Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris.

BACKGROUND: Natural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis. RESULTS: The genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects. CONCLUSIONS: This study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

Genome-Wide Identification of Sorghum Paclobutrazol-Resistance Gene Family and Functional Characterization of SbPRE4 in Response to Aphid Stress.

Sorghum (Sorghum bicolor), the fifth most important cereal crop globally, serves as a staple food, animal feed, and a bioenergy source. Paclobutrazol-Resistance (PRE) genes play a pivotal role in the response to environmental stress, yet the understanding of their involvement in pest resistance remains limited. In the present study, a total of seven SbPRE genes were found within the sorghum BTx623 genome. Subsequently, their genomic location was studied, and they were distributed on four chromosomes. An analysis of cis-acting elements in SbPRE promoters revealed that various elements were associated with hormones and stress responses. Expression pattern analysis showed differentially tissue-specific expression profiles among SbPRE genes. The expression of some SbPRE genes can be induced by abiotic stress and aphid treatments. Furthermore, through phytohormones and transgenic analyses, we demonstrated that SbPRE4 improves sorghum resistance to aphids by accumulating jasmonic acids (JAs) in transgenic Arabidopsis, giving insights into the molecular and biological function of atypical basic helix-loop-helix (bHLH) transcription factors in sorghum pest resistance.

A molecular approach to unravel trophic interactions between parasitoids and hyperparasitoids associated with pecan aphids.

Advances in molecular ecology can overcome many challenges in understanding host-parasitoid interactions. Genetic characterization of the key-players in systems helps to confirm species and identify trophic linkages essential for ecological service delivery by biological control agents; however, relatively few agroecosystems have been explored using this approach. Pecan production consists of a large tree perennial system containing an assortment of seasonal pests and natural enemies. As a first step to characterizing host-parasitoid associations in pecan food webs, we focus on aphid species and their parasitoids. Based on DNA barcoding of field-collected and reared specimens, we confirmed the presence of 3 species of aphid, one family of primary parasitoids, and 5 species of hyperparasitoids. By applying metabarcoding to field-collected aphid mummies, we were able to identify multiple species within each aphid mummy to unravel a complex food web of 3 aphids, 2 primary parasitoids, and upward of 8 hyperparasitoid species. The results of this study demonstrate that multiple hyperparasitoid species attack a single primary parasitoid of pecan aphids, which may have negative consequences for successful aphid biological control. Although further research is needed on a broader spatial scale, our results suggest multiple species exist in this system and may suggest a complex set of interactions between parasitoids, hyperparasitoids, and the 3 aphid species. This was the first time that many of these species have been characterized and demonstrates the application of novel approaches to analyze the aphid-parasitoid food webs in pecans and other tree crop systems.

Preparation and Application of Volatilized Wormwood Essence Derived Naturally into Green Insecticide.

Naturally occurring substances and their derivatives function as vital resources for pesticides that can be used in fields, such as insecticide production and fungicide development. As a botanical entity displaying multifaceted biological functions, wormwood has received thorough scrutiny across multiple sectors. The insect repellency potency combined with antibacterial and antifungal activities of wormwood position it as a potential candidate for prospective development into eco-friendly chemical pesticides. In this research, Wormwood essential oil was procured via ethanol water under ultrasonic scenarios and subsequently diluted with PEG 400 to formulate green chemical pesticides. The defensive efficacy of this green pesticide on plants was validated through 2 weeks of clustered plant growth experiments. Active constituents that exerted their effects were scrutinized by GC-MS. Furthermore, this green pesticide also displays efficacious effects on the prevention and management of aphids, exhibiting a dose-dependent relationship. 4-terpenol, eucalyptol, carvacrol, and L-borneol were identified by GC-MS as the predominant active constituents in this green chemical pesticide. Wormwood can be leveraged to develop green chemical pesticides, which can protect plants without contaminating the environment.

Discovery of Two Novel Viruses of the Willow-Carrot Aphid, Cavariella aegopodii.

The advancement of bioinformatics and sequencing technology has resulted in the identification of an increasing number of new RNA viruses. This study systematically identified the RNA virome of the willow-carrot aphid, Cavariella aegopodii (Hemiptera: Aphididae), using metagenomic sequencing and rapid amplification of cDNA ends (RACE) approaches. C. aegopodii is a sap-sucking insect widely distributed in Europe, Asia, North America, and Australia. The deleterious effects of C. aegopodii on crop growth primarily stem from its feeding activities and its role as a vector for transmitting plant viruses. The virome includes Cavariella aegopodii virga-like virus 1 (CAVLV1) and Cavariella aegopodii iflavirus 1 (CAIV1). Furthermore, the complete genome sequence of CAVLV1 was obtained. Phylogenetically, CAVLV1 is associated with an unclassified branch of the Virgaviridae family and is susceptible to host antiviral RNA interference (RNAi), resulting in the accumulation of a significant number of 22nt virus-derived small interfering RNAs (vsiRNAs). CAIV1, on the other hand, belongs to the Iflaviridae family, with vsiRNAs ranging from 18 to 22 nt. Our findings present a comprehensive analysis of the RNA virome of C. aegopodii for the first time, offering insights that could potentially aid in the future control of the willow-carrot aphid.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

Genome-Wide Comparative Analysis of the Cytochrome P450 Monooxygenase Family in 19 Aphid Species and Their Expression Analysis in 4 Cereal Crop Aphids.

Cytochrome P450 monooxygenases (CYP450s) play a variety of physiological roles, including pesticide resistance, plant allelochemical detoxification, and hormone metabolism catalysis. However, limited information is available on the classification and expression profiles of the CYP450 gene family in aphid species. This is the first study to identify the cytochrome P450 gene family in 19 aphid species at the whole genome level. A total of 1100 CYP450 genes were identified in 19 aphid species. Three hundred CYP450 genes belonged to six cereal crop aphid species, which were further classified into four subfamilies according to the phylogenetic relationship. The conserved motifs, exon-intron structures, and genomic organization of the same subfamilies were similar. Predictions of subcellular localization revealed that the endoplasmic reticulum harbored the majority of CYP450 proteins. In Sitobion avenae and Rhopalosiphum maidis, the increase in the CYP450 gene was primarily caused by segmental duplication events. However, only tandem duplication occurred in the CYP450 gene family of Diuraphis noxia, Rhopalosiphum padi, Schizaphis graminum, and Sitobion miscanthi. Synteny analysis found three continuous colinear CYP450 gene pairs among six cereal crop aphid species. Furthermore, we obtained the expression profiles of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae. Differential expression analysis provided growth stage specificity genes, tissue specificity genes, organ specificity genes and some detoxification metabolic genes among these four cereal crop aphids. Meanwhile, their expression patterns were showed. The related functions and pathways of CYP450s were revealed by GO and KEGG enrichment analysis. Above all, we picked the differentially expressed CYP450 genes from all of the differentially expressed genes (DEGs). These differentially expressed CYP450 genes provided some new potential candidates for aphid control and management. This work establishes the foundation for further investigations into the regulatory functions of the CYP450 gene family in aphid species and beyond.

microRNA-3037 targeting CYP6CY2 confers imidacloprid resistance to Sitobion miscanthi (Takahashi).

The wheat aphid Sitobion miscanthi is a dominant and destructive pest in agricultural production. Insecticides are the main substances used for effective control of wheat aphids. However, their extensive application has caused severe resistance of wheat aphids to some insecticides; therefore, exploring resistance mechanisms is essential for wheat aphid management. In the present study, CYP6CY2, a new P450 gene, was isolated and overexpressed in the imidacloprid-resistant strain (SM-R) compared to the imidacloprid-susceptible strain (SM-S). The increased sensitivity of S. miscanthi to imidacloprid after knockdown of CYP6CY2 indicates that it could be associated with imidacloprid resistance. Subsequently, the posttranscriptional regulation of CYP6CY2 in the 3' UTR by miR-3037 was confirmed, and CYP6CY2 participated in imidacloprid resistance. This finding is critical for determining the role of P450 in relation to the resistance of S. miscanthi to imidacloprid. It is of great significance to understand this regulatory mechanism of P450 expression in the resistance of S. miscanthi to neonicotinoids.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to the defense against bacterial infection in the pea aphid.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is activated by infections of bacteria, fungi, viruses and parasites and mediated cellular and humoral immune responses. In the pea aphid Acyrthosiphon pisum little is known about the function of JAK/STAT signaling in its immune system. In this study, we first showed that expression of genes in the JAK/STAT signaling, including the receptors Domeless1/2, Janus kinase (JAK) and transcriptional factor Stat92E, is up-regulated upon bacteria Escherichia coli and Staphylococcus aureus and fungus Beauveria bassiana infections. After knockdown of expression of these genes by means of dsRNA injection, the aphids harbored more bacteria and suffered more death after infected with E. coli and S. aureus, but showed no significant change after B. bassiana infection. Our study suggests the JAK/STAT signaling contributes to the defense against bacterial infection in the pea aphid.

Salivary gland transcriptomics of the cotton aphid Aphis gossypii and comparative analysis with other sap-sucking insects.

Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.

Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

Genome-Wide Transcriptomic and Metabolomic Analyses Unveiling the Defence Mechanisms of Populus tremula against Sucking and Chewing Insect Herbivores.

Plants and insects coevolved as an evolutionarily successful and enduring association. The molecular arms race led to evolutionary novelties regarding unique mechanisms of defence and detoxification in plants and insects. While insects adopt mechanisms to conquer host defence, trees develop well-orchestrated and species-specific defence strategies against insect herbivory. However, current knowledge on the molecular underpinnings of fine-tuned tree defence responses against different herbivore insects is still restricted. In the current study, using a multi-omics approach, we unveiled the defence response of Populus tremula against aphids (Chaitophorus populialbae) and spongy moths (Lymantria dispar) herbivory. Comparative differential gene expression (DGE) analyses revealed that around 272 and 1203 transcripts were differentially regulated in P. tremula after moth and aphid herbivory compared to uninfested controls. Interestingly, 5716 transcripts were differentially regulated in P. tremula between aphids and moth infestation. Further investigation showed that defence-related stress hormones and their lipid precursors, transcription factors, and signalling molecules were over-expressed, whereas the growth-related counterparts were suppressed in P. tremula after aphid and moth herbivory. Metabolomics analysis documented that around 37% of all significantly abundant metabolites were associated with biochemical pathways related to tree growth and defence. However, the metabolic profiles of aphid and moth-fed trees were quite distinct, indicating species-specific response optimization. After identifying the suitable reference genes in P. tremula, the omics data were further validated using RT-qPCR. Nevertheless, our findings documented species-specific fine-tuning of the defence response of P. tremula, showing conservation on resource allocation for defence overgrowth under aphid and moth herbivory. Such findings can be exploited to enhance our current understanding of molecular orchestration of tree responses against herbivory and aid in developing insect pest resistance P. tremula varieties.

Salinity stress alters plant-mediated interactions between above- and below-ground herbivores.

Below-ground herbivory impacts plant development and often induces systemic responses in plants that affect the performance and feeding behavior of above-ground herbivores. Meanwhile, pest-damaged root tissue can enhance a plant's susceptibility to abiotic stress such as salinity. Yet, the extent to which herbivore-induced plant defenses are modulated by such abiotic stress has rarely been studied. In this study, we examine whether root feeding by larvae of the turnip moth, Agrotis segetum (Lepidoptera: Noctuidae) affects the performance of the above-ground, sap-feeding aphid Aphis gossypii (Hemiptera: Aphididae) on cotton, and assess whether those interactions are modulated by salinity stress. In the absence of salinity stress, A. segetum root feeding does not affect A. gossypii development. On the other hand, under intense salinity stress (i.e., 600 mM NaCl), A. segetum root feeding decreases aphid development time by 16.1 % and enhances fecundity by 72.0 %. Transcriptome, metabolome and bioassay trials showed that root feeding and salinity stress jointly trigger the biosynthesis of amino acids in cotton leaves. Specifically, increased titers of valine in leaf tissue relate to an enhanced performance of A. gossypii. Taken together, salinity stress alters the interaction between above- and below-ground feeders by changing amino acid accumulation. Our findings advance our understanding of how plants cope with concurrent biotic and abiotic stressors, and may help tailor plant protection strategies to varying production contexts.

Biotic interactions promote local adaptation to soil in plants.

Although different ecological factors shape adaptative evolution in natural habitats, we know little about how their interactions impact local adaptation. Here we used eight generations of experimental evolution with outcrossing Brassica rapa plants as a model system, in eight treatment groups that varied in soil type, herbivory (with/without aphids), and pollination mode (hand- or bumblebee-pollination), to study how biotic interactions affect local adaptation to soil. First, we show that several plant traits evolved in response to biotic interactions in a soil-specific way. Second, using a reciprocal transplant experiment, we demonstrate that significant local adaptation to soil-type evolved in the "number of open flowers", a trait used as a fitness proxy, but only in plants that evolved with herbivory and bee pollination. Whole genome re-sequencing of experimental lines revealed that biotic interactions caused a 10-fold increase in the number of SNPs across the genome with significant allele frequency change, and that alleles with opposite allele frequency change in different soil types (antagonistic pleiotropy) were most common in plants with an evolutionary history of herbivory and bee pollination. Our results demonstrate that the interaction with mutualists and antagonists can facilitate local adaptation to soil type through antagonistic pleiotropy.

Detecting sorghum aphid infestation in grain sorghum using leaf spectral response.

Sorghum aphid, Melanaphis sorghi (Theobald) have become a major economic pest in sorghum causing 70% yield loss without timely insecticide applications. The overarching goal is to develop a monitoring system for sorghum aphids using remote sensing technologies to detect changes in plant-aphid density interactions, thereby reducing scouting time. We studied the effect of aphid density on sorghum spectral responses near the feeding site and on distal leaves from infestation and quantified potential systemic effects to determine if aphid feeding can be detected. A leaf spectrometer at 400-1000 nm range was used to measure reflectance changes by varying levels of sorghum aphid density on lower leaves and those distant to the caged infestation. Our study results demonstrate that sorghum aphid infestation can be determined by changes in reflected light, especially between the green-red range (550-650 nm), and sorghum plants respond systemically. This study serves as an essential first step in developing more effective pest monitoring systems for sorghum aphids, as leaf reflection sensors can be used to identify aphid feeding regardless of infestation location on the plant. Future research should address whether such reflectance signatures can be detected autonomously using small unmanned aircraft systems or sUAS equipped with comparable sensor technologies.

UDP-Glycosyltransferases UGT350C3 and UGT344L7 Confer Tolerance to Neonicotinoids in Field Populations of Aphis gossypii.

The cotton aphid, Aphis gossypii, is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, A. gossypii has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, UGT350C3 and UGT344L7, were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic Drosophila bioassay results indicated that compared with the control lines, the UGT350C3 and UGT344L7 overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of UGT350C3 and UGT344L7 significantly increased A. gossypii sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.

Twenty-nine newly sequenced genomes and a comprehensive genome dataset for the insect endosymbiont Buchnera.

Most phloem-feeding insects face nutritional deficiency and rely on their intracellular symbionts to provide nutrients, and most of endosymbiont genomes have undergone reduction. However, the study of genome reduction processes of endosymbionts has been constrained by the limited availability of genome data from different insect lineages. The obligate relationship between aphids and Buchnera aphidicola (hereafter Buchnera) makes them a classic model for studying insect-endosymbiont interaction. Here, we report 29 newly sequenced Buchnera genomes from 11 aphid subfamilies, and a comprehensive dataset based on 90 Buchnera genomes from 14 aphid subfamilies. The dataset shows a significant genomic difference of Buchnera among different aphid lineages. The dataset exhibits a more balanced distribution of Buchnera (from 14 aphid subfamilies) genome sizes, ranging from 400 kb to 600 kb, which can illustrate the genome reduction process of Buchnera. The new genome data provide valuable insights into the microevolutionary processes leading to genomic reduction of insect endosymbionts.

Structural basis for odorant recognition of the insect odorant receptor OR-Orco heterocomplex.

Insects detect and discriminate a diverse array of chemicals using odorant receptors (ORs), which are ligand-gated ion channels comprising a divergent odorant-sensing OR and a conserved odorant receptor co-receptor (Orco). In this work, we report structures of the ApOR5-Orco heterocomplex from the pea aphid Acyrthosiphon pisum alone and bound to its known activating ligand, geranyl acetate. In these structures, three ApOrco subunits serve as scaffold components that cannot bind the ligand and remain relatively unchanged. Upon ligand binding, the pore-forming helix S7b of ApOR5 shifts outward from the central pore axis, causing an asymmetrical pore opening for ion influx. Our study provides insights into odorant recognition and channel gating of the OR-Orco heterocomplex and offers structural resources to support development of innovative insecticides and repellents for pest control.

S431F mutation on AChE1 and overexpression of P450 genes confer high pirimicarb resistance in Sitobion miscanthi.

Sitobion miscanthi is a destructive wheat pest responsible for significant wheat yield losses. Pirimicarb, one of the most important representatives of N, N-dimethylcarbamate insecticides, is widely used to control wheat aphids. In present work, heterozygous S431F mutation of acetylcholinesterase 1 (AChE1) was identified and verified in three pirimicarb-resistant S. miscanthi populations (two field populations (HA and HS, >955.8-fold) and one lab-selected population (PirR, 486.1-fold)), which has not been reported in S. miscanthi yet. The molecular docking results revealed that AChE1 containing the S431F mutation of S. miscanthi (SmAChE1S431F) showed higher free binding energy to three insecticides (pirimicarb, omethoate, and methomyl) than wild-type AChE1 of S. miscanthi (SmAChE1). Enzyme kinetic and inhibition experiments showed that the recombinant SmAChE1S431F was more insensitive to pirimicarb and omethoate than the recombinant SmAChE1. Furthermore, two overexpression P450 genes (CYP6K1 and CYP6A14) associated with pirimicarb resistance of S. miscanthi were verified by RNAi. These results suggested both target alteration and enhanced metabolism contributed to high pirimicarb resistance of S. miscanthi in the field and laboratory. These findings lay a foundation for further elucidating the mechanism of pirimicarb resistance in S. miscanthi, and have important implications for the resistance management of S. miscanthi control.

Novel program for automatic calculation of EPG variables.

The electrical penetration graph (EPG) technique is the most powerful tool for studying the feeding behavior of pierce-sucking insects. However, calculating EPG variables is often very time-consuming, and consequently, several software programs have been developed for the automatic calculation of EPG variables. Here we present a new user-friendly Excel Workbook that uses a standardized list of EPG variables and follows expert guidelines for calculating them. The program developed in Visual Basic for Applications (VBA) is a step up from the existing software and allows easy data analysis and interpretation. It also includes a novel option for dealing with the common problem of "truncated"-waveforms artificially terminated by the end of recording. The only requirement to run the program is Microsoft Excel software running under a PC environment. The Workbook was validated by calculating variables from EPG recordings of aphids and psyllids and the results obtained were compared with those of existing software such as the Sarria Workbook. Our EPG Workbook provides researchers with a reliable and standardized tool for the automatic calculation of up to 127 EPG variables from phloem-sap-sucking insects.