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The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli . The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

The DNA methyltransferase family contains a conserved set of DNA-modifying enzymatic proteins. They are responsible for epigenetic gene modulation, such as transcriptional silencing, transcription activation, and post-transcriptional modulation. Recent research has revealed that the canonical DNA methyltransferases (DNMTs) biological roles go beyond their traditional functions of establishing and maintaining DNA methylation patterns. Although a complete DNA methylation toolkit is absent in most insect orders, recent evidence indicates the de novo DNA methylation and maintenance function remain conserved. Studies using various molecular approaches provided evidence that DNMTs are multi-functional proteins. However, still in-depth studies on their biological role lack due to the least studied area in insects. Here, we review the DNA methylation toolkit of insects, focusing on recent research on various insect orders, which exhibit DNA methylation at different levels, and for which DNMTs functional studies have become available in recent years. We survey research on the potential roles of DNMTs in the regulation of gene transcription in insect species. DNMTs participate in different physiological processes by interacting with other epigenetic factors. Future studies on insect's DNMTs will benefit to understand developmental processes, responses to various stimuli, and adaptability of insects to different environmental conditions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glycosyltransferases (GTs) catalyse the reaction of glyco-conjugation of various biomolecules by transferring the saccharide moieties from an activated nucleotide sugar to nucleophilic glycosyl acceptor. In insects, GTs show diverse temporal and site-specific expression patterns and thus play significant roles in forming the complex biomolecular structures that are necessary for insect survival, growth and development. Several insects exhibit GT-mediated detoxification as a key defence strategy against plant allelochemicals and xenobiotic compounds, as well as a mechanism for pesticide cross-resistance. Also, these enzymes act as crucial effectors and modulators in various developmental processes of insects such as eye development, UV shielding, cuticle formation, epithelial development and other specialized functions. Furthermore, many of the known insect GTs have been shown to play a fundamental role in other physiological processes like body pigmentation, cuticular tanning, chemosensation and stress response. This review provides a detailed overview of the multifaceted functionality of insect GTs and summarizes numerous case studies associated with it., (© 2020 The Royal Entomological Society.)

Mycotoxins are secondary metabolites produced primarily by filamentous fungi that when consumed cause pathological responses in animal hosts or consumers. Defined functionally rather than structurally, mycotoxins derive from numerous primary metabolic pathways. Through opportunistic or mutualistic associations, insect herbivores inflict damage that can predispose plants to infection by mycotoxin-producing phytopathogens, resulting in economically significant contamination. The few cytochrome P450 subfamilies implicated in mycotoxin detoxification by insects, including CYP6 and CYP9, are also known to detoxify phytochemicals. Some insect P450s bioactivate, rather than detoxify, mycotoxins, suggestive of an 'escalation' in arms-race interactions between these herbivores and fungi. Characterizing insect P450s that detoxify mycotoxins can be useful for developing biological remediation technologies and for ensuring the safety of insects reared for human or livestock consumption., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Insect cytochrome P450-monooxygenases (P450s) are an enzyme superfamily involved in the oxidative transformation of endogenous and exogenous substrates, including insecticides. They were also shown to determine insecticide selectivity in beneficial arthropods such as bee pollinators, and to detoxify plant secondary metabolites. The recent explosion in numbers of P450s due to increased invertebrate genomes sequenced, allowed researchers to study their functional relevance for xenobiotic metabolism by recombinant expression using different expression systems. Troubleshooting strategies, including different systems and protein modifications typically adapted from mammalian P450s, have been applied to improve the functional expression, with partial success. The aim of this mini review is to critically summarize different strategies recently developed and used to produce recombinant insect P450s for xenobiotic metabolism studies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Enzymes in the cytochrome P450 (P450) superfamily have important functions ranging from those that are essential for the physiology and development of the individual to those that mediate interactions between individuals and their biotic environment. Until recently the study of P450s had focused on single functions, substrates, or pathways. Recent advances in sequencing, genome assembly, and phylogenetic methods have returned emphasis to the adaptive value of these enzymes in the context of herbivory. Comparisons of whole repertoires of P450s across related species reveal that P450s capable of metabolizing xenobiotics have an increased rate of gains compared to losses after gene duplications. In plants, studies have focused on enzymes and end-functions that have converged to provide increased resistance to herbivory. This review summarizes the latest findings related to the ecological value of P450s in the interactions between phytophagous insects and their host plants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insect herbivores use phytochemicals as signals to induce expression of their phytochemical-detoxifying cytochrome P450 monooxygenases (P450s). The regulatory cascades that transduce phytochemical signals to enhanced expression of P450s are the focus of this review. At least seven signaling pathways, including RTK/MAPK, GPCR/CREB, GPCR/NFκB, ROS/CncC/Keap1, AhR/ARNT, cytosol NR, and nucleus-located NR, may be involved in phytochemical induction of P450s. Constitutive overexpression, overphosphorylation, and/or activation of one or more effectors in the corresponding pathway are common causes of P450 overexpression that lead to phytochemical or insecticide resistance. Future research should pay more attentions to the starting point of each pathway, the number of pathways and their cross talk for a given phytochemical, and the pathways for downregulation of P450s., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insect cytochrome P450 monooxygenases (P450s) are well known to be involved in metabolic detoxification of xenobiotics, such as phytochemicals, insecticides and environmental pollutants. Enhanced metabolic detoxification is closely associated with the constitutive overexpression and induction of P450s. In general, multiple insect P450s are co-responsible for xenobiotic detoxification. Considering the capacity of P450s to respond to a wide range of xenobiotics, synergistic interactions between natural and synthetic xenobiotics and P450-mediated cross-tolerance/resistance are ubiquitous. Recent studies have indicated that both transcription factors and signaling pathways are involved in the regulation of P450 genes in xenobiotic responses. This article reviews our current understanding of P450-mediated detoxification in insect adaptation to xenobiotics and highlights recent progress in the molecular basis of P450 regulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cytochrome P450 monooxygenases (P450s) play a key role in the detoxification of phytochemicals in arthropod herbivores. We present here an overview of recent progress in understanding the breadth and specificity of gene expression plasticity of P450s in response to phytochemicals. We discuss experimental setups and new findings in mechanisms of P450 regulation. Whole genome transcriptomic analysis of arthropod herbivores, either after direct administration of phytochemicals or after host plant shifts, allowed to integrate various levels of chemical complexity and lead to the unbiased identification of responsive P450 genes. However, despite progress in identification of inducible P450s, the link between induction and metabolism is still largely unexplored, and to what extent the overall response is biologically functional should be further investigated. In the near future, such studies will be more straightforward as forward and reverse genetic tools become more readily available., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Modifications to DNA and core histones influence chromatin organization and expression of the genome. DNA methylation plays a significant role in the regulation of multiple biological processes that regulate behavior and caste differentiation in social insects. Histone modifications play significant roles in the regulation of development and reproduction in other insects. Genes coding for acetyltransferases, deacetylases, methyltransferases, and demethylases that modify core histones have been identified in genomes of multiple insects. Studies on the function and mechanisms of action of some of these enzymes uncovered their contribution to post-embryonic development. The results from studies on epigenetic modifiers could help in the identification of inhibitors of epigenetic modifiers that could be developed to control pests and disease vectors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: α-Amylase inhibitors (α-AIs) belong to the discrete classes, and exhibited differential specificities against α-amylases from various sources. Several α-amylases and their complexes with inhibitors at the molecular level have been studied in detail. Interestingly, some α-AIs depict specific and selective interactions amid different insect α-amylases., Scope of Review: There are studies to understand evolutionary variability and functional differentiation of insect α-amylases and their cognate inhibitors. We have examined sequence, structural, and interaction diversity between various α-amylases and α-AIs. Based on these analyses, we are providing a potential basis for the functional differentiation among certain insect α-amylases concerning mammalian counterparts and their interactions with different proteinaceous α-AIs., Major Conclusions: Insect α-amylases have conserved domain architecture with differences in length, number of disulfide bonds, and secondary structure. Furthermore, few of them exhibit variable characteristics like chloride dependent activity, the presence of N-terminal glutamine residue to protect against proteolytic degradation, and loop variations near the enzyme active site. Conformation of α-AI protein could be an essential factor for their specificity and binding affinities towards target α-amylase(s). Furthermore, variation into the enzyme binding pocket residues might contribute to differential interactions with inhibitors., General Significance: Molecular insights in the interactions between insect α-amylases and plant α-AI will provide the details of mechanisms assisting the inhibitor specificity. Furthermore, this information will help to design potent and effective α-AIs against specific α-amylase., Competing Interests: Declaration of Competing Interest All the authors declared no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The main objective of this article was to present the possibilities of using the enzymatic system of microorganisms and insects to transform small molecules, such as monoterpenes. The most important advantage of this type of reaction is the possibility of obtaining derivatives that are not possible to obtain with standard methods of organic synthesis or are very expensive to obtain. The interest of industrial centers focuses mainly on obtaining particles of high optical purity, which have the desired biological properties. The cost of obtaining such a compound and the elimination of toxic or undesirable chemical waste is important. Enzymatic reactions based on enzymes alone or whole microorganisms enable obtaining products with a specific structure and purity in accordance with the rules of Green Chemistry.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The "blocker residue" theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The "second shell" theory suggests that residues distant (∼8 Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.

Legumes are affected by biotic factors such as insects, molds, bacteria, and viruses. These plants can produce many different molecules in response to the attack of phytopathogens. Protease inhibitors (PIs) are proteins produced by legumes that inhibit the protease activity of phytopathogens. PIs are known to reduce nutrient availability, which diminishes pathogen growth and can lead to the death of the pathogen. PIs are classified according to the specificity of the mechanistic activity of the proteolytic enzymes, with serine and cysteine protease inhibitors being studied the most. Previous investigations have reported the efficacy of these highly stable proteins against diverse biotic factors and the concomitant protective effects in crops, representing a possible replacement of toxic agrochemicals that harm the environment.

Chitinase (EC 3.2.1.14) is an enzyme to breakdown β-1,4-glycosidic bonds in chitin and chitooligosaccharides. The loss of chitinase enzymatic activity in insects results in severe exoskeleton defects and lethality at all developmental stages, indicating that insect chitinases can be promising pesticide targets. However, there are no pesticides known to target chitinases. This perspective will focus on the latest research progress of insect chitinases, paying special attention to crystal structures and chemical biology advances in the field. The physiological importance and unique structural features of insect chitinases may ensure the development of new pesticides through a novel acting mode.

Many hemimetabolous insects produce their own cellulase enzymes from the glycoside hydrolase family 9, first observed in termites and cockroaches. Phasmatodea have multiple cellulases, some of which are multifunctional and can degrade xylan or xyloglucan. To discover when these abilities evolved, we identified cellulases from the Polyneoptera sampled by the 1000 Insect Transcriptome and Evolution (1KITE) project, including all cockroach and termite transcriptomes. We hoped to identify what role enzyme substrate specificities had in the evolution of dietary specification, such as leaf-feeding or wood-feeding. Putative cellulases were identified from the transcriptomes and analysed phylogenetically. All cellulases were amplified from an exemplar set of Polyneoptera species using rapid amplification of cDNA ends PCR and heterologously expressed in an insect cell line, then tested against different polysaccharides for their digestive abilities. We identified several multifunctional xyloglucanolytic enzymes across Polyneoptera, plus a large group of cellulase-like enzymes found in nearly all insect orders with no discernible digestive ability. Multifunctional xylanolytic cellulases remain unique to Phasmatodea. The presence or absence of multifunctional enzymes does not impact dietary specification, but rather having multiple, multifunctional cellulase genes is an ancestral state for Polyneoptera and possibly Insecta. The prevalence of multifunctional cellulases in other animals demands further investigation., (© 2019 The Royal Entomological Society.)

Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates and in some bacterial species. It has been also detected in mammalian cells, although only in trace amounts. Complete understanding of the biophysical and physiological relevance of CPE is still lacking, and its biological role is still an open question. CPE differs in its biosynthetic mechanisms from sphingomyelin, due to the specific CPE synthase in invertebrates. In contrast to well-established sphingomyelin/cholesterol interactions that result in the formation of ordered membrane domains, the formation of ordered CPE/cholesterol domains is not favored. CPE might be crucial for the early development of Drosophila melanogaster, and it might be involved in the developmental stages of Trypanosoma brucei. As a Bacteroidetes-associated sphingolipid, CPE might also be involved in maintenance of these bacteria in their ecological niches. Therefore, efficient detection of CPE in biological systems is needed to better define its distribution and biological role(s)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Food allergies originate from adverse immune reactions to some food components. Ingestion of food allergens can cause effects of varying severity, from mild itching to severe anaphylaxis reactions. Currently there are no clues to predict the allergenic potency of a molecule, nor are cures for food allergies available. Cutting-edge research on allergens is aimed at increasing information on their diffusion and understanding structure-allergenicity relationships. In this context, purified recombinant allergens are valuable tools for advances in the diagnostic and immunotherapeutic fields. Chitinases are a group of allergens often found in plant fruits, but also identified in edible insects. They are classified into different families and classes for which structural analyses and identification of epitopes have been only partially carried out. Moreover, also their presence in common allergen databases is not complete. In this review we provide a summary of the identified food allergenic chitinases, their main structural characteristics, and a clear division in the different classes., Competing Interests: The authors declare no conflict of interest.

Insects are often regarded as the most successful group of animals in the terrestrial environment. Their success can be represented by their huge biomass and large impact on ecosystems. Among the factors suggested to be responsible for their success, we focus on the possibility that the cuticle might have affected the process of insects' evolution. The cuticle of insects, like that of other arthropods, is composed mainly of chitin and structural cuticle proteins. However, insects seem to have evolved a specific system for cuticle formation. Oxidation reaction of catecholamines catalyzed by a copper enzyme, laccase, is the key step in the metabolic pathway for hardening of the insect cuticle. Molecular phylogenetic analysis indicates that laccase functioning in cuticle sclerotization has evolved only in insects. In this review, we discuss a theory on how the insect-specific "laccase" function has been advantageous for establishing their current ecological position as terrestrial animals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-β-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group., Competing Interests: The authors have declared that no competing interests exist.

We use the genomes of 160 insect species to test the hypothesis that the size of detoxifying enzyme families is greater in species using more chemically diverse food resources. Phylogenetically appropriate contrasts in subsamples of the data generally support the hypothesis. We find relatively high numbers of cytochrome P450, glutathione S-transferase and carboxyl/choline esterase genes in omnivores and herbivores feeding on chemically complex tissues and relatively low numbers of these genes in specialists on relatively simple diets, including plant sap, nectar and pollen, and blood. Among Lepidoptera feeding on green plant tissue and Condylognatha feeding on sap we also find more of these genes in highly polyphagous species, many of which are major agricultural pests. These genomic signatures of food resource use are consistent with the hypothesis that some taxa are preadapted for insecticide resistance evolution., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the cleavage of 1,4-glycosidic bonds various plant cell wall polysaccharides and chitin. In contrast to glycoside hydrolases, LPMOs are active on the crystalline regions of polysaccharides and thus synergize with hydrolytic enzymes. This synergism leads to an overall increase in the biomass-degradation activity of enzyme mixtures. Chitin-active LPMOs were discovered in 2010 and are currently classified in families AA10, AA11, and AA15 of the Carbohydrate-Active enZYmes database, which include LPMOs from bacteria, fungi, insects, and viruses. LPMOs have become important enzymes both industrially and scientifically and, in this chapter, we provide a brief introduction to chitin-active LPMOs including a summary of the 20+ chitin-active LPMOs that have been characterized so far. Then, we describe their structural features, catalytic mechanism, and appended carbohydrate modules. Finally, we show how chitin-active LPMOs can be used to perform chemo-enzymatic modification of chitin substrates.

Serine proteases (SPs) and serine protease homologs (SPHs) play essential roles in insect physiological processes including digestion, defense and development. Studies of insect genomes, transcriptomes and proteomes have generated a vast amount of information on these proteins, dwarfing the biological data acquired from a few model species. The large number and high diversity of homologous sequences makes it a challenge to use the limited functional information for making predictions across a broad taxonomic group of insects. In this work, we have extensively updated the framework of knowledge on the SP-related proteins in Drosophila melanogaster by identifying 52 new SPs/SPHs, classifying the 257 proteins into four groups (CLIP, gut, single- and multi-domain SPs/SPHs), and detecting inherent connections among phylogenetic relationships, genomic locations and expression profiles for 99 of the genes. Information on the existence of specific proteins in eggs, larvae, pupae and adults is presented to facilitate future research. More importantly, we have developed an approach to reveal close homologous or orthologous relationships among SPs/SPHs from D. melanogaster, Anopheles gambiae, Apis mellifera, Manduca sexta, and Tribolium castaneum thus inspiring functional studies in these and other holometabolous insects. This approach is useful for tackling similar problems on large and diverse protein families in other groups of organisms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Industrialising edible insects goes along with quality control and hazard analysis and critical control points. One of those steps is assessing heat treatment. For the present contribution, the potential of enzymatic heat assessment tests used in the dairy industry (alkaline phosphatase and lactoperoxidase) to detect heat treatment in several insect species ( Acheta domesticus, Gryllus assimilis, Gryllus bimaculatus, Locusta migratoria, Schistocerca gregaria, Chilecomadia moorei, Galleria mellonella, Bombyx mori, Pachnoda marginata, Tenebrio molitor, Zophobas atratus, Apis mellifera, and Hermetia illucens) was evaluated. Insect material was homogenised, diluted, and the enzymatic tests (Lactognost®, Peroxtesmo®) were carried with these liquids as if they were milk. All species but C. moorei, B. mori, P. marginata, and A. mellifera showed alkaline phosphatase activity in raw samples and none in heated (10 min at 100 ℃) ones, while only G. mellonella, T. molitor, and Z. atratus reacted accordingly with lactoperoxidase. In trial 2 focusing only on alkaline phosphatase activity, inactivation of the enzyme after 5, 10, and 15 min of heating occurred species specific within a range of 60-86 ℃, i.e. within ordinary pasteurisation schemes. Thus and for the time being, heat treatment in many edible insect species can be assessed using alkaline phosphatase activity test kits. In contrast to milk samples, positive results may display bluish or greenish colours, and the time until a reliable reading is possible is extended to 1-1.5 h (24 h in the case of Gryllidae).

The study of insect midgut features has been made possible by the recent availability of transcriptome datasets. These data uncovered the preferential expression of mucus-forming mucins at midgut regions that require protection (e.g. the acidic middle midgut of Musca domestica) or at sites of enzyme immobilization, particularly around the peritrophic membrane of Spodoptera frugiperda. Coleoptera lysosomal peptidases are directed to midgut lumen when over-expressed and targeted to lysosomes by a mechanism other than the mannose 6-phosphate-dependent pathway. We show that this second trend is likely conserved across Annelida, Mollusca, Nematoda, and Arthropoda. Furthermore, midgut transcriptomes of distantly related species reveal a general overexpression of xenobiotic detoxification pathways. In addition to attenuating toxicity of plant-derived compounds and insecticides, we also discuss a role for these detoxification pathways in regulating host-microbiota interactions by metabolizing bacterial secondary metabolites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The π-cation interaction that differs from the cation-π interaction is a valuable concept in molecular design of pharmaceuticals and pesticides. In this Perspective we present an up-to-date review (from 1995 to 2017) on bioactive molecules involving π-cation interactions with the recognition site, and categorize into systems of inhibitor-enzyme, ligand-receptor, ligand-transporter, and hapten-antibody. The concept of π-cation interactions offers use of π systems in a small molecule to enhance the binding affinity, specificity, selectivity, lipophilicity, bioavailability, and metabolic stability, which are physiochemical features desired for drugs and pesticides.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.

This work aims to synthesize new trehalase inhibitors selective towards the insect trehalase versus the porcine trehalase, in view of their application as potentially non-toxic insecticides and fungicides. The synthesis of a new pseudodisaccharide mimetic 8 , by means of a stereoselective α-glucosylation of the key pyrrolizidine intermediate 13 , was accomplished. The activity of compound 8 as trehalase inhibitor towards C. riparius trehalase was evaluated and the results showed that 8 was active in the μM range and showed a good selectivity towards the insect trehalase. To reduce the overall number of synthetic steps, simpler and more flexible disaccharide mimetics 9 - 11 bearing a pyrrolidine nucleus instead of the pyrrolizidine core were synthesized. The biological data showed the key role of the linker chain's length in inducing inhibitory properties, since only compounds 9 (α,β-mixture), bearing a two-carbon atom linker chain, maintained activity as trehalase inhibitors. A proper change in the glucosyl donor-protecting groups allowed the stereoselective synthesis of the β-glucoside 9 β, which was active in the low micromolar range (IC 50 = 0.78 μM) and 12-fold more potent (and more selective) than 9 α towards the insect trehalase., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state. Both challenges can be addressed by creating fusion proteins that combine the P450 and CPR functions while eliminating membrane anchors, allowing the production and purification of soluble multifunctional polypeptides suitable for industrial applications. Here we discuss several strategies for the construction of fusion enzymes combining insect P450 with CPRs.

Facultative parthenogenetic species, in which females can alternate between sex and parthenogenesis, are useful models to investigate the costs and benefits of sex and parthenogenesis, an ongoing issue in biology. The necessary empirical studies comparing the outcomes of alternative reproductive modes on life history traits are rare and focus mainly on traits directly associated with reproductive fitness. Immune function determines the ability of individuals to defend themselves against injury and disease and is therefore likely to have a significant impact on fitness. Here, we used the facultatively parthenogenetic Australian phasmatid, Extatosoma tiaratum, to investigate the effect of both maternal and offspring mode of conception (sexual or parthenogenetic) on offspring immune function (haemocyte concentration, lytic activity and phenoloxidase activity). We show that when parthenogenesis persists beyond one generation, it has negative effects on immune response in terms of haemocyte concentration and lytic activity. Phenoloxidase activity positively correlates with the level of microsatellite heterozygosity. Moreover, immune response decreases across consecutive sampling weeks, suggesting there are physiological constraints with respect to mounting immune responses in close time intervals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interactions between pesticides at common molecular targets and detoxification systems often determine their effectiveness and safety. Compounds with the same mode of action or target are candidates for cross resistance and restrictions in their recommended uses. Discovery research is therefore focused on new mechanisms and modes of action. Interactions in detoxification systems also provide cross resistance and synergist and safener mechanisms illustrated with serine hydrolases and inhibitors, cytochrome P450 and insecticide synergists, and glutathione S-transferases and herbicide safeners. Secondary targets are also considered for inhibitors of serine hydrolases, aldehyde dehydrogenases, and transporters. Emphasis is given to the mechanistic aspects of interactions, not the incidence, which depends on potency, exposure, ratios, and timing. The benefits of pesticide interactions are the additional levels of chemical control to achieve desired organismal effects. The risks are the unpredictable interactions of complex interconnected biological systems. However, with care, two can be better than one.

The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Biomarkers have been widely employed in ecotoxicology as early warning indicators of exposure to toxicants. Very often, they are used to compare reference and polluted sites, or to analyse time trends. However, very few studies focus on the natural variability range of biomarkers in the environment, which is pivotal to understand if the detected differences are actually determined by any adverse effects due to pollution. This work assesses the natural spatio-temporal variability of some enzymatic levels, frequently used as biomarkers, in freshwater benthic invertebrates. The influence of some environmental parameters on the enzymatic levels was also evaluated. Three families of insect larvae (Perlidae, Baetidae, and Heptageniidae) were sampled in three pristine streams and in eight different dates. Four enzymes (acetylcholinesterase, glutathione-S-transferase, alkaline phosphatase, and catalase) were measured. The natural variability of enzymatic levels was often significant in all considered species across both space and time. The observed pattern was poorly explained by the monitored environmental parameters. The results of this work show that great care should be paid when interpreting monitoring data in which biomarker levels are measured and compared among sites or dates. Presuming that measured differences are due to anthropogenic factors can be misleading, when other potentially influencing factors have not been accounted for.

Commercial applications of nanometal oxides have increased concern about their release into natural waters and consequent risks to aquatic biota and the processes they drive. In forest streams, the invertebrate shredder Allogamus ligonifer plays a key role in detritus food webs by transferring carbon and energy from plant litter to higher trophic levels. We assessed the response profiles of oxidative and neuronal stress enzymatic biomarkers in A. ligonifer after 96h exposure to nanoCuO at concentration ranges 30 . To better understand the contribution of ionic form in nanoCuO-induced stress, Cu 2+ released from nanoCuO was quantified and the enzymatic responses to Cu 2+ exposure at similar effective concentrations were compared. The highest activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed at concentrations 5 . The enzymatic activities decreased at effective concentrations between LC 10 and LC 30 . GR activity remained higher than in control at all concentrations. The activity of glutathione S-transferase (GST) increased whereas that of catalase (CAT) decreased at concentrations between LC 10 and LC 30 . The response patterns suggested that antioxidant enzymes could prevent oxidative stress at low concentrations (10 ) of nanoCuO, thereby contributing to the survival of A. ligonifer. At concentrations between LC 10 and LC 30 , effects of nanoparticulate or released ionic copper on enzyme activities were concentration-dependent, and led to oxidative stress and even to animal death. The activity of acetylcholinesterase (AChE) was strongly inhibited even at concentrations 10 , suggesting neuronal stress in A. ligonifer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection., (© 2016 Federation of European Biochemical Societies.)

The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hydrolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly- and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. Such enzymes are highly unusual for Metazoa, for which no xyloglucanases had been reported. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Telomerase and telomerase-generated telomeric DNA sequences are widespread throughout eukaryotes, yet they are not universal. Neither telomerase nor the simple DNA repeats associated with telomerase have been found in some plant and animal species. Telomerase was likely lost from Diptera before the divergence of Diptera and Siphonaptera, some 260 million years ago. Even so, Diptera is one of the most successful animal orders, making up 11% of known animal species. In addition, many species of Coleoptera and Hemiptera seem to lack canonical telomeric repeats at their chromosome ends. These and other insects that appear to lack canonical terminal repeat sequences account for another 10-15% of animal species. Conversely, the silk moth Bombyx mori maintains canonical telomeric sequences at its chromosome ends but seems to lack a functional telomerase. We speculate that a telomere-specific capping complex that recognizes the telomeric repeats and protects chromosome ends is the determining factor in maintaining canonical telomeric sequences and that telomerase is an early and efficacious mechanism for satisfying the needs of capping complex. There are alternate mechanisms for maintaining chromosome ends that do not depend on telomerase, such as recombination found in some human cancer cells and yeast mutants. These mechanisms may maintain the canonical telomeric repeats or allow the terminal sequence to evolve when specificity of the capping complex for terminal repeat sequences is weak.

The size of gene families associated with xenobiotic detoxification in insects may be associated with the complexity of their diets and their propensities to develop insecticide resistance. We test these hypotheses by collating the annotations of cytochrome P450, carboxyl/cholinesterase and glutathione S-transferase genes in 65 insect species with data on their host use and history of insecticide resistance. We find 2-4 fold variation across the species in the numbers of these genes and, in some orders, especially the Hymenoptera, there is a clear relationship between the numbers of genes and feeding preferences. However in other orders, in particular the Lepidoptera, no such relationship is apparent. The size of these three gene families also tend to correlate with insecticide resistance propensity but this may not be an independent effect because species with broader host ranges are more likely to be pests that are heavily sprayed with insecticides., (Copyright © 2016 Elsevier Inc. All rights reserved.)

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs., (Copyright © 2015. Published by Elsevier Inc.)

The European earwig Forficula auricularia is an effective predator in apple orchards. It is therefore crucial to study whether insecticides affect this natural pest control agent. Predation activity, i.e., the number of aphids eaten in 24 h, was determined under laboratory conditions after exposure of fourth-instar nymphs and adult earwigs to widely used insecticides (acetamiprid, chlorpyrifos-ethyl, deltamethrin, and spinosad), which were applied at the normal application rates. Inhibition of acetylcholinesterase and carboxylesterase activities were also measured as indicators of pesticide exposure. Predation activity decreased significantly in nymphs exposed to spinosad (62%) and chlorpyrifos-ethyl (98%) compared with controls. A similar response was found for both esterase activities. Spinosad had a stronger effect on AChE (-33%) whereas chlorpyrifos-ethyl affected CbE activity preferentially (-59%). Spinosad (20% of controls), acetamiprid (28%), and chlorpyrifos-ethyl (66%) also significantly decreased the predation behavior of adult male but not female (5 to 40%) earwigs. Adult AChE and CbE activities were also significantly reduced (28 to 67% of controls) in pesticide-exposed earwigs. Our results suggest that earwigs should be included in the environmental risk assessment framework for authorization of newly marketed plant protection products. Their predation behavior appears to be a sensitive and complementary biomarker.

Lipases are key enzymes involved in lipid digestion, storage and mobilization of reserves during fasting or heightened metabolic demand. This is a highly conserved process, essential for survival. The genomes of five marine invertebrate species with distinctive digestive system were screened for the six major lipase families. The two most common families in marine invertebrates, the neutral an acid lipases, are also the main families in mammals and insects. The number of lipases varies two-fold across analyzed genomes. A high degree of orthology with mammalian lipases was observed. Interestingly, 19% of the marine invertebrate lipases have lost motifs required for catalysis. Analysis of the lid and loop regions of the neutral lipases suggests that many marine invertebrates have a functional triacylglycerol hydrolytic activity as well as some acid lipases. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these families of enzymes in marine invertebrates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Studies examining phylogenetic community structure have become increasingly prevalent, yet little attention has been given to the influence of the input phylogeny on metrics that describe phylogenetic patterns of co-occurrence. Here, we examine the influence of branch length, tree reconstruction method, and amount of sequence data on measures of phylogenetic community structure, as well as the phylogenetic signal (Pagel's λ) in morphological traits, using Trichoptera larval communities from Churchill, Manitoba, Canada. We find that model-based tree reconstruction methods and the use of a backbone family-level phylogeny improve estimations of phylogenetic community structure. In addition, trees built using the barcode region of cytochrome c oxidase subunit I (COI) alone accurately predict metrics of phylogenetic community structure obtained from a multi-gene phylogeny. Input tree did not alter overall conclusions drawn for phylogenetic signal, as significant phylogenetic structure was detected in two body size traits across input trees. As the discipline of community phylogenetics continues to expand, it is important to investigate the best approaches to accurately estimate patterns. Our results suggest that emerging large datasets of DNA barcode sequences provide a vast resource for studying the structure of biological communities.

P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing., (© 2014 Society of Chemical Industry.)