The genome-wide response of Dermatophagoides pteronyssinus to cystatin A, a peptidase inhibitor from human skin, sheds light on its digestive physiology and allergenicity.

The digestive physiology of house dust mites (HDMs) is particularly relevant for their allergenicity since many of their allergens participate in digestion and are excreted into faecal pellets, a main source of exposure for allergic subjects. To gain insight into the mite dietary digestion, the genome of the HDM Dermatophagoides pteronyssinus was screened for genes encoding peptidases (n = 320), glycosylases (n = 77), lipases and esterases (n = 320), peptidase inhibitors (n = 65) and allergen-related proteins (n = 52). Basal gene expression and transcriptional responses of mites to dietary cystatin A, a cysteine endopeptidase inhibitor with previously shown antinutritional effect on mites, were analysed by RNAseq. The ingestion of cystatin A resulted in significant regulation of different cysteine endopeptidase and glycosylase genes. One Der p 1-like and two cathepsin B-like cysteine endopeptidase genes of high basal expression were induced, which suggests their prominent role in proteolytic digestion together with major allergen Der p 1. A number of genes putatively participating in the interaction of mites with their microbiota and acquired by horizontal gene transfer were repressed, including genes encoding the peptidase Der p 38, two 1,3-beta-glucanases, a lysozyme and a GH19 chitinase. Finally, the disruption of mite digestion resulted in the regulation of up to 17 allergen and isoallergen genes. Altogether, our results shed light on the putative role of specific genes in digestion and illustrate the connection between the digestive physiology of HDM and allergy.

Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant Ceratitis capitata Phenotypes.

The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.

Monitoring Insect Resistance to Bt Maize in the European Union: Update, Challenges, and Future Prospects.

Transgenic maize producing the Cry1Ab toxin of Bacillus thuringiensis (Bt maize) was approved for cultivation in the European Union (EU) in 1998 to control the corn borers Sesamia nonagrioides (Lefèbvre) and Ostrinia nubilalis (Hübner). In the EU since then, Cry1Ab is the only Bt toxin produced by Bt maize and Spain is the only country where Bt maize has been planted every year. In 2021, about 100,000 hectares of Bt maize producing Cry1Ab were cultivated in the EU, with Spain accounting for 96% and Portugal 4% of this area. In both countries, Bt maize represented less than 25% of all maize planted in 2021, with a maximum regional adoption of 64% Bt maize in northeastern Spain. Insect resistance management based on the high-dose/refuge strategy has been implemented in the EU since 1998. This has been accompanied by monitoring to enable early detection of resistance. The monitoring data from laboratory bioassays show no decrease in susceptibility to Cry1Ab had occurred in either pest as of 2021. Also, control failures have not been reported, confirming that Bt maize producing Cry1Ab remains effective against both pests. Conditions in the EU preventing approval of new genetically modified crops, including maize producing two or more Bt toxins targeting corn borers, may limit the future effectiveness of resistance management strategies.

Expanding the Medfly Virome: Viral Diversity, Prevalence, and sRNA Profiling in Mass-Reared and Field-Derived Medflies.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly's fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.

Current situation and forecasting of resistance evolution to lambda-cyhalothrin in Spanish medfly populations.

BACKGROUND: The control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) in Spanish field populations mainly relies on the insecticides lambda-cyhalothrin and spinosad as bait sprays. However, their sustainable used is compromised by the development of lambda-cyhalothrin resistance and the detection of spinosad resistant alleles. In addition, the use of lure-and-kill traps covered with deltamethrin has increased in the last years. It is thus urgent to predict the impact that the combination of both pyrethroids will have in the evolution of lambda-cyhalothrin resistance and how they could be combined with spinosad so as to establish proper resistance management programs. RESULTS: Toxicity bioassays were performed to analyze the current levels of lambda-cyhalothrin resistance in field populations, proving that it has remained stable in the last decade. An evolutionary model was established to explore the weight of selected parameters in the evolution of lambda-cyhalothrin resistance in C. capitata and to forecast resistance development under different resistance management scenarios. Our results highlight the importance of fitness cost and inheritance to fit the experimental results. The analyses predicted that the rotation of lambda-cyhalothrin and spinosad, when deltamethrin traps are also deployed in the field, will slow down the evolution of resistance, especially when cross-resistance between both pyrethroids is considered. CONCLUSION: Lambda-cyhalothrin resistance has not increased in the last decade, probably due to the alternation of this insecticide with spinosad. Our modelling results indicate that the best option to avoid an increase in lambda-cyhalothrin resistant alleles, considering that deltamethrin use is growing, would be to continue combining their use with spinosad. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Contribution of cysteine and serine proteases to proteolytic digestion in an allergy-eliciting house dust mite.

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.

Early Molecular Responses of Tomato to Combined Moderate Water Stress and Tomato Red Spider Mite Tetranychus evansi Attack.

Interaction between plants and their environment is changing as a consequence of the climate change and global warming, increasing the performance and dispersal of some pest species which become invasive species. Tetranychus evansi also known as the tomato red spider mite, is an invasive species which has been reported to increase its performance when feeding in the tomato cultivar Moneymaker (MM) under water deficit conditions. In order to clarify the underlying molecular events involved, we examined early plant molecular changes occurring on MM during T. evansi infestation alone or in combination with moderate drought stress. Hormonal profiling of MM plants showed an increase in abscisic acid (ABA) levels in drought-stressed plants while salicylic acid (SA) levels were higher in drought-stressed plants infested with T. evansi, indicating that SA is involved in the regulation of plant responses to this stress combination. Changes in the expression of ABA-dependent DREB2, NCED1, and RAB18 genes confirmed the presence of drought-dependent molecular responses in tomato plants and indicated that these responses could be modulated by the tomato red spider mite. Tomato metabolic profiling identified 42 differentially altered compounds produced by T. evansi attack, moderate drought stress, and/or their combination, reinforcing the idea of putative manipulation of tomato plant responses by tomato red spider mite. Altogether, these results indicate that the tomato red spider mite acts modulating plant responses to moderate drought stress by interfering with the ABA and SA hormonal responses, providing new insights into the early events occurring on plant biotic and abiotic stress interaction.

Inheritance, Fitness Cost, and Management of Lambda-Cyhalothrin Resistance in a Laboratory-Selected Strain of Ceratitis capitata (Wiedemann).

The management of the medfly, Ceratitis capitata, in Spanish citrus crops relies mainly on the use of insecticides and the release of sterile males. However, the development of resistance to different insecticides in field populations, including lambda-cyhalothrin, implies a threat for the sustainable control of this pest. The inheritance, fitness cost, and management of lambda-cyhalothrin resistance were examined in the laboratory-selected W-1Kλ strain. We have demonstrated that lambda-cyhalothrin resistance in W-1Kλ is autosomic, completely dominant, and polygenic. In addition, individuals from W-1Kλ showed a lower embryo to pupal viability, a slower developmental time from egg to pupae, and an increase in adults' weight and longevity. We did not find significant trade-offs in the activity of digestive hydrolytic enzymes, with the exception of higher α-amylase activity in W-1Kλ females. A comparative study with different insecticide treatment strategies showed that lambda-cyhalothrin resistance increased when several consecutive treatments with this insecticide were applied. However, the alternation of this insecticide with spinosad was enough to delay the development of resistance. Our results indicate that the rotation of lambda-cyhalothrin with spinosad-a practice already used in some fields-may contribute to prevent the development of resistance.

Field detection and predicted evolution of spinosad resistance in Ceratitis capitata.

BACKGROUND: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6). RESULTS: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad-resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented. CONCLUSION: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multiple mutations in the nicotinic acetylcholine receptor Ccα6 gene associated with resistance to spinosad in medfly.

Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata. Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα63aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα63aAG>AT containing a point mutation in the 5' splicing site of exon 3a (3aAG > AT); and (iii) Ccα63aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

Plant-Mediated Effects of Water Deficit on the Performance of Tetranychus evansi on Tomato Drought-Adapted Accessions.

Climate change is expected to increase drought periods and the performance and dispersal of some invasive species such as Tetranychus evansi, which has been reported to take advantage of the nutritional changes induced by water-shortage on the tomato cultivar Moneymaker (MM). We have examined the implications for mite's biology of four accessions of the drought-adapted tomatoes, "Tomàtiga de Ramellet" (TR), under moderate drought stress. Mite performance was enhanced by drought in two accessions (TR61 and TR154), but not in the other two accessions (TR58 and TR126). We selected one accession of each outcome (i.e., TR154 and TR126) to further analyze plant nutritional parameters. We found that free sugars and most essential amino acids for mites were induced by drought and/or mite infestation on MM and TR154 plants, whereas sugars were not altered and a reduced number of essential amino acids were induced by drought in TR126. Remarkably, mite performance was enhanced by leaf infiltration of free sugars, essential amino acids mixture, and L-proline on well-watered MM and by free sugars on drought-stressed TR126 plants. These results indicate a positive link between the induction of soluble carbohydrates and amino acids used by the plant for osmotic adjustment and mite performance. The effects of drought and/or mite infestation on the defense response of plants was analyzed at three levels: phytohormone accumulation, the transcript levels of marker genes linked to jasmonates (JAs), salicylic acid (SA), and abscisic acid (ABA) pathways, and the activity of defense proteins. The ability of T. evansi to downregulate the accumulation of defense-related phytohormones was noted on MM and the two TR accessions analyzed (TR126 and TR154), though differences in the induction of protein defense genes and activities by drought and/or mite infestation were observed among them. These results emphasize the importance of studying plant biotic and abiotic stress factors in combination and provides an experimental framework for screening drought-tolerant tomato accessions that will be also resistant to herbivore mites.

Weeds and ground-dwelling predators' response to two different weed management systems in glyphosate-tolerant cotton: A farm-scale study.

The use of glyphosate, as a post-emergence broad-spectrum herbicide in genetically modified glyphosate-tolerant (GT) cotton, supposes a big change in weed management programs with respect to a conventional regime. Thus, alterations in arable flora and arthropod fauna must be considered when evaluating their potential impacts. A 3-year farm-scale study was conducted in a 2-ha GT cotton crop, in southern Spain, to compare the effects of conventional and glyphosate herbicide regimes on weed abundance and diversity and their consequences for ground-dwelling predators. Surveys reveal that weed density was relatively low within all treatments with a few dominant species, with significantly higher weed densities and modifications of the floristic composition in glyphosate-treated plots that led to an increase in the abundance of Portulaca oleracea and to a reduction in plant diversity. The activity-density of the main predatory arthropod taxa (spiders, ground beetles, rove beetles and earwigs) varied among years, but no significant differences were obtained between conventional and glyphosate herbicide regimes. However, significant differences between treatments were obtained for ground beetles species richness and diversity, being higher under the glyphosate herbicide regime, and a positive correlation with weed density could be established for both parameters. The implications of these findings to weed control in GT cotton are discussed.

Monitoring of Sesamia nonagrioides resistance to MON 810 maize in the European Union: lessons from a long-term harmonized plan.

BACKGROUND: Use of MON 810 maize (Zea mays), which expresses the insecticidal protein Cry1Ab from Bacillus thuringiensis (Bt maize), is a highly effective method to control Sesamia nonagrioides (Lefèbvre), a key maize pest in Mediterranean countries. Monitoring programs to assess the potential development of resistance of target pests to Bt maize are mandatory in the European Union (EU). Here we report the results of the S. nonagrioides resistance monitoring plan implemented for MON 810 maize in the EU between 2004 and 2015 and reassess the different components of this long-term harmonized plan. RESULTS: No major shifts in the susceptibility of S. nonagrioides to the Cry1Ab protein have occurred over time. The reassessment of this long-term program has identified some practical and technical constraints, allowing us to provide specific recommendations for improvement: use reference strains instead of susceptibility baselines as comparators for field-collected populations; shift from dose-response bioassays to diagnostic concentrations; and focus monitoring on areas with high adoption rates, such as the Ebro basin in Spain. CONCLUSION: There are no signs of field resistance of S. nonagrioides to the Cry1Ab protein of MON 810 maize. Specific recommendations for improvement are provided, based on the knowledge and experience accumulated through the implementation of this unique EU-wide harmonized plan. © 2017 Society of Chemical Industry.

Farm-scale evaluation of the impact of Cry1Ab Bt maize on canopy nontarget arthropods: a 3-year study.

The cultivation of Cry1Ab-expressing genetically modified MON810 (Bt maize) has led to public concern in Europe, regarding its impact on nontarget arthropods (NTAs). We have assessed the potential effects of DKC 6451 YG (MON810) maize on canopy NTAs in a farm-scale study performed in Central Spain during 3 years. The study focused on hemipteran herbivores (leafhoppers and planthoppers) and hymenopteran parasitic wasps (mymarids) collected by yellow sticky traps, which accounted for 72% of the total number of insects studied. The dynamics and abundance of these groups varied among years, but no significant differences were found between Bt and non-Bt maize, indicating that Bt maize had no negative effect on these taxa. Nonetheless, the Cry1Ab toxin was detected in 2 different arthropods collected from Bt maize foliage, the cicadellids Zyginidia scutellaris and Empoasca spp. A retrospective power analysis on the arthropod abundance data for our field trials has determined that Z. scutellaris and the family Mymaridae have high capacity to detect differences between the Bt maize and its isogenic counterpart. The use of these canopy NTAs as surrogates for assessing environmental impacts of Bt maize is discussed.

Drought stress promotes the colonization success of a herbivorous mite that manipulates plant defenses.

Climate change is expected to bring longer periods of drought and this may affect the plant's ability to resist pests. We assessed if water deficit affects the tomato russet mite (TRM; Aculops lycopersici), a key tomato-pest. TRM thrives on tomato by suppressing the plant's jamonate defenses while these defenses typically are modulated by drought stress. We observed that the TRM population grows faster and causes more damage on drought-stressed plants. To explain this observation we measured several nutrients, phytohormones, defense-gene expression and the activity of defensive proteins in plants with or without drought stress or TRM. TRM increased the levels of total protein and several free amino acids. It also promoted the SA-response and upregulated the accumulation of jasmonates but down-regulated the downstream marker genes while promoting the activity of cysteine-but not serine-protease inhibitors, polyphenol oxidase and of peroxidase (POD). Drought stress, in turn, retained the down regulation of JA-marker genes and reduced the activity of serine protease inhibitors and POD, and altered the levels of some free-amino acids. When combined, drought stress antagonized the accumulation of POD and JA by TRM and synergized accumulation of free sugars and SA. Our data show that drought stress interacts with pest-induced primary and secondary metabolic changes and promotes pest performance.

MATI, a Novel Protein Involved in the Regulation of Herbivore-Associated Signaling Pathways.

The defense response of the plants against herbivores relies on a complex network of interconnected signaling pathways. In this work, we characterized a new key player in the response of Arabidopsis against the two-spotted spider mite Tetranychus urticae, the MATI (Mite Attack Triggered Immunity) gene. This gene was differentially induced in resistant Bla-2 strain relative to susceptible Kon Arabidopsis accessions after mite attack, suggesting a potential role in the control of spider mites. To study the MATI gene function, it has been performed a deep molecular characterization of the gene combined with feeding bioassays using modified Arabidopsis lines and phytophagous arthropods. The MATI gene belongs to a new gene family that had not been previously characterized. Biotic assays showed that it confers a high tolerance not only to T. urticae, but also to the chewing lepidopteran Spodoptera exigua. Biochemical analyses suggest that MATI encodes a protein involved in the accumulation of reducing agents upon herbivore attack to control plant redox homeostasis avoiding oxidative damage and cell death. Besides, molecular analyses demonstrated that MATI is involved in the modulation of different hormonal signaling pathways, affecting the expression of genes involved in biosynthesis and signaling of the jasmonic acid and salicylic acid hormones. The fact that MATI is also involved in defense through the modulation of the levels of photosynthetic pigments highlights the potential of MATI proteins to be exploited as biotechnological tools for pest control.