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Scanning and transmission electron microscopy are used to elucidate the internal morphology of the feeding structures of the western flower thrips, Frankliniella occidentalis (Thysanoptera : Thripidae). The position of the single mandible... more
Scanning and transmission electron microscopy are used to elucidate the internal morphology of the feeding structures of the western flower thrips, Frankliniella occidentalis (Thysanoptera : Thripidae). The position of the single mandible and paired maxillary stylets, relative to one another within the mouthcone, are shown through sequential thin sectioning. Observation of thin sections of all 3 stylets demonstrate them to be innervated. Within the mandibular stylet are 3 dendrites extending its length, and 2 lateral dendrites in its wall. Each maxillary stylet contains 4 dendritic canals, each with one dendrite. The location of the cibarium within the mouthcone is examined and the attachments of its dilator muscles are shown. One to 3 dendrites, varying in functional type, are found in each paraglossal sensillum. The ultrastructural evidence presented suggests that some paraglossal sensilla have a dual chemosensory and mechanosensory function, while others are apparently solely mechanosensory. The significance of these findings relative to the feeding mechanisms of thrips and the possible function of stylet and paraglossal sensory structures in host location, feeding site selection and host choice are discussed.
Emani, C., Hunter, WB. 2013. Insect pests exhibit a diverse array of genetic-based responses when interacting with crop systems; these changes can be in response to pathogens, symbiotic microbes, host plants, chemicals, and the... more
Emani, C., Hunter, WB. 2013. Insect pests exhibit a diverse array of genetic-based responses when interacting with crop systems; these changes can be in response to pathogens, symbiotic microbes, host plants, chemicals, and the environment. Agricultural research has for decades focused on gathering crucial information on the biochemical , genetic, and molecular realms that deal with plant–insect interactions in changing ecosystems. Environmental conditions, which include the overall conditions of climate change, are a reality that needs to be considered as one of the crucial phenomena of changing ecosystems when planning future crop security and/or pest management strategies. Focused research in documenting the interactions that occur between crop systems and insect pests under changing climates will be a needed addition to current research efforts whose aim is to define future strategies that crop scientists relate to the broader societal concerns of food security in an ever-changing environment. This chapter attempts to integrate the past and present research in classical and molecular breeding, transgenic technology , and pest management within the context of climate change. The integrated approach will direct present research efforts that aim at creating plant–insect pest interaction–climate change models that reliably advise future strategies to develop improved insect-resistant, climate-resilient plant varieties.
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Hail, DA, Dowd, S, Hunter, WB, Bextine, BR. 2010. The potato psyllid is the vector of the α-Proteobacteria Candidatus Liberibacter psyllaurous, the causal agent of Zebra Chip in potatoes. The disease is a major limiting factor in both the... more
Hail, DA, Dowd, S, Hunter, WB, Bextine, BR. 2010. The potato psyllid is the vector of the α-Proteobacteria Candidatus Liberibacter psyllaurous, the causal agent of Zebra Chip in potatoes. The disease is a major limiting factor in both the production and processing of potatoes into chips with no cure. RNAi has the potential to limit the spread and severity of Zebra Chip by reducing the insect load per field per season. Pyrosequencing was used to identify target genes and synthetic dsRNA constructs were designed to block the activity of heat shock protein 70 and heat shock cognate 70. These molecular chaperones are intimately involved in essentially all life processes by folding and stabilizing other proteins. In this study these constructs were tested on potato psyllid cell cultures and morphological evidence showed shriveling and dispersal into the media. We plan to target other genes and deliver RNAi to insects via the plant through a root soaking procedure.
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&amp... more
'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.
... Principal Investigator: Blake Bextine Dept. of Biology University of Texas, Tyler Tyler, TX 75799 bbextine@uttyler.edu Co-Principal Investigator: Wayne Hunter USDA, ARS Fort Pierce, FL 34945 wayne.hunter@ars.usda.gov Researchers:... more
... Principal Investigator: Blake Bextine Dept. of Biology University of Texas, Tyler Tyler, TX 75799 bbextine@uttyler.edu Co-Principal Investigator: Wayne Hunter USDA, ARS Fort Pierce, FL 34945 wayne.hunter@ars.usda.gov Researchers: Chelsea Swatsell Dept. ...
Page 1. Comparison of Feeding Behavior of the Potato Leafhopper Empoasca fabae (Homoptera: Cicadellidae) on Alfalfa and Broad Bean Leaves ELAINE A. BACKUS ANDWAYNE B. HUNTER' Department of Entomology, University of Missouri,... more
Page 1. Comparison of Feeding Behavior of the Potato Leafhopper Empoasca fabae (Homoptera: Cicadellidae) on Alfalfa and Broad Bean Leaves ELAINE A. BACKUS ANDWAYNE B. HUNTER' Department of Entomology, University of Missouri, Columbia, Missouri65211 ...
We successfully developed Asian citrus psyllid cell cultures as a tool to subculture Canidatus Liberibacter asiaticus (Las), which is a fastidious bacterium that reportedly replicates within the psyllid host. This novel approach uses... more
We successfully developed Asian citrus psyllid cell cultures as a tool to subculture Canidatus Liberibacter asiaticus (Las), which is a fastidious bacterium that reportedly replicates within the psyllid host. This novel approach uses psyllid cell cultures as the medium to isolate and culture this bacterium to permit further research. The Asian Citrus Psyllid (ACP), Diaphorina citri, (Hemiptera: Psyllidae) is a highly competent vector of the phloem-inhabiting bacterium (Las), associated with the disease Huanglongbing (HLB). World-wide HLB has become a major limiting factor to the production of citrus. A specific psyllid cell culture medium was developed during the screening of 12 commercially available insect cell culture media. Attempts were made using starter cells from embryos and midgut tissues. Cells and tissues could be maintained for up to 4 months. The developed psyllid culture medium, Hert-Hunter-70, provided the best growth rates with doubling times at ~ten days. Previous attempts to use a lepidopteran cell culture, Spodoptera frugiperda (Sf9), to culture Liberibacter were unsuccessful, suggesting further that a psyllid cell culture would be more permissive. Psyllid cell cultures are also being used to propagate and study psyllid viral pathogens.
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Transmission electron microscopy was used to confirm the presence of picorna-like virus particles presumed to be Homalodisca coagulata virus-1 (HoCV-1) in the midgut region of adult glassy-winged sharpshooters (GWSS). In addition, we... more
Transmission electron microscopy was used to confirm the presence of picorna-like virus particles presumed to be Homalodisca coagulata virus-1 (HoCV-1) in the midgut region of adult glassy-winged sharpshooters (GWSS). In addition, we offer a reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of this virus with a sensitivity of 95 genome equivalents. A survey employing this assay in conjunction with GWSS samples collected throughout the USA including California, Hawaii, Florida Georgia, and Carolinas revealed a fairly widespread pattern of distribution, although potentially restricted to temperate regions, areas with elevated host densities, or to populations of a common origin. The virus was found to naturally infect adults regardless of host plant and was not specific to a particular life stage or sex. Examination of alternate leafhopper species demonstrated that, although  infection is not ubiquitous to all sharpshooter genera, HoCV-1 is not limited to Homalodisca vitripennis (aka. H. coagulata).
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The salivary glands of the Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar 1821, (syn. H. coagulate, Hemiptera: Cicadellidae) were collected and used to produce a cDNA library. Examination by BLASTX analyses identified 2... more
The salivary glands of the Glassy-winged sharpshooter (GWSS),
Homalodisca vitripennis Germar 1821, (syn. H. coagulate, Hemiptera: Cicadellidae) were collected and used to produce a cDNA library. Examination by BLASTX analyses identified 2 viral sequences, one a 610-base pair fragment and a second 839-base pair fragment, both of which had significant homology to viruses within the genus Phytoreovirus . Resequencing of the fragments confirmed sequence validities. These sequences were used for in silico protein translation and BLASTP analysis confirming the established homology. While the GWSS is the primary vector of Pierce’s disease of grapes, this is the first report that GWSS may be a vector of phytoreoviruses.  Phytoreoviruses are transmitted in a propagative manner by cicadellid leafhoppers (Hemiptera: Cicadellidae), which acquire and transmit them during feeding.  phytoreoviruses have been reported from Agallian, Agalliopsis, Nephotettix , and Recilia , genera of leafhoppers, with evidence for transovarial  transmission. The GWSS, although considered to feed primarily from the xylem, ingests from other plant tissues, such as the phloem and mesophyll during probing similar to other leafhoppers. Monitoring for an increase of Phytoreovirus spread in graminaceous crops that are in proximity to vineyards or tree crop orchards, where GWSS occurs, such as in southern California, will provide a better understanding of the potential role of the GWSS as a disease vector in the spread of phytoreoviruses and other plant pathogens. Sequences have been deposited in NCBI database under accession numbers (EF058280) for GWSS-V1, WHSg013C11 and (EF058281) for GWSS-V2, WHSg024H02.
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The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt... more
The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5′-proximal ORF (ORF1, nt 420–5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3′ ORF2 (5990–8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.
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Two methods used for the clearing of gut contents for plant-feeding hemipterans were evaluated: (i) non-viral host feeding and (ii) artificial diet feeding by monitoring the permanence of ingested plant mRNA transcripts within... more
Two methods used for the clearing of gut contents for plant-feeding  hemipterans were evaluated: (i) non-viral host
feeding and (ii) artificial diet feeding by monitoring the
permanence of ingested plant mRNA transcripts within
non-viruliferous and viruliferous insects. Results show that typical RNA transcripts are more stable in the alimentary canal of phloem feeders such as whiteflies (and potentially other hemipterans) than was previous thought.
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Begomoviruses have complex associations with their whitefly vecotrs. Analyses showed that Tomato Yellow Leaf Curl Virus, replicated within the whitefly Bemisia tabaci, while the Tomato mottle virus, did not. TYLCV also reduce the fitness... more
Begomoviruses have complex associations with their whitefly vecotrs. Analyses showed that Tomato Yellow Leaf Curl Virus, replicated within the whitefly Bemisia tabaci, while the Tomato mottle virus, did not.  TYLCV also reduce the fitness of B. tabaci, but ToMoV did not.
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We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and... more
We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide,  polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5Vproximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3Vproximal ORF2 (nucleotides 4390–7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st–2nd) and late (3rd–4th) instars, worker pupae, workers, sexual pupae, alates (h and U), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavatedfrom the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.
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The botanical product RD-Repelin, was highly repellent to pea aphids at 1%, 4%, or 10% concentrations. Repellency occurred prior to leaf contact by aphids at all concentrations. RD-Repelin delayed symptom expression of ZYMV in 81% of... more
The botanical product RD-Repelin, was highly repellent to pea aphids at 1%, 4%, or 10% concentrations. Repellency occurred prior to leaf contact by aphids at all concentrations. RD-Repelin delayed symptom expression of ZYMV in 81% of plants treated with 1% concentrations, although virus transmission was not prevented. The effects of RD-Repellin on aphid settling behavior, symptom expression and potenital for pest management are discussed.
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Probing behaviors of the potato leafhopper Empoasca fabae (Harris), were studied using an AC electronic monitor. Observations on two artificial media, water-sucrose and agar-sucrose, revealed three waveforms correlated with probing... more
Probing behaviors of the potato leafhopper Empoasca fabae (Harris), were studied using an AC electronic monitor.  Observations on two artificial media, water-sucrose and agar-sucrose, revealed three waveforms correlated with probing behaviors. These results plus thin-layer chromatography of the host plants and insects show that the potato leafhopper like many other typhlocybines, does not ingest strictly from the phloem but also from the mesophyll.
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The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly... more
The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. To better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing project of cDNA clones from alate aphids. Similar large-scale expressed sequence tag (EST) sequencing projects from other insects have provided a vehicle for answering biological questions relating to development and physiology. Although there is a growing database in GenBank of ESTs from insects, most are from Drosophila melanogaster and Anopheles gambiae, with relatively few specifically derived from aphids. However, important morphogenetic processes are exclusively associated with piercing-sucking insect development and sap feeding insect metabolism. In this paper, we describe the first public data set of ESTs from the brown citrus aphid, T. citricida. The cDNA library was derived from alate adults due to their significance in spreading viruses (e.g., citrus tristeza virus). Over 5180 cDNA clones were sequenced, resulting in 4263 high-quality ESTs. Contig alignment of these ESTs resulted in 2124 total assembled sequences, including both contiguous sequences and singlets. Approximately 33% of the ESTs currently have no significant match in either the non-redundant protein or nucleic acid databases. Sequences returning matches with an E-value of ≤ -10 using BLASTX, BLASTN, or TBLASTX were annotated based on their putative molecular function and biological process using the Gene Ontology classification system. These data will aid research efforts in the identification of important genes within insects, specifically aphids and other sap feeding insects within the Order Hemiptera
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Scanning electron microscopy was used to elucidate the morphology and sequential movement of thrips feeding structures in the 2 species, Frankliniella occidentalis and F. schultzei, (Thysanoptera : Thripidae). The mouthcone consists of... more
Scanning electron microscopy was used to elucidate the morphology and sequential movement of thrips feeding structures in the 2 species, Frankliniella occidentalis and F. schultzei, (Thysanoptera : Thripidae). The mouthcone consists of paired paraglossae, and fringed labral pad, a single, apically fused mandible and a pair of interlocking maxillae. The maxillae are open apically and form a feeding tube. Ten pairs of sensory pegs of 3 distinct morphological types (sensilla basiconica with a cuticular collar, sensilla basiconica without a cuticular collar, and sensilla trichoidea) were found on the paraglossae. The possible function of these sensory structures in host finding and choice are discussed. No structure for rasping the leaf's surface was found on the mouthcone or the ventral surface of the insect. Live specimens were observed feeding through Parafilm on artificial media (sucrose solution) and lettuce leaf tissue. These observations support earlier findings that thrips feed by piercing leaf cells with the mandible and ingesting cell contents through the feeding tube formed by the maxillary stylets. Based on these findings, we suggest that thrips be classified as piercing-sucking rather than rasping-sucking insects.
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An in vitro feeding method for acquisistion of impatiens necrotic spot tospovirus and tomato spotted wilt tospovirus by western flower thrips (Frankliniella occidentalis (Pegande) was develped. This is the first system developed in which... more
An in vitro feeding method for acquisistion of impatiens necrotic spot tospovirus and tomato spotted wilt tospovirus by western flower thrips (Frankliniella occidentalis (Pegande) was develped. This is the first system developed in which tospovirus viability was maintained in an in vitro technique specifically designed for thips feeding. Thrips were caged within a tube cage, with parafilm covering both ends. Leaf extracts from virus infected N. benthamiana or Datura stramoniium were prepared.  Following one cycle of differential centrifugation, the extract was placed on the membrane and a second membrane placed over the liquid, thus securing the plant extract between two parafilm membranes. After 24 h acquisition access feeding period thrips were kept on green bean pods until 10 d old. ELISA analyses showed that acquisition feeding of virus suspensions prepared from infected plants resulted in positively detected virus in thrips.
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The location of tomato mottle virus (ToMoV) and cabbage leaf curl virus (CabLCV) (Geminiviridae, genus Begomovirus) in the whitefly vector Bemisia tabaci B-biotype (Homoptera: Aleyrodidae) was elucidated using a novel technique... more
The location of tomato mottle virus (ToMoV) and cabbage leaf curl virus (CabLCV) (Geminiviridae, genus Begomovirus) in the whitefly vector Bemisia tabaci B-biotype (Homoptera: Aleyrodidae) was elucidated using a novel technique incorporating indirect immunofluorescent labeling in freshly dissected whiteflies. Begomoviruses were visualized in the whitefly by indirect-fluorescent-microscopy. Polyclonal and monoclonal primary antibodies were used to successfully detect both ToMoV and CabLCV. Both begomoviruses were located in the anterior region of the midgut and filter-chamber of adult whiteflies, with ToMoV detected in the salivary glands. CabLCV was detected at a greater frequency than ToMoV, with a positive detection of 16% (89 out of 560) for CabLCV and 3% (25 out of 840) for ToMoV. Possible sites involved in geminivirus transport from the gut lumen of whiteflies into the hemocoel were located in the filter-chamber and anterior portion of the midgut. The location of these begomoviruses suggests a possible scenario of virus movement through the whitefly.
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Invertebrate iridescent virus 6 (IIV6) was evaluated for mode of transmission and ability to cause infection in the root weevil, Diaprepes abbreviatus (L.). This is the first evidence of IIV6 infection in D. abbreviatus, which caused both... more
Invertebrate iridescent virus 6 (IIV6) was evaluated for mode of transmission and ability to cause infection in the root weevil,
Diaprepes abbreviatus (L.). This is the first evidence of IIV6 infection in D. abbreviatus, which caused both patent and sub-lethal covert infections in both larvae and adults. Adults and larvae were successfully infected with IIV6 by puncture, injection and per os. Transmission of IIV6 was demonstrated between infected and healthy individuals regardless of gender. Virus was detected in egg masses produced by virus-infected females suggesting IIV6 is transmitted transovarially. Virus particles were observed in the cytoplasm of weevil cells, and were shown to infect fat bodies, muscle, and nerve tissues, as visualized using TEM. Patent infections resulted in death of individuals within 3 to 4 days post infection. Individuals with covert infections tested positive for virus infection on day 7 by polymerase chain reaction analysis. Sequencing of PCR amplicons confirmed virus infection. Discovery of new pathogens against root weevils may provide new management tools for development of control strategiesbased on induced epizootics. This is the first report of a virus infecting D. abbreviatus.
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Whitefly cells were inoculated with IIV-6 developed cytopathic effects (CPE) which indicated virus replication, the BtB-2.97 cell line, Infected cells became more rounded and developed inclusions that increased in size over the infection... more
Whitefly cells were inoculated with IIV-6 developed cytopathic
effects (CPE) which indicated virus replication, the BtB-2.97 cell line, Infected cells became more rounded and developed inclusions that increased in size over the infection period.
Examination of infected cells by electron microscopy revealed that an electron-dense virogenic stroma had formed and that the icosahedral virus accumulated only in the cytoplasm, which is characteristic of iridoviruses.  Immunofluorescent staining was used to detect virus structural proteins within the cells. Whitefly cells were grown on coverslips and infected with IIV-6 (multiplicity of infection (moi) of 10). The coverslips were removed at various times postinfection, fixed in 3.7% formaldehyde, and permeabilized for 10 min in acetone
at 220°C. The cells were probed with an anti-IIV-6 antibody (a gift from Dr. J. Kalmakoff) for 30 min (1:500 dilution), then probed with an anti-rabbit antibody labeled with Alexa Fluor 488 (Molecular Probes Inc.) (30 min, 1:200 dilution), and counterstained with 300 nM 49,6-diamidino-2-phenylindole.
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Report on the development and viral infection of a Diaprepes root weevil cell culture. Embryonic tissues of the root weevil were used to establish cell cultures for use in screening viral pathogens as potential biological control agents.... more
Report on the development and viral infection of a Diaprepes root weevil cell culture. Embryonic tissues of the root weevil were used to establish cell cultures for use in screening viral pathogens as potential biological control agents. Tissues were seeded into a prepared solution of insect medium and kept at a temperature of 24 C. The cell culture had primarily fibroblast-like morphology with some epithelial monolayers. Root weevil cells were successfully infected in vitro with a known insect virus, Invertebrate Iridescent Virus 6. Potential uses of insect cell cultures and insect viruses are discussed.
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The whitefly Bemisia tabaci (Gennadius) is a pest of many important food and fiber crops. This whitefly is also a vector of more than 70 plant-infecting viruses. A cell line was established in vitro using embryonic tissues from the eggs... more
The whitefly Bemisia tabaci (Gennadius) is a pest of many important food and fiber crops. This whitefly is also a vector of more than 70 plant-infecting viruses. A cell line was established in vitro using embryonic tissues from the eggs of Bemisia
tabaci (Gennadius), B biotype (pseudonym B. argentifolii
Bellows & Perring), and referred to as ‘Btb(Ba)97, Hunter-Polston’. Cell cultures were successfully inoculated with begomovirus (BGMV and ToMoV)-infected tomato plant sap. Embryonic tissues were seeded into Kimura’s modified medium and kept at a temperature of 24°C. Continuous cell cultures were established and have since undergone 92 passages in 25-cm2 flasks. Cell doubling time is approximately 3 days and the cells have been successfully revived after 1 year after storage at 280°C. The cell population is monolayers of
predominately fibroblast with some epithelial cells. Begomoviruses (bean golden mosaic begomovirus, BGMV, and tomato mottle begomovirus, ToMoV) were inoculated to the cell cultures independently and detected by labeling by an indirect immunofluorescence technique. The viruses were detected bound to the cell membranes and within the cell cytoplasm. This is the first report of a continuous cell line established from a species of whitefly and its inoculation with two begomoviruses. The successful inoculation of whitefly cell
cultures with begomoviruses shown in our results represents
great promise for the development of systems that allow researchers to achieve a better understanding of the complex relationship between begomoviruses and their whitefly vectors.
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First report of a pathogenic virus in citrus aphids, and its subsequent transovarial transmission. Aphids are important horticultural pests which have few biological control agents available for use in their management. A known... more
First report of a pathogenic virus in citrus aphids, and its subsequent transovarial transmission. Aphids are important horticultural pests which have few biological control agents available for use in their management. A known entomopathogenic virus, insect iridescent virus 6 (IIV6), was
evaluated for pathogenicity to the brown citrus aphid, Toxoptera citricida (Kirkaldy). The virus, an Iridovirus, is a dsDNA virus, with an icosahedral symmetry and is ~120 nm in diameter. Although invertebrate iridescent iridoviruses are known to infect insects, this is the first report of an iridovirus infection in citrus aphids that is pathogenic. Modes of transmission for iridoviruses in other insect systems have been shown to be through oral ingestion and cuticular wounding. We demonstrated virus acquisition by aphids through oral ingestion via artificial membrane feeding experiments, and show the first evidence of vertical, transovarial transmission of an iridovirus, as confirmed by PCR analysis of virus-infected nymphs. Virus fed aphids had a decrease in longevity and reduced fecundity over control aphids. At 19 d post-treatment, the control aphid population had a 30% mortality rate, while the virus-fed aphid adults had a 68.3% mortality rate. Virus-fed aphids had a lower fecundity rate, producing on average 1.6 nymphs per day, while control aphids produced an average of 3 nymphs per day. Entomopathogenic viruses in aphids need to be re-examined for use in developing molecular tools and for their potential as biological control agents.
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Adult whiteflies, Bemisia tabaci (Gennadius), collected from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected... more
Adult whiteflies, Bemisia tabaci (Gennadius), collected
from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected whiteflies were applied to cell cultures and checked for cytopathic effects (CPE). Cells were observed to develop cytoplasmic inclusions and to have a change in morphology. Cells displaying CPE were observed using a transmission electron microscope and
found to be infected with a virus. The virus particles had
an icosahedral shape and an approximate size of 120–130 nm. The virus was observed in defined areas of the cytoplasm adjacent to the cell nucleus. Analysis using PCR reaction, Southern blot hybridization, and DNA sequencing confirmed that the virus discovered infecting the whitefly cell cultures was an iridovirus. Sequence analysis showed that the amplimer (893
bp) had a 95% homology to the invertebrate iridescent virus type 6 major capsid protein gene. Discovery of new viruses of whiteflies may provide renewed interest in using pathogens in the development of innovative management strategies. This is the first report of an iridescent virus isolated from whiteflies, B. tabaci, collected from the field.
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Internal anatomy of the anterior alimentary canal of the whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae) B-biotype, was examined by light, scanning, and transmission electron microscopy to elucidate the location and number of... more
Internal anatomy of the anterior alimentary canal of the whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae) B-biotype, was examined by light, scanning, and transmission electron microscopy to elucidate the location and number of precibarial and cibarial gustatory sensilla. Elucidation of the epipharyngeal organ complex within the precibarium revealed 10 precibarial sensilla located proximal to where the paired maxillary stylets diverge on their retraction. The sensory organ complex within the cibarium consists of 8 sensilla, 6 on the epipharyngeal sclerite with 2 found within the hypopharyngeal sclerite. Fine structure investigation revealed the individual neurons to terminate at sensillar pores, which allow direct contact with passing fluids, thus supporting a chemosensory function.
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The internal anatomy of the mouthcone of the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera : Thripidae), was examined by scanning and transmission electron microscopy to show location, number and fine... more
The internal anatomy of the mouthcone of the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera : Thripidae), was examined by scanning and transmission electron microscopy to show location, number and fine structure of the precibarial and cibarial chemosensilla. Four precibarial sensilla are located approximately 12 um from the tip of the mouthcone, proximal to where the maxillary stylets diverge on their retraction. The cibarium has 20 sensilla, 18 on the epipharyngeal sclerite and 2 on the hypopharyngeal sclerite. The fine structure of individual neurons and their termination at a pore opening into the precibarium and cibarium, respectively, supports a chemosensory function.
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A technique that allows the staining and observation of whole leafhopper salivary sheaths left behind in plant tissue is described. A derivation of McBride's acid fuchsin stain for fungal hyphae was used, followed by clearing of the plant... more
A technique that allows the staining and observation of whole leafhopper salivary sheaths left behind in plant tissue is described. A derivation of McBride's acid fuchsin stain for fungal hyphae was used, followed by clearing of the plant tissue for observation under a dissecting microscope. Data gathered included the shape and location of sheaths, and the orientation of sheaths to veins. The technique simultaneously stained eggs present in the plant tissue. Thus, measures of both feeding and oviposition were acquired from the same plant. This method is reliable in recovering known sheaths and is applicable to several hemipteran sheath-feeders. The method should be useful to researchers studying host plant resistance and other aspects of the feeding of haustellate insects.
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The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world’s food production through pollination of crops. Since fall 2006, honey bees... more
The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world’s food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus, placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNAi technology has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.
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A new viral pathogen of the glassy-winged sharpshooter, GWSS, (Homalodisca vitripennis, Germar, aka H. coagulata) was discovered and characterized. Few pathogens of leafhoppers have been discovered which have potential for use as a... more
A new viral pathogen of the glassy-winged sharpshooter, GWSS, (Homalodisca vitripennis, Germar, aka H. coagulata) was discovered and characterized. Few pathogens of leafhoppers have been discovered which have potential for use as a biological control agent. To identify new pathogens of GWSS we used a genomic approach to isolate, sequence, and identify expressed sequence tags, ESTs, produced from field collected GWSS populations. Viral sequences were identified out of the initial 9,620 ESTs generated from single-pass 5’ end sequencing of the GWSS expression library. Processing produced 8,795 ESTs which had lengths greater than 100 nucleotides post quality and vector trimming. The ESTs had an average read length of 689 bp, and an average inset size of 899 bp. Sequences shorter than 200 contiguous bases were removed from analyses. After assembly there were 3,008 sequences, 799 contigs with an average length of 1,113 bp, and 2,209 singlets. Using these sequences to get a start, it was possible to completely sequence the full virus genome, and the virus was labeled, HoCV-1. Further analyses and characterization of HoCV-1 demonstrated that it infected and crossed the midgut barrier of GWSS. The virus was classified as a member of the family Dicistroviridae, which are single-stranded RNA viruses which do not have a DNA stage. Two other viruses were also identified which are currently being characterized and which were taxonomically unrelated to HoCV-1. These leafhopper viral pathogens appear to induce increased mortality, 40% or more, during the nymphal stages of leafhopper development and may have further applications in the management of leafhopper pests to reduce the spread of Pierce’s disease of grapes.
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Asian citrus psyllid, ACP, Diaphorina citri, Kuwayama (Hemiptera: Psyllidae) is considered the primary vector of Huanglongbing (HLB), which is a serious plant pathogenic bacterium, Candidatus Liberibacter asiaticus, that is strongly... more
Asian citrus psyllid, ACP, Diaphorina citri, Kuwayama (Hemiptera: Psyllidae) is considered the primary vector of Huanglongbing (HLB), which is a serious plant pathogenic bacterium, Candidatus Liberibacter asiaticus, that is strongly associated with severe losses in citrus production worldwide. We elected to fill a void in psyllid genomics by producing expressed sequence tags, ESTs, to adult D. citri. Use of ESTs provides immediate information on the genes being expressed within insects under set conditions. This information enables the identification of many of the genes and proteins playing a role in psyllid survival.
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Short interfering RNAs which can be used to reduce gene expression, or ‘silence’ this expression in a sequence specific manner is call RNA interference, RNAi. In the case of hemipteran pests, RNAi has only recently been shown to disrupt... more
Short interfering RNAs which can be used to reduce gene expression, or ‘silence’ this expression in a sequence specific
manner is call RNA interference, RNAi. In the case of hemipteran pests, RNAi has only recently been shown to disrupt their biology. We propose that RNAi has the potential to be applied in an area wide management strategy, thereby suppressing the pest populations to reduce disease spread. Previously we generated leafhopper genetic datasets from three species of known vectors of Xylella fastidiosa (Xf), the plant infecting bacterium which causes Pierce’s disease (PD) of grapevine. Using genomic analyses we identified a subset of potential genetic targets which may be used to suppress these sharpshooter leafhoppers. The primary disease vector, the glassy-winged sharpshooter (GWSS; Homalodisca vitripennis) vectors the Xf xylem-limited bacteria that causes PD in grapevine as well as ‘Scorch-like’ pathogens of other woody agricultural fruit, nut, and ornamental crops. But many other species of  sharp-shooters are also capable of transmitting Xf. Therefore, we chose to produce two constructs of dsRNA to Arginine kinase, one from the GWSS leafhopper, and one for the Asian citrus psyllid.  Both insects feed on citrus and transmit different bacterial pathogens. We treated several host plants (Grapevine, Citrus, Okra, and Chrysanthemum), using cut flush, to seedlings, to mature grapevines and citrus trees for this study. The dsRNA treated plants caused an increase in mortality for both the GWSS and ACP. However, when each insect fed upon the dsRNA specific to the other insect no increase in mortality was observed. RNAi due to this specificity may be an excellent treatment to reduce insect pests while protecting the non-target beneficial organisms.
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Go to AGRIS search. NATO ASI series. Series A, Life sciences (1995). Establishing thrips cell cultures to study Tospoviruses. ...
Primary cell cultures of immunocytes have been developed from the three psyllid species Cacopsylla melanoneura, Cacopsylla pyri (vectors of 'Candidatus Phytoplasma mali' and 'Candidatus Phytoplasma pyri', respectively) and... more
Primary cell cultures of immunocytes have been developed from the three psyllid species Cacopsylla melanoneura, Cacopsylla pyri (vectors of 'Candidatus Phytoplasma mali' and 'Candidatus Phytoplasma pyri', respectively) and Cacopsylla crataegi. The medium most suitable of those evaluated was Hert-Hunter 70 (HH70) psyllid medium. In fact, good survival and proliferation of the Cacopsylla immunocytes for over 60 d were observed, with mitosis activities starting at 15-d post culture. Moreover, adhesion and phagocytosis activities were confirmed for all the psyllid cell cultures by functionality tests. Morphological examination of cultured immunocytes revealed the presence of different cell types in all the three psyllid species in accordance to published data about insect immunocytes. The in vitro maintenance of psyllid immunocytes represents a powerful tool for a wide range of applications, especially for psyllid cell biology. In particular, in-depth studies on the biol...
The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly... more
The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. To better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing project of cDNA clones from alate aphids. Similar large-scale expressed sequence tag (EST) sequencing projects from other insects have provided a vehicle for answering biological questions relating to development and physiology. Although there is a growing database in GenBank of ESTs from insects, most are from Drosophila melanogaster and Anopheles gambiae, with relatively few specifically derived from aphids. However, important morphogenetic processes are exclusively associated with pierc...
... 1981a ; Stopka et al., 1999 ). Growth-promoting effects have also been more recently reported for pigs ( Kratky et al., 1997 ) and Japanese quails ( Koudela et al., 1995 ; Sl?ma et al., 1996 ). In many instances however, these ...
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Background Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium “Candidatus Liberibacter asiaticus” (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of... more
Background Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium “Candidatus Liberibacter asiaticus” (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of CLas and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to CLas infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from CLas-positive and CLas-negative D. citri using the chromosomal length D. citri genome assembly. Results Each organ had a unique transcriptome profile and response to CLas infection. Though most psyllids were infected with the bac...
First report of a pathogenic virus in citrus aphids, and its subsequent transovarial transmission. Aphids are important horticultural pests which have few biological control agents available for use in their management. A known... more
First report of a pathogenic virus in citrus aphids, and its subsequent transovarial transmission. Aphids are important horticultural pests which have few biological control agents available for use in their management. A known entomopathogenic virus, insect iridescent virus 6 (IIV6), was evaluated for pathogenicity to the brown citrus aphid, Toxoptera citricida (Kirkaldy). The virus, an Iridovirus, is a dsDNA virus, with an icosahedral symmetry and is ~120 nm in diameter. Although invertebrate iridescent iridoviruses are known to infect insects, this is the first report of an iridovirus infection in citrus aphids that is pathogenic. Modes of transmission for iridoviruses in other insect systems have been shown to be through oral ingestion and cuticular wounding. We demonstrated virus acquisition by aphids through oral ingestion via artificial membrane feeding experiments, and show the first evidence of vertical, transovarial transmission of an iridovirus, as confirmed by PCR analysis of virus-infected nymphs. Virus fed aphids had a decrease in longevity and reduced fecundity over control aphids. At 19 d post-treatment, the control aphid population had a 30% mortality rate, while the virus-fed aphid adults had a 68.3% mortality rate. Virus-fed aphids had a lower fecundity rate, producing on average 1.6 nymphs per day, while control aphids produced an average of 3 nymphs per day. Entomopathogenic viruses in aphids need to be re-examined for use in developing molecular tools and for their potential as biological control agents.
The Agricultural Research Service (ARS) is the intramural research agency of the United States Department of Agriculture (USDA) which addresses basic scientific questions and develops applied solutions to a range of agricultural problems,... more
The Agricultural Research Service (ARS) is the intramural research agency of the United States Department of Agriculture (USDA) which addresses basic scientific questions and develops applied solutions to a range of agricultural problems, and in doing so protects national food security and supports international trade. The damage to agricultural commodities inflicted by insects and other arthropod pest species causes a reduction in producer output and profitability, thereby affecting product quality, such that the development of novel and effective arthropod control tactics remains a research challenge at USDA ARS. Additionally, USDA ARS conducts research into arthropod control within urban settings, where damage to dwellings, and ornamental and shade plants are of concern to homeowners and businesses alike. These goals of controlling pests must be balanced with environmental concerns, including the protection of pollinators and other beneficial species. The recent development of RN...
Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. In this study, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri. This psyllid... more
Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. In this study, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri. This psyllid vector has come to prominence in the last decade due to its role in the transmission of the devastating bacterial pathogen, Candidatus Liberibacter asiaticus (CLas). Infection of citrus crops by this pathogen causes Huanglongbing (HLB or citrus greening disease) and results in the eventual death of citrus trees. The identification and correct annotation of these genes in D. citri will be useful for functional genomic studies that aid in the development of RNAi-based management strategies aimed at reducing the spread of HLB. Investigating the effects of CLas infection on the expression of ubiquitin-proteasome pathway genes may provide new information regarding the role that these genes play in the acquisition and transmission of CLas by D. citri.
Citrus greening disease is caused by the pathogenCandidatusLiberibacter asiaticus, which is transmitted by the Asian citrus psyllid,Diaphorina citri. There is no curative treatment or significant prevention mechanism for this detrimental... more
Citrus greening disease is caused by the pathogenCandidatusLiberibacter asiaticus, which is transmitted by the Asian citrus psyllid,Diaphorina citri. There is no curative treatment or significant prevention mechanism for this detrimental disease that causes continued economic losses from reduced citrus production. A high quality genome ofD. citriis being manually annotated to provide accurate gene models required to identify novel control targets and increase understanding of this pest. Here, we annotated genes involved in glycolysis, gluconeogenesis, and trehaloneogenesis in theD. citrigenome, as these are core metabolic pathways and suppression could reduce this pest. Specifically, twenty-five genes were identified and annotated in the glycolysis and gluconeogenesis pathways and seven genes for the trehaloneogenesis pathway. Comparative analysis showed that the glycolysis genes inD. citriare highly conserved compared to orthologs in other insect systems, but copy numbers vary inD....
The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earth's rotation on its axis. This report presents the... more
The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earth's rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.
BackgroundDiaphorina citriis a vector of “CandidatusLiberibacter asiaticus” (CLas), the gram-negative bacterial pathogen associated with citrus greening disease. Control measures rely on pesticides with negative impacts on the... more
BackgroundDiaphorina citriis a vector of “CandidatusLiberibacter asiaticus” (CLas), the gram-negative bacterial pathogen associated with citrus greening disease. Control measures rely on pesticides with negative impacts on the environment, natural ecosystems and human and animal health. In contrast, gene-targeting methods have the potential to specifically target the vector species and/or reduce pathogen transmission.ResultsTo improve the genomic resources needed for targeted pest control, we assembled aD. citrigenome based on PacBio long reads followed by proximity ligation-based scaffolding. The 474 Mb genome has 13 chromosomal-length scaffolds. 1,036 genes were manually curated as part of a community annotation project, composed primarily of undergraduate students. We also computationally identified a total of 1,015 putative transcription factors (TFs) and were able to infer motifs for 337 TFs (33 %). In addition, we produced a genome-independent transcriptome and genomes forD. c...
BackgroundArthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive... more
BackgroundArthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods.ResultsUsing 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appear...
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus. It is caused by (1)CandidatusLiberibacter africanus, transmitted byTrioza erytreaeand (2)C.L. asiaticus andC.L. americanus, transmitted... more
Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus. It is caused by (1)CandidatusLiberibacter africanus, transmitted byTrioza erytreaeand (2)C.L. asiaticus andC.L. americanus, transmitted byDiaphorina citri. As part of a multidisciplinary project onD. citri(www.citrusgreening.org), we made a detailed study, using micro-computed tomography, of the female abdominal terminalia, reproductive system (ovaries, accessory glands, spermatheca, colleterial (= cement) gland, connecting ducts, and ovipositor) and bacteriome, which we present here. New terms and structures are introduced and described, particularly concerning the spermatheca, ovipositor and bacteriome. The quality of images and bacteriome reconstructions are comparable, or clearer, than those previously published using a synchrotron or fluorescencein situhybridisation (FISH). This study: reviews knowledge of the female reproductive system and bacteriome organ inD. citri; represe...
The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. which causes Huanglongbing (HLB) (citrus greening disease); this is considered to be the most serious... more
The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. which causes Huanglongbing (HLB) (citrus greening disease); this is considered to be the most serious bacterial disease of citrus plants. Here we detail an anatomical study of the external and internal anatomy (excluding the reproductive system) using micro-computed tomography (micro-CT). This is the first complete 3D micro-CT reconstruction of the anatomy of a psylloid insect and includes a 3D reconstruction of an adult feeding on a citrus leaf that can be used on mobile devices. Detailed rendered images and videos support first descriptions of coxal and scapus antennal glands and sexual differences in the internal anatomy (hindgut rectum, mesothoracic ganglion and brain). This represents a significant advance in our knowledge of ACP anatomy, and of psyllids in general. Together the images, videos and 3D model constitute a unique anatomical atlas and are u...
Development, evaluation, and efficient delivery of antisense oligonucleotide FANA (2′-deoxy-2′-fluoro-arabinonucleotide) RNA-targeting technology into citrus trees and potato plants for management of bacterial pathogens and arthropod... more
Development, evaluation, and efficient delivery of antisense oligonucleotide FANA (2′-deoxy-2′-fluoro-arabinonucleotide) RNA-targeting technology into citrus trees and potato plants for management of bacterial pathogens and arthropod pests. The FANA ASO technology is a single nucleotide strand of 20–24 nt in length that incorporates 2′F- chemically modifications of nucleotides, along with a phosphorothioate backbone and modified flanking nucleotides in their structure called “gapmers,” (AUM LifeTech., Inc). The unique modifications of FANA “triggers” enables gymnotic activity (self-delivers into cells) moving systemically in treated plants and insects, results in significant suppression of their RNA targets. Reported is the FANA suppression of two plant-infecting bacterium Candidatus Liberibacter asiaticus, CLas (in citrus trees) and CLso (in potato and tomato), as well as in their psyllid vectors.
The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. bacteria which cause Huanglongbing (HLB) (Citrus Greening Disease); This is the most serious lethal disease of... more
The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. bacteria which cause Huanglongbing (HLB) (Citrus Greening Disease); This is the most serious lethal disease of citrus trees. Here we detail an anatomical study of the external and internal anatomy using micro-computed tomography (micro-CT). This is the first complete 3D micro-CT reconstruction of the anatomy of a hemipteran, and advances psylloid anatomical knowledge by producing 3D reconstructions of an adult while feeding one a citrus leaf. Software that can be used on electronic devices enables rotation and digital slicing through the anatomical digital specimen at any angle. This produces thousands of usable, opensource, imaging at significant savings for researchers and educators. Detailed rendered images and videos support first descriptions of coxal and scapus antennal glands and sexual differences in the internal anatomy (hindgut rectum, mesothoracic ganglion and brain). This represents a significant advance in our knowledge of ACP anatomy, and of psyllids in general. Together the images, videos and 3D model constitute a unique anatomical atlas and are useful tools for future research and as teaching aids.
The Asian citrus psyllid (ACP), Diaphorina citri, is a major vector of the bacteria Candidatus Liberibacter asiaticus and C.L. americanus, which cause Huanglongbing disease (aka Citrus Greening Disease), considered the most serious... more
The Asian citrus psyllid (ACP), Diaphorina citri, is a major vector of the bacteria Candidatus Liberibacter asiaticus and C.L. americanus, which cause Huanglongbing disease (aka Citrus Greening Disease), considered the most serious disease of citrus trees. As part of a multidisciplinary project on psyllid biology (www.AgriVectors.org), the results presented here concern a detailed anatomical study of the male reproductive system (testes, seminal vesicles, accessory glands, sperm pump, connecting ducts, and aedeagus) using micro-computed tomography (micro-CT). The study summarizes current knowledge on psyllids male reproductive system and represents significant advancement in the knowledge of psyllid anatomy.
Reproduction is a critical feature in the search for means to manage the Asian citrus psyllid, vector of a devastating bacterial pathogen of citrus called Huanglongbing. The importance of accuracy in functional, anatomical descriptions... more
Reproduction is a critical feature in the search for means to manage the Asian citrus psyllid, vector of a devastating bacterial pathogen of citrus called Huanglongbing. The importance of accuracy in functional, anatomical descriptions and interpretations for use by other disciplines, particularly molecular genetics, cannot be overstressed. The term 'sperm pump' was coined by classical authors on observational appearance of the endoskeleton of the male reproductive apparatus. They described a thimble-shaped cuticle with smooth, cylindrical columns, interpreted as muscles, that ran longitudinally around a central cylinder. They detected transverse lines on the cylinder giving the false impression of a coiled spring. These features fostered the teleological interpretation that the device is a contractile pump. Now obsolete, the term is replaced by 'drum/spout complex'. It is a hypodermis with a sclerotized cuticle that houses the phallus which transports seminal fluid through its lumen to the female for insemination. Between the spout and the external genitalia is a spout extension, conferring flexibility to the apparatus. Approximately 21 longitudinal columns extend circumferentially around the cylinder's hemolymph-side, from the thimble's basal plate to its apical plate. These columns are muscle cells, and reinterpreted to exude a lipaceous, lubricating substance for mating.
Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus, now in over 40 citrus producting countries. It is caused by bacteria infections: 1) Candidatus Liberibacter africanus, transmitted by... more
Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus, now in over 40 citrus producting countries. It is caused by bacteria infections: 1) Candidatus Liberibacter africanus, transmitted by Trioza erytreae, or 2) C.L. asiaticus and C.L. americanus, transmitted by Diaphorina citri, the Asian citrus psyllid. As part of a multidisciplinary project on D. citri (www.AgriVectors.org), we made a detailed study, using micro-computed tomography, of the female psyllid reproductive system (ovaries, accessory glands, spermatheca, colleterial gland, connecting ducts, ovipositor) and bacteriome. New terms and structures are introduced and described, particularly concerning the spermatheca, ovipositor and bacteriome. The quality of images and bacteriome reconstructions are comparable, or clearer, than those previously published using a synchrotron or fluorescence in situ hybridisation (fiSH). This study: reviews knowledge of the female reproductive system and bacteriome organ in D. citri; represents the first detailed morphological study of D. citri to use micro-ct; and extensively revises existing morphological information relevant to psylloids, hemipterans and insects in general. High quality images and supplementary videos represent a significant advance in knowledge of psylloid anatomy and are useful tools for future research and as educational aids. Huanglongbing (HLB) or citrus greening disease is considered to be the most serious disease threatening the citrus industry 1. HLB is a bacterial disease caused by causing fruit loss, small, bitter, unpalatable fruit, and eventually tree death. To date, HLB has spread to over 40 countries in Asia, Oceania, North America and South America. There are two species of psylloids known to be vectors of the disease 4-6 : (1) Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) which vectors C. L. africanus, and (2) Diaphorina citri, Kuwayama (Hemiptera: Liviidae) which vectors C. L. asiaticus, and C. L. americanus [https://www.cabi.org/isc/datasheet/16565]. Since D. citri was first discovered in Taiwan in 1907 7 , it has become the major vector of HLB in Asia and USA. This psylloid predominantly acquires the bacterium during nymphal feeding on infected citrus plants 8. When D. citri is infected with C. Liberibacter its fecundity increases and it produces more offspring 9. A number of studies have described the female anatomy of psylloid species 10-23. However, few studies have focused on the female reproductive system of D. citri 24,25. Those studies that have been done, have used dissected specimens and descriptions based on light and electron microscopy photographs with some schematic drawings. Many insects, and in particular different groups of sap-sucking hemipterans, have endosymbiotic microorganisms located within a specialized organ, the bacteriome (also known as the mycetome). While the microorganisms contained within the bacteriome seem to primarily provide essential amino acids and vitamins, they
The expression of essential genes in the psyllid vector, Diaphorina citri (Hemiptera), in the endosymbiont, Wolbachia, wDi, and the bacterial pathogen, Candidatus Liberibacter asiaticus, causing Huanglongbing of citrus were suppressed in... more
The expression of essential genes in the psyllid vector, Diaphorina citri (Hemiptera), in the endosymbiont, Wolbachia, wDi, and the bacterial pathogen, Candidatus Liberibacter asiaticus, causing Huanglongbing of citrus were suppressed in response to morpholino, PPMO treatments. This study reports the first formal report for  morpholino delivery into citrus trees to target plant-infecting bacteria, and in plant-delivered PPMO to target insects and their endosymbionts as novel management strategies for bacterial pathogens of fruit trees, such as CLas in citrus induced Huanglongbing.
The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees... more
The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage in the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) was used successfully to silence a virus gene critical for replication, in infected honeybees. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory and field conditions. This first-of-a-kind study is the largest field trial of a RNAi therapeutic for animal health (Honeybees). IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This multi-year controlled study included a total of 160 bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.
Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage... more
Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We outline non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as a clay-slow-release system that can deliver dsRNA to plants for over 9 months for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed to induce significant suppression of targeted transcripts, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and pest management.
Research on the development of RNA-suppressing technologies, which include RNAi, and applications of RNA suppression using cell-penetrating peptide Morpholinos, PPMO, for suppressing pests and bacterial pathogens are presented. Due to the... more
Research on the development of RNA-suppressing technologies, which include RNAi, and applications of RNA suppression using cell-penetrating
peptide Morpholinos, PPMO, for suppressing pests and bacterial pathogens are presented. Due to the extensive nature of RNAi and antisense oligonucleotide research, readers are directed to this review to obtain a more in-depth understanding of each field of study. Genomic resources and websites for dataset downloads and free access are listed to facilitate a broader community collaboration to develop solutions for this complex Liberibacter pathosystem. Strategies for gene drive and psyllid population modification or replacement, with Wolbachia or CRISPR/Cas9-based alterations, have continued to gain interest and are being developed for insect pests.
Innovative gene targeting strategies are often limited in application across arthropod species due to problems with successful delivery. In hemipterans, embryonic injections used to deliver CRISPR components fail due to nearly complete... more
Innovative gene targeting strategies are often limited in application across arthropod species due to problems with successful delivery. In hemipterans, embryonic injections used to deliver CRISPR components fail due to nearly complete embryo mortality.  Presented is the first successful gene delivery system in a the plant feeding hemipteran, the Asian citrus psyllid, Diaphorina citri, Kuwayama, (Hemiptera: Liviidae). This vector spread a pathogenic bacterium, C. Liberibacter asiaticus, CLas, that is lethal to citrus trees after infection. The disease, called Huanglongbing is devastating the global citrus industries. To develop management therapeutics aimed at stopping pathogen spread by the vector, the new method successfully used adult and nymphal stages to produce heritable gene edits.  The method opens transformation technology for all hemipteran and insect species.
Innovative technologies, like RNA suppression by RNAi, morpholino oligos, or gene editing tools, like CRISPR/Cas9, all provide non-transgenic strategies, as well as transgenic solutions to manage arthropod vectors, pests and pathogens.... more
Innovative technologies, like RNA suppression by RNAi, morpholino oligos, or gene editing tools, like CRISPR/Cas9, all provide non-transgenic strategies, as well as transgenic solutions to manage arthropod vectors, pests and pathogens. Innovative breakthroughs enable gene-editing in psyllids, using BAPC-assisted-CRISPR/Cas9 System, injection of adult females near ovaries produces heritable germline edits. Development of these safer biopesticides and non-GMO uses continue to support sustainable agriculture in the USA.
Hunter WB, Cooper WR, Sandoval-Mojica AF, McCollum G, Aishwarya V, Pelz-Stelinski KS (2021) Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides... more
Hunter WB, Cooper WR, Sandoval-Mojica AF, McCollum G,
Aishwarya V,  Pelz-Stelinski KS (2021)  Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides (FANA).
Front. Agron. 3:675247.  doi: 10.3389/fagro.2021.675247
The classification of a novel picorna-like virus in L. fagarae, with evolutionary characteristics similar to picorna-like viruses infecting Bactericera cockerelli and D. citri, provides an opportunity to examine virus host specificity, as... more
The classification of a novel picorna-like virus in L. fagarae, with
evolutionary characteristics similar to picorna-like viruses infecting Bactericera cockerelli and D. citri, provides an opportunity to examine virus host specificity, as well as identify critical components of the virus’ genome required for successful transmission, infection, and replication. This bioinformatic classification allows for further insight into a novel virus species, and aids in the research of a closely related virus of the invasive psyllid, D. citri, a major pest of Floridian citriculture. The potential use of viral pathogens as expression vectors to manage the spread D. citri is an area that requires additional research; however, it may bring forth an effective control strategy to reduce the transmission of Candidatus Liberibacter asiaticus (CLas), the causative agent of Huanglongbing
(HLB).
Detailed annotation and comparative analysis were performed on the Asian citrus psyllid (ACP), Diaphorina citri, vacuolar-type ATP synthase (V-ATPase) to support the biological understanding and development of novel therapeutics to manage... more
Detailed annotation and comparative analysis were performed on the Asian citrus psyllid (ACP), Diaphorina citri, vacuolar-type ATP synthase (V-ATPase) to support the biological understanding and development of novel therapeutics to manage psyllid vectors. D. citri is a hemipteran insect that vectors the causative agent, the bacteria Candidatus Liberibacter asiaticus (CLas), of the citrus greening disease, Huanglongbing (HLB). Millions of citrus trees have been destroyed by citrus greening and every grove in Florida has been directly impacted. In eukaryotic organisms, V-ATPase is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Manual curation was completed on 15 putative V-ATPase genes in the D. citri genome. Comparative genomic analysis reveals that the D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily doma...
Detection of pathogens is critical to monitoring their distribution and spread, and is a key component in the prediction and management of disease epidemiology. Monitoring for pathogens as bioaerosols requires developing techniques which... more
Detection of pathogens is critical to monitoring their distribution and spread, and is a key component in the prediction and management of disease epidemiology. Monitoring for pathogens as bioaerosols requires developing techniques which are sensitive, affordable, and time saving before they will have widespread impact. This approach also overcomes private property issues, which are a major pitfall in monitoring diseases in complex agricultural and urban settings. In this study, we have applied an emerging technology of electrostatic sampling to the detection of an insect-transmitted plant pathogen as a bioaerosol. Where insects aggregate in large numbers, as with whiteflies, leafhoppers, psyllids and honey bees, the pathogen (ie. virus or bacteria) becoming aerosolized as thousands of excreta droplets fall from the plants during feeding. Agricultural systems have not fully measured the impact of bioaerosols on disease epidemiology. Electrostatic sampling provides a valuable, afford...
Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular... more
Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e. avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules vitroand in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their therapeutic applications.

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Ag-biotechnology along and gene-editing technologies can modifying gene structure, or gene expression, revolutionizing research fields across: plant and animal breeding, pathology, crop improvement, virology, microbiology, entomology,... more
Ag-biotechnology along and gene-editing technologies can modifying gene structure, or gene expression, revolutionizing research fields across: plant and animal breeding, pathology, crop improvement, virology, microbiology, entomology, biopharma, and biomedical research. RNA interference (RNAi) and CRISPR-based technologies are pivotal in the development of new varieties of foods, livestock, and therapeutics leading to safer crop improvement; management of arthropod pests and viral pathogens. This review examines the genetic diversity from weeds and distantly related plants to currently grown crops, and how they provide solutions to a wide set of local and global problems in agriculture linked to drought, insect pests,  biofuel production, and human health challenges. Bio-techniques are built upon the understanding of the genetic process in natural mechanisms, used by plants, insects, or microbes, provide plants safer for human and animal consumption.