Architecture and potential roles of a delta-class glutathione S-transferase in protecting honey bee from agrochemicals.

The European honey bee, Apis mellifera, serves as the principle managed pollinator species globally. In recent decades, honey bee populations have been facing serious health threats from combined biotic and abiotic stressors, including diseases, limited nutrition, and agrochemical exposure. Understanding the molecular mechanisms underlying xenobiotic adaptation of A. mellifera is critical, considering its extensive exposure to phytochemicals and agrochemicals present in the environment. In this study, we conducted a comprehensive structural and functional characterization of AmGSTD1, a delta class glutathione S-transferase (GST), to unravel its roles in agrochemical detoxification and antioxidative stress responses. We determined the 3-dimensional (3D) structure of a honey bee GST using protein crystallography for the first time, providing new insights into its molecular structure. Our investigations revealed that AmGSTD1 metabolizes model substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrophenyl acetate (PNA), phenylethyl isothiocyanate (PEITC), propyl isothiocyanate (PITC), and the oxidation byproduct 4-hydroxynonenal (HNE). Moreover, we discovered that AmGSTD1 exhibits binding affinity with the fluorophore 8-Anilinonaphthalene-1-sulfonic acid (ANS), which can be inhibited with various herbicides, fungicides, insecticides, and their metabolites. These findings highlight the potential contribution of AmGSTD1 in safeguarding honey bee health against various agrochemicals, while also mitigating oxidative stress resulting from exposure to these substances.

Potential risk to pollinators from neonicotinoid applications to host trees for management of spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae).

Neonicotinoid insecticides are used to manage spotted lanternfly (Lycorma delicatula (White); hereafter SLF), a recently introduced pest in the United States. Neonicotinoids can harm nontargets, such as pollinators potentially exposed via floral resources of treated plants. We quantified neonicotinoid residues in whole flowers of two SLF host plant species, red maple (Acer rubrum L. [Sapindales: Sapindaceae]) and tree-of-heaven (Ailanthus altissima (Mill.) [Sapindales: Simaroubaceae]), treated with post-bloom imidacloprid or dinotefuran applications that differed in timing and method of application. In red maple flowers, dinotefuran residues from fall applications were significantly higher than summer applications, while imidacloprid residues from fall applications were significantly lower than summer applications. Residues did not differ between application methods or sites. In tree-of-heaven flowers, dinotefuran residues were only detected in one of 28 samples at a very low concentration. To assess acute mortality risk to bees from oral exposure to residues in these flowers, we calculated risk quotients (RQ) using mean and 95% prediction interval residue concentrations from treatments in this study and lethal concentrations obtained from acute oral bioassays for Apis mellifera (L. (Hymenoptera: Apidae)) and Osmia cornifrons (Radoszkowski (Hymenoptera: Megachilidae)), then compared these RQs to a level of concern. For A. mellifera, only one treatment group, applied at 2X maximum label rate, had an RQ that exceeded this level. However, several RQs for O. cornifrons exceeded the level of concern, suggesting potential acute risk to solitary bees. Further studies are recommended for more comprehensive risk assessments to nontargets from neonicotinoid use for SLF management.

Roles of insect odorant binding proteins in communication and xenobiotic adaptation.

Odorant binding proteins (OBPs) are small water-soluble proteins mainly associated with olfaction, facilitating the transport of odorant molecules to their relevant receptors in the sensillum lymph. While traditionally considered essential for olfaction, recent research has revealed that OBPs are engaged in a diverse range of physiological functions in modulating chemical communication and defense. Over the past 10 years, emerging evidence suggests that OBPs play vital roles in purifying the perireceptor space from unwanted xenobiotics including plant volatiles and pesticides, potentially facilitating xenobiotic adaptation, such as host location, adaptation, and pesticide resistance. This multifunctionality can be attributed, in part, to their structural variability and effectiveness in transporting, sequestering, and concealing numerous hydrophobic molecules. Here, we firstly overviewed the classification and structural properties of OBPs in diverse insect orders. Subsequently, we discussed the myriad of functional roles of insect OBPs in communication and their adaptation to xenobiotics. By synthesizing the current knowledge in this field, our review paper contributes to a comprehensive understanding of the significance of insect OBPs in chemical ecology, xenobiotic adaptation, paving the way for future research in this fascinating area of study.

MicroRNAs miR-14 and miR-2766 regulate tyrosine hydroxylase to control larval-pupal metamorphosis in Helicoverpa armigera.

BACKGROUND: The cotton bollworm, Helicoverpa armigera, is a worldwide polyphagous pest, causing huge economic losses in vegetable, cotton and corn crops, among others. Owing to long-term exposure to Bacillus thuringiensis (Bt) toxins, evolution of resistance has been detected in this pest. As a conservative and effective neurotransmitter, dopamine (DA) has an important role in insect growth and development. In this study, we investigated the regulatory functions of DA and its associated non-coding RNA in metamorphosis in H. armigera. RESULTS: Expression profiles indicated that DA and DA pathway genes were highly expressed during larval-pupal metamorphosis in H. armigera. RNA interference and pharmacological experiments confirmed that tyrosine hydroxylase (TH), dopa decarboxylase, vesicular amine transporter and DA receptor 2 are critical genes related to the development of H. armigera from larvae to pupae. We also found that miR-14 and miR-2766 targeted the 3' untranslated region to post-transcriptionally regulate HaTH function. Application of miR-2766 and miR-14 antagomirs significantly increased levels of HaTH transcripts and proteins, while injection of miR-2766 and miR-14 agomirs not only suppressed messenger RNA and protein levels of HaTH, but also resulted in defective pupation in H. armigera. CONCLUSION: These results suggest that DA deficiency inhibits larval-pupal metamorphosis in H. armigera. Potentially, DA pathway genes and their microRNAs could be used as a novel target for H. armigera management. © 2022 Society of Chemical Industry.

Glutaredoxins and thioredoxin peroxidase involved in defense of emamectin benzoate induced oxidative stress in Grapholita molesta.

Glutaredoxins (Grxs) and thioredoxin peroxidases (Tpxs) are major antioxidant enzyme families involved in regulating cellular redox homeostasis and in defense of enhanced oxidative stress through scavenging reactive oxygen species (ROS). However, the functions of these enzymes have not been reported in the oriental fruit moth, Grapholita molesta (Busck), a worldwide pest of stone and pome fruits. Here, we identified four new antioxidant genes, GmGrx, GmGrx3, GmGrx5, and GmTpx which were induced by exposure with emamectin benzoate, a commonly used biopesticide for G. molesta control. Other environmental factors (low and high temperatures, Escherichia coli and Metarhizium anisopliae) also significantly induced the expression of these genes. After GmGrx or GmTpx silenced by RNA interference (RNAi), the percentage of larval survival to emamectin benzoate were significantly decreased, demonstrating that GmGrx and GmTpx are involved in protecting G. molesta from stresses induced by emamectin benzoate. Furthermore, silenced GmGrx, GmGrx3, GmGrx5, or GmTpx significantly enhanced the enzymatic activities of superoxide dismutase (SOD) (except GmTpx) and peroxidase (POD), as well as the contents of hydrogen peroxide and metabolites ascorbate. Taken together, our results suggest that GmGrx, GmGrx3, GmGrx5, and GmTpx may play critical roles in antioxidant defense. Specially, GmGrx and GmTpx contribute to the defense of oxidative damage induced by exposure to emamectin benzoate through scavenging excessive ROS in G. molesta. Our findings provided a theoretical basis for understanding functions of insect glutaredoxin and peroxidase systems.

Odorant degrading carboxylesterases modulate foraging and mating behaviors of Grapholita molesta.

Odorant degrading carboxylesterases (CXEs) play key roles in the process of odor signal reception via degrading ester odorants. But the functional mechanisms of CXEs in modulating insect behaviors are unclear. Herein, we studied the roles that CXEs played in mating, foraging, and signal receptions of sex pheromones and host volatiles in Grapholita molesta. As a result, 23 candidate CXEs were identified by transcriptome analysis of G. molesta. The GmolCXE1 and 5 highly expressed in the antennae of male moths and GmolCXE14 and 21 abundantly expressed in larval heads, were significantly upregulated after exposure with odors from female adults or fresh ripe fruits respectively. After knockdown of GmolCXE1 and 5, or GmolCXE14 and 21 by RNA interference, the behavioral responses of G. molesta to ester sex pheromones or host volatiles were decreased, by exhibiting an inhibited searching behavior of G. molesta for females or fruits, respectively. Then evidence form GC-MS analysis, showed that the protein GmolCXE1 and GmolCXE5 could metabolize the sex pheromone components (Z/E)-8-dodecenyl acetate to their metabolites products (Z/E)-8-dodecenol, and that GmolCXE14 and GmolCXE21 could metabolize ethyl butanoate and ethyl hexanoate of ripe pears. In addition, fluorescent binding assays verified that GmolCXEs could degrade the free ester odor molecules, but not degrade the odor molecules protected by odorant-binding proteins. Our study not only demonstrated CXEs modulated the mating and foraging behaviors of G. molesta through inactivation of ester sex pheromone and host volatiles, but also discovered great potential molecular targets to develop behavioral inhibitors for pest management.

Illumina Sequencing of 18S/16S rRNA Reveals Microbial Community Composition, Diversity, and Potential Pathogens in 17 Turfgrass Seeds.

The increasing need for turfgrass seeds is coupled with the high risk of dangerous microbial pathogens being transmitted through the domestic and international trade of seeds. Concerns continue to be raised about seed safety and quality. Here, we show that next-generation sequencing (NGS) of DNA represents an effective and reliable tactic to monitor the microbial communities within turfgrass seeds. A comparison of DNA sequence data with reference databases revealed the presence of 26 different fungal orders. Among them, serious plant disease pathogens such as Bipolaris sorokiniana, Boeremia exigua, Claviceps purpurea, and Rhizoctonia zeae were detected. Seedborne bacteria, including Erwinia persicina and Acidovorax avenae, were identified from different bacterial orders. Our study indicated that the traditional culturing method and the NGS approach for pathogen identification complement each other. The reliability of culturing and NGS methods was further validated by PCR with specific primers. The combination of these different techniques ensures maximum sensitivity and specificity for turfgrass seed pathogen testing assay.

Physiological functions of a methuselah-like G protein coupled receptor in Lymantria dispar Linnaeus.

Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest.

Crystal Structure of Phosphoserine BlaC from Mycobacterium tuberculosis Inactivated by Bis(Benzoyl) Phosphate.

Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide. The class A serine ß-lactamase BlaC confers Mycobacterium tuberculosis resistance to conventional ß-lactam antibiotics. As the primary mechanism of bacterial resistance to ß-lactam antibiotics, the expression of a ß-lactamase by Mycobacterium tuberculosis results in hydrolysis of the ß-lactam ring and deactivation of these antibiotics. In this study, we conducted protein X-ray crystallographic analysis of the inactivation of BlaC, upon exposure to the inhibitor bis(benzoyl) phosphate. Crystal structure data confirms that serine ß-lactamase is phosphorylated at the catalytic serine residue (Ser-70) by this phosphate-based inactivator. This new crystallographic evidence suggests a mechanism for phosphorylation of BlaC inhibition by bis(benzoyl) phosphate over acylation. Additionally, we confirmed that bis(benzoyl) phosphate inactivated BlaC in a time-dependent manner.

Bis(benzoyl) phosphate inactivators of beta-lactamase C from Mtb.

The class A ß-lactamase BlaC is a cell surface expressed serine hydrolase of Mycobacterium tuberculosis (Mtb), one of the causative agents for Tuberculosis in humans. Mtb has demonstrated increased susceptibility to ß-lactam antibiotics upon inactivation of BlaC; thus, making BlaC a rational enzyme target for therapeutic agents. Herein, we present the synthesis and structure-activity-relationship data for the 1st-generation library of bis(benzoyl) phosphates (1-10). Substituent effects ranged from σp = -0.27 to 0.78 for electronic and π = -0.41 to 1.98 for hydrophobic parameters. Compounds 1, 4 and 5 demonstrated the greatest inhibitory potency against BlaC in a time-dependent manner (kobs = 0.212, 0.324, and 0.450 mn-1 respectively). Combined crystal structure data and mass spectrometric analysis of a tryptic digest for BlaC inactivated with 4 provided evidence that the mechanism of inactivation by this bis(benzoyl) phosphate scaffold occurs via phosphorylation of the active-site Ser-70, ultimately leading to an aged form of the enzyme.

Ocular Albinism Type 1 Regulates Deltamethrin Tolerance in Lymantria dispar and Drosophila melanogaster.

The ocular albinism type 1 (OA1), a pigment cell-specific integral membrane glycoprotein, is a member of the G-protein-coupled receptor (GPCR) superfamily that binds to heterotrimeric G proteins in mammalian cells. We aimed to characterize the physiological functions an insect OA1 from Lymantria dispar (LdOA1) employs in the regulation of insecticide tolerance. In the present study, we investigated the roles of LdOA1 in response to deltamethrin exposure in both L. dispar and Drosophila melanogaster. LdOA1 was expressed at the lowest level during the 4th instar stage, while LdOA1 was significantly upregulated in the 5th instar and male stages. Knockdown of LdOA1 by injecting dsRNA of LdOA1 into gypsy moth larvae caused a 4.80-fold higher mortality than in control larvae microinjected with dsRNA of GFP under deltamethrin stress. Nine out of 11 L. dispar CYP genes were significantly downregulated under deltamethrin stress in LdOA1 silenced larvae as compared to control larvae. Moreover, the LdOA1 gene was successfully overexpressed in D. melanogaster using transgenic technique. The deltamethrin contact assay showed that the LdOA1 overexpression in flies significantly enhanced the tolerance to deltamethrin compared to the control flies. Furthermore, the downstream Drosophila CYP genes were upregulated in the LdOA1 overexpression flies, suggesting LdOA1 may play a master switch role in P450-mediated metabolic detoxification. This study is the first report of an insect OA1 gene regulating insecticide tolerance and potentially playing a role in the regulation of downstream cytochrome P450 expression. These results contribute to the future development of novel insecticides targeting insect GPCRs.

Molecular parallelism in fast-twitch muscle proteins in echolocating mammals.

Detecting associations between genomic changes and phenotypic differences is fundamental to understanding how phenotypes evolved. By systematically screening for parallel amino acid substitutions, we detected known as well as novel cases (Strc, Tecta, and Cabp2) of parallelism between echolocating bats and toothed whales in proteins that could contribute to high-frequency hearing adaptations. Our screen also showed that echolocating mammals exhibit an unusually high number of parallel substitutions in fast-twitch muscle fiber proteins. Both echolocating bats and toothed whales produce an extremely rapid call rate when homing in on their prey, which was shown in bats to be powered by specialized superfast muscles. We show that these genes with parallel substitutions (Casq1, Atp2a1, Myh2, and Myl1) are expressed in the superfast sound-producing muscle of bats. Furthermore, we found that the calcium storage protein calsequestrin 1 of the little brown bat and the bottlenose dolphin functionally converged in its ability to form calcium-sequestering polymers at lower calcium concentrations, which may contribute to rapid calcium transients required for superfast muscle physiology. The proteins that our genomic screen detected could be involved in the convergent evolution of vocalization in echolocating mammals by potentially contributing to both rapid Ca2+ transients and increased shortening velocities in superfast muscles.

Characterization of Class III Peroxidases from Switchgrass.

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.

A specialist herbivore pest adaptation to xenobiotics through up-regulation of multiple Cytochrome P450s.

The adaptation of herbivorous insects to their host plants is hypothesized to be intimately associated with their ubiquitous development of resistance to synthetic pesticides. However, not much is known about the mechanisms underlying the relationship between detoxification of plant toxins and synthetic pesticides. To address this knowledge gap, we used specialist pest Colorado potato beetle (CPB) and its host plant, potato, as a model system. Next-generation sequencing (454 pyrosequencing) was performed to reveal the CPB transcriptome. Differential expression patterns of cytochrome P450 complement (CYPome) were analyzed between the susceptible (S) and imidacloprid resistant (R) beetles. We also evaluated the global transcriptome repertoire of CPB CYPome in response to the challenge by potato leaf allelochemicals and imidacloprid. The results showed that more than half (51.2%) of the CBP cytochrome P450 monooxygenases (P450s) that are up-regulated in the R strain are also induced by both host plant toxins and pesticide in a tissue-specific manner. These data suggest that xenobiotic adaptation in this specialist herbivore is through up-regulation of multiple P450s that are potentially involved in detoxifying both pesticide and plant allelochemicals.

Variation in time and magnitude of immune response and viremia in experimental challenges with Porcine circovirus 2b.

BACKGROUND: Porcine circovirus 2 is the primary agent responsible for inducing a group of associated diseases known as Porcine Circovirus Associated Diseases (PCVAD), which can have detrimental effects on production efficiency as well as causing significant mortality. The objective of this study was to evaluate variation in viral replication, immune response and growth across pigs (n = 974) from different crossbred lines. The approach used in this study was experimental infection with a PCV2b strain of pigs at an average of 43 days of age. RESULTS: The sequence of the PCV2b isolate used in the challenge was similar with a cluster of PCV2b isolates known to induce PCVAD and increased mortality rates. The swine leukocyte antigen class II (SLAII) profile of the population was diverse, with nine DQB1 haplotypes being present. Individual viremia and antibody profiles during challenge demonstrate variation in magnitude and time of viral surge and immune response. The correlations between PCV2 specific antibodies and average daily gain (ADG) were relatively low and varied between - 0.14 to 0.08 for IgM and -0.02 and 0.11 for IgG. In contrast, PCV2 viremia was an important driver of ADG decline following infection; a moderate negative correlation was observed between viral load and overall ADG (r = - 0.35, P < 0.001). The pigs with the lowest 10% level of viral load maintained a steady increase in weekly ADG (P < 0.0001) compared to the pigs that had the 10% greatest viral load (P < 0.55). In addition, the highly viremic group expressed higher IgM and IgG starting with d 14 and d 21 respectively, and higher tumor necrosis factor - alpha (TNF-α) at d 21 (P < 0.005), compared to low viremic group. CONCLUSIONS: Molecular sources of the observed differences in viremia and immune response could provide a better understanding of the host factors that influence the development of PCVAD and lead to improved knowledge of swine immunity.

Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum.

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 µM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol.

Integrated analysis of cytochrome P450 gene superfamily in the red flour beetle, Tribolium castaneum.

BACKGROUND: The functional and evolutionary diversification of insect cytochrome P450s (CYPs) shaped the success of insects. CYPs constitute one of the largest and oldest gene superfamilies that are found in virtually all aerobic organisms. Because of the availability of whole genome sequence and well functioning RNA interference (RNAi), the red flour beetle, Tribolium castaneum serves as an ideal insect model for conducting functional genomics studies. Although several T. castaneum CYPs had been functionally investigated in our previous studies, the roles of the majority of CYPs remain largely unknown. Here, we comprehensively analyzed the phylogenetic relationship of all T. castaneum CYPs with genes in other insect species, investigated the CYP6BQ gene cluster organization, function and evolution, as well as examined the mitochondrial CYPs gene expression patterns and intron-exon organization. RESULTS: A total 143 CYPs were identified and classified into 26 families and 59 subfamilies. The phylogenetic trees of CYPs among insects across taxa provided evolutionary insight for the genetic distance and function. The percentage of singleton (33.3%) in T. castaneum CYPs is much less than those in Drosophila melanogaster (52.5%) and Bombyx mori (51.2%). Most members in the largest CYP6BQ gene cluster may make contribution to deltamethrin resistance in QTC279 strain. T. castaneum genome encodes nine mitochondrial CYPs, among them CYP12H1 is only expressed in the final instar larval stage. The intron-exon organizations of these mitochondrial CYPs are highly diverse. CONCLUSION: Our studies provide a platform to understand the evolution and functions of T. castaneum CYP gene superfamily which will help reveal the strategies employed by insects to cope with their environment.