Our ultimate goal is to provide beekeepers with timely information to help them decide what would be the best alternative for a long-term control of the mites in their apiaries. Our data show that there is a significant variation in the expected efficacy of coumaphos and pyrethroids across the region, indicating the presence of a different ratio of resistant individuals to these acaricides in each population. On the other hand, the expected efficacy of amitraz was more consistent, though slightly below the expected efficacy according to the label.

IPM-recommended insecticides harm beneficial insects through contaminated honeydew.

The use of some systemic insecticides has been banned in Europe because they are toxic to beneficial insects when these feed on nectar. A recent study shows that systemic insecticides can also kill beneficial insects when they feed on honeydew. Honeydew is the sugar-rich excretion of hemipterans and is the most abundant carbohydrate source for beneficial insects such as pollinators and biological control agents in agroecosystems. Here, we investigated whether the toxicity of contaminated honeydew depends on i) the hemipteran species that excretes the honeydew; ii) the active ingredient, and iii) the beneficial insect that feeds on it. HPLC-MS/MS analyses demonstrated that the systemic insecticides pymetrozine and flonicamid, which are commonly used in Integrated Pest Management programs, were present in honeydew excreted by the mealybug Planococcus citri. However, only pymetrozine was detected in honeydew excreted by the whitefly Aleurothixus floccosus. Toxicological studies demonstrated that honeydew excreted by mealybugs feeding on trees treated either with flonicamid or pymetrozine increased the mortality of the hoverfly Sphaerophoria rueppellii, but did not affect the parasitic wasp Anagyrusvladimiri. Honeydew contaminated with flonicamid was more toxic for the hoverfly than that contaminated with pymetrozine. Collectively, our data demonstrate that systemic insecticides commonly used in IPM programs can contaminate honeydew and kill beneficial insects that feed on it, with their toxicity being dependent on the active ingredient and hemipteran species that excretes the honeydew.

Assessing the resistance to acaricides in Varroa destructor from several Spanish locations.

Varroosis is the disease caused by the ectoparasitic mite Varroa destructor, one of the most destructive diseases of honeybees. In Spain, there is great concern because there are many therapeutic failures after acaricide treatments intended to control varroosis outbreaks. In some of these cases it is not clear whether such failures are due to the evolution of resistance. Therefore, it is of high interest the development of methodologies to test the level of resistance in mite populations. In this work, a simple bioassay methodology was used to test whether some reports on low efficacy in different regions of Spain were in fact related to reduced Varroa sensitivity to the most used acaricides. This bioassay proved to be very effective in evaluating the presence of mites that survive after being exposed to acaricides. In the samples tested, the mortality caused by coumaphos ranged from 2 to 89%; for tau-fluvalinate, it ranged from 5 to 96%. On the other hand, amitraz caused 100% mortality in all cases. These results suggest the presence of Varroa resistant to coumaphos and fluvalinate in most of the apiaries sampled, even in those where these active ingredients were not used in the last years. The bioassay technique presented here, either alone or in combination with other molecular tools, could be useful in detecting mite populations with different sensitivity to acaricides, which is of vital interest in selecting the best management and/or acaricide strategy to control the parasite in apiaries.

Plant guttation provides nutrient-rich food for insects.

Plant guttation is a fluid from xylem and phloem sap secreted at the margins of leaves from many plant species. All previous studies have considered guttation as a water source for insects. Here, we hypothesized that plant guttation serves as a reliable and nutrient-rich food source for insects with effects on their communities. Using highbush blueberries as a study system, we demonstrate that guttation droplets contain carbohydrates and proteins. Insects from three feeding lifestyles, a herbivore, a parasitic wasp and a predator, increased their longevity and fecundity when fed on these guttation droplets compared to those fed on control water. Our results also show that guttation droplets, unlike nectar, are present on leaves during the entire growing season and are visited by numerous insects of different orders. In exclusion-field experiments, the presence of guttation modified the insect community by increasing the number of predators and parasitic wasps that visited the plants. Overall, our results demonstrate that plant guttation is highly reliable, compared to other plant-derived food sources such as nectar, and that it increases the communities and fitness of insects. Therefore, guttation represents an important plant trait with profound implications on multi-trophic insect-plant interactions.

Next-generation biological control: the need for integrating genetics and genomics.

Biological control is widely successful at controlling pests, but effective biocontrol agents are now more difficult to import from countries of origin due to more restrictive international trade laws (the Nagoya Protocol). Coupled with increasing demand, the efficacy of existing and new biocontrol agents needs to be improved with genetic and genomic approaches. Although they have been underutilised in the past, application of genetic and genomic techniques is becoming more feasible from both technological and economic perspectives. We review current methods and provide a framework for using them. First, it is necessary to identify which biocontrol trait to select and in what direction. Next, the genes or markers linked to these traits need be determined, including how to implement this information into a selective breeding program. Choosing a trait can be assisted by modelling to account for the proper agro-ecological context, and by knowing which traits have sufficiently high heritability values. We provide guidelines for designing genomic strategies in biocontrol programs, which depend on the organism, budget, and desired objective. Genomic approaches start with genome sequencing and assembly. We provide a guide for deciding the most successful sequencing strategy for biocontrol agents. Gene discovery involves quantitative trait loci analyses, transcriptomic and proteomic studies, and gene editing. Improving biocontrol practices includes marker-assisted selection, genomic selection and microbiome manipulation of biocontrol agents, and monitoring for genetic variation during rearing and post-release. We conclude by identifying the most promising applications of genetic and genomic methods to improve biological control efficacy.

Identification of new viral variants specific to the honey bee mite Varroa destructor.

Large-scale colony losses among managed Western honey bees have become a serious threat to the beekeeping industry in the last decade. Multiple factors contribute to these losses, but the impact of Varroa destructor parasitism is by far the most important, along with the contribution of some pathogenic viruses vectored by the mite. So far, more than 20 viruses have been identified infecting the honey bee, most of them RNA viruses. They may be maintained either as covert infections or causing severe symptomatic infections, compromising the viability of the colony. In silico analysis of available transcriptomic data obtained from mites collected in the USA and Europe, as well as additional investigation with new samples collected locally, allowed the description of three RNA viruses, two of them variants of the previously described VDV-2 and VDV-3 and the other a new species reported here for the first time. Our results showed that these viruses were widespread among samples and that they were present in the mites as well as in the bees but with differences in the relative abundance and prevalence. However, we have obtained strong evidence showing that these three viruses were able to replicate in the mite, but not in the bee, suggesting that they are selectively infecting the mite. This opens the door to future applications that may help controlling the mite through biological control approaches.

Neonicotinoids in excretion product of phloem-feeding insects kill beneficial insects.

Pest control in agriculture is mainly based on the application of insecticides, which may impact nontarget beneficial organisms leading to undesirable ecological effects. Neonicotinoids are among the most widely used insecticides. However, they have important negative side effects, especially for pollinators and other beneficial insects feeding on nectar. Here, we identify a more accessible exposure route: Neonicotinoids reach and kill beneficial insects that feed on the most abundant carbohydrate source for insects in agroecosystems, honeydew. Honeydew is the excretion product of phloem-feeding hemipteran insects such as aphids, mealybugs, whiteflies, and psyllids. We allowed parasitic wasps and pollinating hoverflies to feed on honeydew from hemipterans feeding on trees treated with thiamethoxam or imidacloprid, the most commonly used neonicotinoids. LC-MS/MS analyses demonstrated that both neonicotinoids were present in honeydew. Honeydew with thiamethoxam was highly toxic to both species of beneficial insects, and honeydew with imidacloprid was moderately toxic to hoverflies. Collectively, our data provide strong evidence for honeydew as a route of insecticide exposure that may cause acute or chronic deleterious effects on nontarget organisms. This route should be considered in future environmental risk assessments of neonicotinoid applications.

Genetic variability and pyrethroid susceptibility of the parasitic honey bee mite Varroa destructor (Acari: Varroidae) in Iran.

The ectoparasitic honey bee mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is one of the major concerns for worldwide beekeeping. The use of synthetic pyrethroids for controlling the mite was among the most popular treatments until resistance evolved in the mid 1990's. In Iran, beekeepers are dealing with the parasite and they also used pyrethroids for controlling the mite for a long time. After the evolution of resistance to pyrethroids, they based mite control mostly on treatments with amitraz, organic acids and several management practices. Here we conducted a comprehensive characterization of V. destructor populations parasitizing Apis mellifera in Iran. We determined the genetic variability of mites collected from 28 localities distributed throughout the country. The haplotype of V. destructor was determined by PCR-RFLP, analyzing a fragment of the mitochondrial cox1 gene. It was found that only the Korean haplotype was present in samples from all localities. DNA fragments from cox1, atp6, cox3 and cytb mitochondrial genes were sequenced and the results showed that all samples were identical to the K1-1 or the K1-2 V. destructor haplotypes. Moreover, as it has been reported that resistance to pyrethroids in V. destructor is associated with mutations at position 925 of the voltage-gated sodium channel, a TaqMan®-based allelic discrimination assay was conducted to genotype the mites collected. The results showed that all the mites tested were homozygous for the wild-type allele and, therefore, susceptible to treatment with pyrethroids.

Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

Digestive proteases in bodies and faeces of the two-spotted spider mite, Tetranychus urticae.

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC-nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.

Predictive 3D modelling of the interactions of pyrethroids with the voltage-gated sodium channels of ticks and mites.

BACKGROUND: The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honeybee colonies. RESULTS: We applied in silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. CONCLUSIONS: Three-dimensional modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species.

An amino acid substitution (L925V) associated with resistance to pyrethroids in Varroa destructor.

The Varroa mite, Varroa destructor, is an important pest of honeybees and has played a prominent role in the decline in bee colony numbers over recent years. Although pyrethroids such as tau-fluvalinate and flumethrin can be highly effective in removing the mites from hives, their intensive use has led to many reports of resistance. To investigate the mechanism of resistance in UK Varroa samples, the transmembrane domain regions of the V. destructor voltage-gated sodium channel (the main target site for pyrethroids) were PCR amplified and sequenced from pyrethroid treated/untreated mites collected at several locations in Central/Southern England. A novel amino acid substitution, L925V, was identified that maps to a known hot spot for resistance within the domain IIS5 helix of the channel protein; a region that has also been proposed to form part of the pyrethroid binding site. Using a high throughput diagnostic assay capable of detecting the mutation in individual mites, the L925V substitution was found to correlate well with resistance, being present in all mites that had survived tau-fluvalinate treatment but in only 8 % of control, untreated samples. The potential for using this assay to detect and manage resistance in Varroa-infected hives is discussed.

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation.

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.

Prospects for the biological control of Tuta absoluta in tomatoes of the Mediterranean basin.

Since its detection in the Mediterranean basin at the end of 2006 and later in other European countries, the South American tomato pinworm, Tuta absoluta (Meyrick), has become a serious threat to tomato crops. In newly infested areas, it is especially problematic during the first years of its presence. Nevertheless, after 2-3 years, the incidence of T. absoluta has become less severe in certain areas. There are several factors contributing to this decline, such as the increase in growers' knowledge of pest behaviour and biology and the correct application of integrated pest control strategies. The impact of opportunistic native natural enemies (fortuitous biological control) should be considered as one of the key factors in this decline. In this review, available information on indigenous natural enemies is updated, and the current pest management approaches used against T. absoluta are addressed. Finally, future scenarios for biological control of this pest are discussed.