The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium.

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Bacterial Pesticidal Protein Mpp51Aa1 Delivered via Transgenic Citrus Severely Impacts the Fecundity of Asian Citrus Psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri vectors the causative agent of citrus greening disease that has the capacity to decimate citrus production. As an alternative and more sustainable approach to manage D. citri than repeated application of chemical insecticides, we investigated the potential use of the bacteria-derived pesticidal protein, Mpp51Aa1, when delivered by transgenic Citrus sinensis cv. Valencia sweet orange or Citrus paradisi cv. Duncan grapefruit. Following confirmation of transcription and translation of mpp51aa1 by transgenic plants, no impact of Mpp51Aa1 expression was seen on D. citri host plant choice between transgenic and control Duncan grapefruit plants. A slight but significant drop in survival of adult psyllids fed on these transgenic plants was noted relative to those fed on control plants. In line with this result, damage to the gut epithelium consistent with that caused by pore-forming proteins was only observed in a minority of adult D. citri fed on the transgenic Duncan grapefruit. However, greater impacts were observed on nymphs than on adults, with a 40% drop in the survival of nymphs fed on transgenic Duncan grapefruit relative to those fed on control plants. For Valencia sweet orange, a 70% decrease in the number of eggs laid by adult D. citri on transgenic plants was noted relative to those on control plants, with a 90% drop in emergence of progeny. These impacts that contrast with those associated with other bacterial pesticidal proteins and the potential for use of Mpp51Aa1-expressing transgenic plants for suppression of D. citri populations are discussed. IMPORTANCE Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri. This insect is particularly important as the vector of the causative agent of citrus greening, or huanglongbing disease, which severely impacts global citrus production. In this study, we investigated the potential of transgenic citrus plants that produce the pesticidal protein Mpp51Aa1. While adult psyllid mortality on transgenic plants was modest, the reduced number of eggs laid by exposed adults and the decreased survival of progeny was such that psyllid populations dropped by more than 90%. These results provide valuable insight for potential deployment of Mpp51Aa1 in combination with other control agents for the management of D. citri.

Influence of 'Candidatus Liberibacter asiaticus' infection on the susceptibility of Asian citrus psyllid, Diaphorina citri to Bacillus thuringiensis pesticidal proteins, Mpp51Aa1 and Cry1Ba1.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) transmits the Gram-negative bacterium 'Candidatus Liberibacter asiaticus' that causes citrus greening disease. While chemical control has been the main management strategy for limiting D. citri, the widespread usage of chemical sprays has decreased the susceptibility of D. citri to most insecticides. Pesticidal proteins produced by the bacterium Bacillus thuringiensis (Bt) are active against a wide variety of insects and provide a more sustainable approach to insect control. Herein, we investigated the impact of 'Ca. L. asiaticus' infection of D. citri on the toxicity of two Bt proteins (Mpp51Aa1 and Cry1Ba1). Proteins were delivered to healthy and 'Ca. L. asiaticus'-infected D. citri via topical feeding application. The LC50 values of Mpp51Aa1 and Cry1Ba1 were calculated for both nymphs and adults. Additionally, we evaluated the effect of each protein on the survival probability and life span of healthy and 'Ca. L. asiaticus'-infected D. citri. The LC50 values indicated that adults and nymphs were more susceptible to Mpp51Aa1 than to Cry1Ba1 in both healthy and 'Ca. L. asiaticus'-infected D. citri. 'Ca. L. asiaticus'-infected adults and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than healthy insects, and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than adults. Moreover, we found that Mpp51Aa1 had a greater impact than Cry1Ba1 on the survival and lifespan of adults, and 'Ca. L. asiaticus'-infected insects were more affected by these pesticidal proteins than healthy adults. These results have important implications for the use of pesticidal proteins in D. citri management in Florida and elsewhere given the widespread presence of 'Ca. L. asiaticus' in the D. citri population. In this era of eco-friendly control strategies, Bt-derived pesticidal proteins provide a promising avenue to reducing the application of chemical insecticides for D. citri management.

Genomic sequencing of fourteen bacillus thuringiensis isolates: insights into geographic variation and phylogenetic implications.

OBJECTIVE: This work was performed in support of a separate study investigating the activity of pesticidal proteins produced by Bacillus thuringiensis against the Asian citrus psyllid, Diaphorina citri. The fourteen Bacillus isolates chosen were selected from a large, geographically diverse collection that was characterized only by biochemical phenotype and morphology of the parasporal crystal, hence, for each isolate it was desired to determine the specific pesticidal proteins produced, assign each to a Bacillus cereus multilocus sequence type (ST), and predict their placement within the classical Bt serotyping system. In addition, phylogenetic distances between the isolates and Bacillus thuringiensis serovar type strains were determined by calculating digital DNA-DNA hybridization (dDDH) values among the isolates. RESULTS: Based on the assembled sequence data, the isolates were found to be likely representatives of the Bt serovars kurstaki (ST 8), pakistani (ST 550), toumanoffi (ST 240), israelensis (ST 16), thuringiensis (ST 10), entomocidus (ST 239), and finitimus (ST 171). In cases where multiple isolates occurred within a predicted serovar, pesticidal protein profiles were found to be identical, despite the geographic diversity of the isolates. As expected, the dDDH values calculated for pairwise comparisons of the isolates and their apparent corresponding Bt serovar type strains were quite high (> 98%), however dDDH comparisons of the isolates with other serovar type strains were often surprisingly low (< 70%) and suggest unrecognized taxa within Bt and the Bacillus cereus sensu lato.

Bacteria-derived pesticidal proteins active against hemipteran pests.

Hemipteran pests are among the most important threats to agricultural production. Losses associated with these insects result from both feeding-associated damage and the transmission of plant pathogens by some species. Key among hemipteran pests of agricultural importance are stink bugs, whitefly, aphids and psyllids. While bacteria provide an excellent resource for identification of environmentally benign pesticidal proteins for use against pest insects, relatively few with activity against hemipteran species have been identified. In this comprehensive review including the patent literature, we describe physiological features unique to Hemiptera that may restrict the toxicity of bacterial pesticidal proteins, provide an overview of Hemiptera-active pesticidal proteins and associated structural classes, and summarize biotechnological strategies used for optimization of toxicity against target hemipteran species.

Engineering of Cry3Bb1 provides mechanistic insights toward countering western corn rootworm resistance.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

Composition and abundance of midgut surface proteins in the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the vector of Candidatus Liberibacter asiaticus (CLas), the presumed causative agent of citrus greening disease. For successful transmission, CLas must cross the gut barrier, requiring interaction with proteins on the midgut epithelium. We compared the relative abundance of gut surface proteins for both adult and nymph D. citri, as nymphs are particularly susceptible to CLas infection. To enrich for gut surface proteins, brush border membrane vesicles were prepared from dissected guts, and proteins identified from triplicate samples run on a timsTOF mass spectrometer. A total of 1516 and 1219 proteins were identified from D. citri adults and nymphs respectively. Based on bioinformatics analysis software and manual curation, 112 adult and 87 nymph proteins were predicted to localize to the surface of the microvilli and were further categorized into integral membrane and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteins exploited by insect pathogens such as aminopeptidase, alkaline phosphatase, cadherin, ABC transporters, and carboxypeptidase were among the most abundant proteins on the gut surface. In addition to providing insights into hemipteran gut physiology, the D. citri gut surface proteome will inform novel approaches to interfere with CLas interaction with the psyllid gut to prevent the spread of citrus greening. BIOLOGICAL SIGNIFICANCE: The Asian citrus psyllid (ACP), D. citri is one of the most serious pests of citrus worldwide. ACP transmits the pathogenic bacterium that causes citrus greening or huanglongbing (HLB), which has resulted in severe economic losses in global citriculture. The putative causative agent of this disease, the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), is vectored by the Asian citrus psyllid, D. citri, in a persistent and circulative manner. CLas must interact with gut surface proteins in order to enter midgut epithelial cells. However, the specific proteins exploited by CLas have yet to be identified. The characterization of the most abundant proteins on the surface of the D. citri gut provides insight into candidate receptors for CLas and other pathogens of D. citri. We hypothesize that pathogens of D. citri exploit the most abundant proteins on the surface of the gut for entry into the host insect. Importantly, the abundant gut surface proteins will provide the basis for novel approaches to disrupt CLas-D. citri interactions, with the goal of preventing further economic loss to the citrus industry.

BPPRC database: a web-based tool to access and analyse bacterial pesticidal proteins.

Pesticidal proteins derived from the bacterium Bacillus thuringiensis, have provided the bases for a diverse array of pest management tools ranging from natural products used in organic agriculture, to modern biotechnological approaches. With advances in genome sequencing technologies and protein structure determination, an increasing number of pesticidal proteins from myriad bacterial species have been identified. The Bacterial Pesticidal Protein Resource Center (BPPRC) has been established to provide informational and analytical resources on the wide range of pesticidal proteins derived from bacteria that have potential utility for arthropod management. In association with a revised nomenclature for these proteins, BPPRC contains a database that allows users to browse and download sequences. Users can search the database for the best matches to sequences of interest and can incorporate their own sequences into basic informatic analyses. These analyses include the ability to draw and export guide trees from either whole protein sequences or, in the case of the three-domain Cry proteins, from individual domains. The associated website also provides a portal for users to submit protein sequences for naming. The BPPRC provides a single authoritative source of information to which all stakeholders can be referred including academics, government regulatory bodies and research and development personnel in the industrial sector. The database provides information on more than 1060 pesticidal proteins derived from 13 species of bacteria, including insecticidal activities for a subset of these proteins. Database URL: www.bpprc.org and www.bpprc-db.org/.

Anopheles gambiae strain (Ag55) cultured cells originated from Anopheles coluzzii and are phagocytic with hemocyte-like gene expression.

Anopheles gambiae and Anopheles coluzzii are closely related species that are predominant vectors of malaria in Africa. Recently, A. gambiae form M was renamed A. coluzzii and we now conclude on the basis of a diagnostic PCR-restriction fragment length polymorphism assay that Ag55 cells were derived from A. coluzzii. We established an Ag55 cell transcriptome, and KEGG pathway analysis showed that Ag55 cells are enriched in phagosome pathway transcripts. The Ag55 transcriptome has an abundance of specific transcripts characteristic of mosquito hemocytes. Functional E. coli bioparticle uptake experiments visualized by fluorescence microscopy and confocal microscopy and quantified by flow cytometry establish the phagocytic competence of Ag55 cells. Results from this investigation of Ag55 cell properties will guide researchers in the use and engineering of the Ag55 cell line to better enable investigations of Plasmodium, other microbes, and insecticidal toxins.

Streamlined phage display library protocols for identification of insect gut binding peptides highlight peptide specificity.

Phage display libraries have been used to isolate insect gut binding peptides for use as pathogen transmission blocking agents, and to provide artificial anchors for increased toxicity of bacteria-derived pesticidal proteins. Previously, phage clones displaying enriched peptides were sequenced by Sanger sequencing. Here we present a streamlined protocol for identification of insect gut binding peptides, using insect-appropriate feeding strategies, with next generation sequencing and tailored bioinformatics analyses. The bioinformatics pipeline is designed to eliminate poorly enriched and false positive peptides, and to identify peptides predicted to be stable and hydrophilic. In addition to developing streamlined protocols, we also sought to address whether candidate gut binding peptides can bind to insects from more than one order, which is an important consideration for safe, practical use of peptide-modified pesticidal proteins. To this end, we screened phage display libraries for peptides that bind to the gut epithelia of two pest insects, the Asian citrus psyllid, Diaphorina citri (Hemiptera) and beet armyworm, Spodoptera exigua (Lepidoptera), and one beneficial insect, the western honey bee, Apis mellifera (Hymenoptera). While unique peptide sequences totaling 13,427 for D. citri, 89,561 for S. exigua and 69,053 for A. mellifera were identified from phage eluted from the surface of the insect guts, final candidate pools were comprised of 53, 107 and 1423 peptides respectively. The benefits of multiple rounds of biopanning, along with peptide binding properties in relation to practical use of peptide-modified pesticidal proteins for insect pest control are discussed.

A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins.

In 1998 a nomenclature for the growing list of pesticidal proteins from Bacillus thuringiensis (Bt) was derived based solely on protein sequence comparisons. This nomenclature was widely adopted and provided a robust framework for the naming and classification of the proteins. The success of these proteins in integrated pest management schemes prompted an increased effort to find others with improved or more diverse activities. These discovery activities led to the characterization of proteins from a wider range of bacteria and with a variety of different protein folds. Since most of these new proteins were grouped together as Cry proteins it became apparent that the existing nomenclature had limitations in representing the diverse range of proteins that had been identified. This revised nomenclature retains the basic principles of the 1998 version but provides specific mnemonics to represent different structural groups. For the purposes of consistency, the vast majority of the proteins have either retained their name or have a new name that clearly references the previous one. Other pesticidal proteins not previously included in the nomenclature have been incorporated into this version.

High throughput sequencing reveals Drosophila suzukii responses to insecticides.

Global climate change and acquired resistance to insecticides are threats to world food security. Drosophila suzukii, a devastating invasive pest in many parts of the world, causes substantial economic losses to fruit production industries, forcing farmers to apply broad-spectrum insecticides frequently. This could lead to the development of insecticide resistance. We determined the Lethal Concentration 50 (median lethal concentration, LC50 ) values of zeta-cypermethrin, spinosad, and malathion insecticides against D. suzukii colonies established from Clarke and Pierce county Georgia, United States. The LC50 values were 3 fold higher in the Pierce county population for all insecticide treatments. We then used RNA sequencing to analyze the responses of Pierce and Clarke population flies surviving a LC50 treatment of the 3 insecticides. We identified a high number of differentially expressed genes that are likely involved in detoxification and reduced cuticular penetration, especially in the Pierce population, with extensive overlap in differentially expressed genes between the 3 insecticide treatments. Finally, we predicted fewer nonsynonymous single nucleotide variants having deleterious effects on protein function among detoxification, insecticide target, and cuticular protein encoding genes in Pierce flies. Thus a combination of increased gene expression and fewer deleterious single nucleotide variants highlights molecular mechanisms underlying the higher LC50 values for Pierce population flies.

Anopheles gambiae Ag55 cell line as a model for Lysinibacillus sphaericus Bin toxin action.

Binary toxin (Bin) produced by Lysinibacillus sphaericus is toxic to Culex and Anopheles mosquito larvae. It has been used world-wide for control of mosquitoes that vector disease. The Bin toxin interacts with the glucosidase receptor, Cpm1, in Culex and its orthologue, Agm3, in Anopheles mosquitoes. However, the exact mechanism of its mode of action is not clearly understood. It is essential to understand mode of action of Bin toxin to circumvent the resistance that develops over generations of exposure. A suitable model cell line will facilitate investigations of the molecular action of Bin toxin. Here we report Bin toxin activity on Ag55 cell line that has been derived from an actual target, Anopheles gambiae larvae. The Bin toxin, both in pro and active forms, kills the Ag55 cells within 24h. Bin toxin internalizes in Ag55 cells and also induces vacuolation as tracked by Lysotracker dye. The dose response studies showed that 1.5nM of Bin toxin is sufficient to induce vacuolation and Ag55 cell death. Presence of α-glucosidase gene (Agm3) expression in the Ag55 cells was also confirmed. Thus, Ag55 cells constitute an appropriate model system to decipher the mode of Bin action in mosquito larvae.