Carbohydrate-active enzymes (CAZymes) are proteins that assemble, remodel, and depolymerize complex carbohydrates. They are ubiquitous across the tree of life, underpinning energy capture, cell-wall architecture, microbiome interactions, and host-pathogen dynamics. CAZymes are modular in structure and function, being tightly related to the overall enzyme architecture. Domain architecture constrains folding, substrate range, and integration into metabolic pathways. Arthropods, the most species-rich animal phylum, offer a powerful system to study enzymatic modularity because their enzymatic machineries must function in extremely diverse ranges of diet, niche, and abiotic conditions. Here we built a genome-scale framework to characterize CAZyme modular organization across Arthropoda and to test how domain architecture is influenced by diets, life histories and clades while revealing emergent functional relationships. We find that unimodular architectures dominate CAZyme repertoires, while multimodular and class-exclusive arrangements remain rare but highly variable. We identify herbivory as responsible for the discrimination on three CAZyme structural strategies (saprophagous, leaf-stem-root, and pollen-nectar feeders) differing in class composition, modular complexity, and catalytic emphasis. Correlation and co-occurrence analyses at genomic and protein levels show that expansions of glycosyltransferases, glycoside hydrolases, carbohydrate-binding modules, and auxiliary activities are tightly coupled, especially in myriapods and crustaceans, and that intra-protein networks of CAZyme families are denser in hexapods, holometabolans, and herbivores. Within these networks, GT2 and hydrolytic domains occupy central positions in co-occurrence clusters. Together, our findings indicate that arthropod CAZymes evolve as integrated macromolecular systems whose modularity and class balance track ecological demands, providing a scalable framework for engineering CAZyme networks for biotechnological and pest-management applications.

BACKGROUND:Malathion resistance in the Mediterranean fruit fly, Ceratitis capitata, is associated with: (i) the mutation G328A in the target acetylcholinesterase gene (Ccace2) (R allele); and (ii) a heterogeneous duplication of the Ccace2 gene (RS haplotype, one of the copies bearing the mutation G328A and the other copy nonmutated). RESULTS:Bioassays with the resistant (R/R and RS/RS) and susceptible (S/S) genotypes, F1 crosses and back-crosses indicated that resistance was inherited in both cases as a semi-dominant trait. We have also demonstrated that the R allele imposes a higher fitness cost than RS haplotype by three different lines of evidence: (i) individuals with genotype R/R had a reduced performance compared to RS/RS and S/S individuals for some biological traits; (ii) the stability of the RS haplotype was higher than that of the R allele under laboratory conditions in the absence of insecticide selection; and (iii) the frequency of the R allele declined in field populations, when assessed in the same seven localities in the Comunitat Valenciana (Spain) at 1, 4 and 6 years after malathion withdrawal, whereas the RS haplotype maintained its frequency during the same period. CONCLUSION:An evolutionary model supported that the fitness cost of the different genotypes in field populations was higher for those containing the R allele than for those containing the RS haplotype, and was used to explore which resistance management strategies should be implemented if malathion or other insecticides showing cross-resistance were authorized in the future. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Laboratory assessment of environmental stressors on wild bumblebees typically rely on workers from laboratory colonies founded by wild-collected queens. In Spain, where Bombus terrestris lusitanicus and Bombus terrestris terrestris coexist, accurately identifying the subspecies of wild individuals used in these experiments remains challenging. Since the 1990s, commercial colonies of B. t. terrestris have been widely used in Spain for greenhouse pollination, and previous studies have documented the escape and naturalisation of individuals in areas where B. t. lusitanicus is native. The present study integrates three methodologies—morphological traits, mitochondrial 16S rRNA gene, and microsatellites—to achieve accurate colony-level identification, enabling the differentiation of the two subspecies and their hybrids. Laboratory colonies were established with wild queens from Almería (southeastern Spain; high greenhouse density) and Madrid (central Spain; minimal greenhouse infrastructure), allowing the analysis of both queens and offspring. This approach allowed to determine colony subspecies composition for studies involving the use of wild individuals. Overall, our findings show widespread hybridization between both subspecies, with higher levels observed in areas of high greenhouse density, which may be associated with commercial use. Furthermore, the inclusion of commercial and historical specimens showed that introgression among wild bumblebees predated commercial colony use, though hybridization is currently more pronounced.

In plants, jasmonic acid (JA) is traditionally seen as the key regulator of plant defense against spider mites although indirect evidence suggests that abscisic acid (ABA) might also be involved. This study examined this possibility using an ABA-deficient tomato genotype (notabilis) and the specialist T. evansi which is known to suppress JA-dependent defenses. Wild type cv. Lukullus showed enhanced leaf damage than notabilis, showing that ABA influences susceptibility to specialist spider mites potentially modulating jasmonate levels and dependent defenses. Among the upregulated genes, cv. Lukullus displayed GO enrichment in JA-dependent defense, regulation of ROS production, SA-biosynthesis and the negative regulation of defense whereas in notabilis, these mechanisms were less prominent and all GO terms related to photosynthesis were severely repressed, suggesting that these could be crucial in restricting progression of T. evansi infestation in tomato. At the metabolic level, reduced susceptibility to T. evansi in notabilis accompanied an increased accumulation of free fatty acids and phenylpropanoids mirrored by enhanced gene expression of lipid, phenylpropanoid and lignin metabolic genes as well as genes related to ABA/dehydration and JA signaling. A gene co-expression analysis identified modules correlated with ABA and SA levels which included defense and photosynthesis responses among the up- and downregulated genes, respectively, suggesting their potential crosstalk in the regulation of tomato responses to T. evansi infestation. This extent was further investigated with feeding assays in tomato mutants spr2 (JA-deficient) and NahG (SA-deficient) with T. evansi confirming their role as positive and negative regulators of susceptibility to spider mites, respectively.

Bumblebees are important pollinators widely used to meet crop pollination demands worldwide. In such cropping systems, the use of different active ingredients for pest control may result in pollinator exposure to a variety of pesticide mixtures. Traditional risk assessment has focused on single-pesticide toxicity to honeybees. However, it is now known that responses vary among bee species, and that interactions between pesticide mixtures can occur, highlighting the need to identify pesticide combinations that are safer for bees. To this end, we evaluated the acute toxicity of three pesticide mixtures -consisting on the neonicotinoid thiacloprid in combination with three different SBI fungicides- on Bombus terrestris bumblebees. Subsequently, their transcriptomic response to dietary exposure to thiacloprid and tebuconazole both individual and in combination was evaluated. Our experiments revealed synergistic toxic effects in bumblebee mortality, as well as on their gene expression profiles. Particularly, we found that the expanded CYP6AQ subfamily of P450 detoxification enzymes changed its expression in response to fungicide treatment. Moreover, recombinant expression of the members of this enzyme subfamily revealed that at least three of them interact in vitro with thiacloprid and that their activity was differentially inhibited depending on the SBI fungicide tested. Finally, the metabolism of thiacloprid by recombinantly expressed P450s was assessed, revealing that CYP6AQ26 generates 4-hydroxy-thiacloprid and six additional tentative thiacloprid metabolites, while CYP6AQ27 and CYP6AQ28 also contribute to its metabolism, but the specific mechanisms remain unresolved. Our findings provide evidence of the different molecular interactions and regulatory responses underlying multiple-pesticide exposure in bees.

BACKGROUND:House dust mites (HDMs) such as Dermatophagoides pteronyssinus are major allergy elicitors worldwide, yet their gene expression across developmental stages remains underexplored. Herein, we report a comprehensive RNAseq analysis of larvae, nymphs, and adult males and females, mapped to a recently published high-quality genome with extended functional annotations. RESULTS:Analysis of differentially expressed genes (DEG) revealed that female-biased expression was the most prevalent profile (16% of genes), while males exhibited the highest fold-change differences. DEG data, combined with network clustering and functional enrichment analysis, highlighted distinct genes and biological processes for each stage and sex: females showed upregulation of genes related to cell division and oogenesis, with vitellogenins among the most abundant transcripts; males exhibited increased expression of genes encoding putative seminal fluid proteins (e.g. endopeptidases, serpins, antimicrobial peptides), and those involved in reproductive regulation (e.g. testis-specific serine kinases); while juveniles displayed enhanced expression of genes related to energy metabolism and growth. Further analysis of endocrine pathways revealed non-canonic mechanisms compared to insect models, particularly in ecdysteroid and sesquiterpenoid biosynthesis and regulation. Expression patterns in genes involved in cuticle formation were also identified, reflecting their role in developmental transitions and sexual differentiation. Allergen and allergen-related gene expression showed an overall increase in feeding juveniles, as well as sex-biased expression, with Der p 27 upregulated in females. These findings provide insight into the physiological roles of allergens in digestion, immunity, and muscle formation, among other functions. Additionally, seven new horizontally transferred genes, including a DNA-repair photolyase linked to females, and novel multigene families (e.g. 119 male-specific beta-propeller proteins, 70 hypothetical cuticular proteins, 23 tetraspanin-like proteins, 5 female-associated putative odorant-binding proteins) were identified. CONCLUSIONS:This study provides the first genome-wide transcriptomic analysis of a HDM across life stages and sexes, expanding our understanding of the molecular mechanisms underlying mite development, sexual reproduction, and allergen expression. The generated data, fully available via supplementary spreadsheet and the ORCAE online platform, provide a valuable foundation for future allergy research and the development of new mite control strategies.

BACKGROUND:Field resistance to malathion was reported for Mediterranean fruit fly (medfly), Ceratitis capitata, populations collected in Spain in 2004 and 2005, when medfly control mainly relied on malathion bait sprays. The mutation G328A in the acetylcholinesterase (AChE) gene (Ccace2) was then identified as the main resistance mechanism in a field-derived resistant strain. However, outdoor plant protection products containing malathion were withdrawn from the European Union in 2009 and other insecticides gained importance, such as spinosad and pyrethroids, though other organophosphates were occasionally used for medfly control for a few years. RESULTS:We have: (i) provided evidences of a novel malathion resistant mechanism in Ceratitis capitata, mediated by a heterogeneous duplication of the Ccace2 gene (RS haplotype, one of the copies bearing the mutation G328A and the other copy non-mutated); (ii) found that individuals bearing the G328A mutation (R allele) and/or the RS haplotype were widely distributed in Spanish medfly populations during the years that malathion was used; and (iii) showed that malathion resistance reverted in field populations when analysed 8-13 years after malathion was withdrawn, but the frequencies of the genotypes containing the RS haplotype remained stable (RS/RS) or declined less (S/RS) than those containing the R allele (R/R, R/S, R/RS). CONCLUSION:This represents a scenario where the R allele and the RS haplotype are present in the field at low frequencies, but resistance may rapidly evolve if malathion or other organophosphates were used in the absence of appropriate management resistance strategies. © 2025 Society of Chemical Industry.

Background: Arthropods represent the largest and most diverse phylum on Earth, playing a pivotal role in the biosphere. One key to their evolutionary success is their ability to feed on plant material. However, their endogenous enzymatic repertoire, which contributes to plant digestion, remains largely unexplored and poorly understood. Results: We analyzed 815 arthropod proteomes and identified a total of 268,171 carbohydrate-active modules. Our findings revealed a strong correlation between enzymatic content and feeding habits, with herbivorous species possessing significantly higher enzyme levels. We identified widespread carbohydrate-active families across the AA, CBM, GH, and GT classes, and observed a progressive increase in taxa-exclusive families in more recent arthropod lineages. Notably, we highlighted the impact of the transition from ametabolous to holometabolous development on carbohydrate metabolism, as well as the ecological adaptations of different species groups. By reconstructing the ancestral enzymatic profiles of arthropods, we identified significant fluctuations in 10 carbohydrate-active families over time. Conclusions: Our analysis advances the understanding of the evolutionary mechanisms utilized by the megadiverse phylum Arthropoda. We emphasize the critical role of herbivory as a selective force shaping enzymatic strategies, particularly those involved in carbohydrate metabolism. The distribution and exclusivity of carbohydrate-active families across different arthropod groups provide insights into their evolutionary trajectories and offer a clearer picture of the metabolic pathways that led their ancestors to their present forms.

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.

The control of the Mediterranean fruit fly (Medfly), Ceratitis capitata , in citrus orchards in Spain is mainly based in three insecticides (spinosad, lambda-cyhalothrin and deltamethrin) and the liberation of sterile males. However, Medfly control is compromised by the development of lambda-cyhalothrin resistance and the detection of spinosad-resistant alleles in field populations. We report here, for the first time, resistance to deltamethrin in populations collected in fields under different management strategies, including MagnetMed™ traps coated with this insecticide and/or spinosad and lambda-cyhalothrin used as bait sprays, and even in populations obtained from non-treated fields. Two deltamethrin-resistant strains (BP-delta and Rfg-delta) were generated from the descendants of some of the field populations that showed lower susceptibility to deltamethrin. Both strains showed low susceptibility to MagnetMed™ traps, moderate susceptibility to Ceratipack traps, and lacked cross-resistance to spinosad and lambda cyhalothrin. Our data suggest that deltamethrin resistance was mediated by P450 enzymes, since bioassays with synergists showed that PBO reverted resistance in a field population and the laboratory strains, whereas the effect of DEF and DEM was minor and no mutations were found in the VGSC gene. The inheritance of resistance for both strains was completely recessive, autosomic and did not fit the mortality expected for a recessive character under a monogenic or digenic model. We also found that deltamethrin resistance presented a fitness cost in terms of males’ weight, males’ and females’ longevity and lifetime fecundity, with a more pronounced effect in the BP-strain than in the Rfg-delta strain. Our results highlight the need to implement insecticide resistance management strategies to prevent control failures.

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.

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.

EDITORIAL article Front. Plant Sci., 12 January 2022Sec. Plant Pathogen Interactions Volume 12 - 2021 | https://doi.org/10.3389/fpls.2021.773439

AbstractBACKGROUNDThe 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.RESULTSToxicity 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.CONCLUSIONLambda‐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.

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.

The sterile insect technique (SIT) is widely used in integrated pest management programs for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata . The genetic interactions between the released individuals from the genetic sexing strains (GSS), used for SIT applications worldwide, and wild individuals have not been studied. Under the hypothesis that a number of Vienna GSS individuals released to the field might not be completely sterile and may produce viable offspring, we have analyzed medfly Spanish field populations to evaluate the presence of Vienna strain genetic markers. To this goal, we have used contrasted nuclear and mitochondrial genetic markers, and two novel sets of nuclear polymorphisms with the potential to be markers to discriminate between Vienna and wild individuals. Nuclear Vienna markers located on the 5th chromosome of Vienna males have been found in 2.2% (19 from 875) of the Spanish wild medfly females captured at the area where SIT is applied. In addition, a female-inherited mitochondrial Vienna marker has been found in two from the 19 females showing nuclear Vienna markers. The detection of several of these markers in single individuals represents evidence of the introgression of Vienna strain into natural populations. However, alternative explanations as their presence at low frequency in wild populations in the studied areas cannot be fully discarded. The undesired release of non-fully sterile irradiated GSS individuals into the field and their interactions with wild flies, and the potential environmental implications should be taken into account in the application of the SIT.

Centro de Investigaciones Biologicas Margarita Salas, CSIC, Madrid, Spain, 2 KeyGene N.V., Wageningen, Netherlands, Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Japan, Department of Agricultural Development, Faculty of Agricultural Sciences and Forestry, Democritus University of Thrace, Komotini, Greece, Department of Plants and Crops, Ghent University, Ghent, Belgium, Centro de Biotecnología y

EDITORIAL article Front. Plant Sci., 11 August 2021Sec. Plant Pathogen Interactions https://doi.org/10.3389/fpls.2021.730290

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.

Abstract 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.