Maintenance of primary cell cultures of immunocytes from Cacopsylla spp. psyllids: a new in vitro tool for the study of crop pest insects.

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 biology of psyllids as vector insects as well as analyses to understand the mechanisms behind the interactions with pathogens and symbionts are now possible. These cultures can be used as an in vitro model to study psyllid humoral immune responses, which also will allow in-depth investigations on the abilities of psyllids as vectors of phytoplasmas. All these applications provide new opportunities to develop more focused and specific pest control strategies.

Overview of worldwide diversity of Diaphorina citri Kuwayama mitochondrial cytochrome oxidase 1 haplotypes: two Old World lineages and a New World invasion.

Relationships among worldwide collections of Diaphorina citri (Asian citrus psyllid) were analyzed using mitochondrial cytochrome oxidase I (mtCOI) haplotypes from novel primers. Sequences were produced from PCR amplicons of an 821bp portion of the mtCOI gene using D. citri specific primers, derived from an existing EST library. An alignment was constructed using 612bps of this fragment and consisted of 212 individuals from 52 collections representing 15 countries. There were a total of eight polymorphic sites that separated the sequences into eight different haplotypes (Dcit-1 through Dcit-8). Phylogenetic network analysis using the statistical parsimony software, TCS, suggests two major haplotype groups with preliminary geographic bias between southwestern Asia (SWA) and southeastern Asia (SEA). The recent (within the last 15 to 25 years) invasion into the New World originated from only the SWA group in the northern hemisphere (USA and Mexico) and from both the SEA and SWA groups in the southern hemisphere (Brazil). In only one case, Reunion Island, did haplotypes from both the SEA and SWA group appear in the same location. In Brazil, both groups were present, but in separate locations. The Dcit-1 SWA haplotype was the most frequently encountered, including ~50% of the countries sampled and 87% of the total sequences obtained from India, Pakistan and Saudi Arabia. The second most frequently encountered haplotype, Dcit-2, the basis of the SEA group, represented ~50% of the countries and contained most of the sequences from Southeast Asia and China. Interestingly, only the Caribbean collections (Puerto Rico and Guadeloupe) represented a unique haplotype not found in other countries, indicating no relationship between the USA (Florida) and Caribbean introductions. There is no evidence for cryptic speciation for D. citri based on the COI region included in this study.

Effects of invertebrate iridescent virus 6 in Phyllophaga vandinei and its potential as a biocontrol delivery system.

Invertebrate iridescent virus 6 (IIV6) was determined to cause infection in Phyllophaga vandinei Smyth (Coleoptera: Scarabaeidae) through a range of modes of transmissions. This is the first evidence of IIV6 infection in P. vandinei that caused both patent and sub-lethal infections in larvae and adults. Mortality rates were determined to be ~30% when virus inoculum was injected into larvae or adults. Adults injected with virus showed dramatically altered behavior; injected beetles were not observed feeding or mating compared with adults injected with buffer or adults that were not injected. Tissue collected from infected adults resulted in infection when injected into healthy adults, as confirmed with PCR. PCR also confirmed that frass of infected larvae and adults contained virus, and when reconstituted frass from infected individuals was injected into healthy adults or larvae they become infected. Healthy adults could be infected by coming into contact with soil or plant material that had been exposed to infected adults as much as two weeks prior to introduction of nonvirus exposed adults. Although relatively low mortality resulted when adults or larvae were injected with the virus, the demonstration of horizontal transmission, potentially through frass of infected individuals, identifies a mode of transmission that may be exploited as a potential management tool to reduce P. vandinei.

Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenoptera: Pteromalidae).

We report three novel small RNA viruses uncovered from cDNA libraries from parasitoid wasps in the genus Nasonia. The genome of this kind of virus is a positive-sense single-stranded RNA with a 3' poly(A), which facilitates cloning from cDNAs. Two of the viruses, NvitV-1 and NvitV-2, possess a RNA-dependent RNA polymerase that associates them with the family Iflaviridae of the order Picornavirales. A third virus, NvitV-3, is most similar to the Nora virus from Drosophila. A reverse transcription-PCR method developed for NvitV-1 indicates that it is a persistent commensal infection of Nasonia.

Behavioral model for Homalodisca vitripennis (Hemiptera: Cicadellidae): optimization of host plant utilization and management implications.

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), (Hemiptera: Cicadellidae), is a xylophagous leafhopper native to the southeastern United States and northern Mexico, with recent introductions into California, Arizona, French Polynesia, and Hawaii. It is a primary vector of the xylem-limited bacterium, Xylella fastidiosa Wells et al., the causative agent of Pierce's disease of grape, citrus variegated chlorosis, phony peach, and numerous leaf scorch diseases. H. vitripennis uses several hundred species of host plants for feeding, development, and reproduction. Variation in host utilization allows H. vitripennis to respond to diurnal and seasonal changes in its nutrient-poor food source, xylem fluid, as well as changing nutritional requirements of each leafhopper developmental stage. Here we provide a conceptual model that integrates behavior, life history strategies, and their associated risks with the nutritional requirements of adult and nymphal stages of H. vitripennis. The model is a useful heuristic tool that explains patterns of host plant use, describes insect behavior and ecology, suggests new associations among the ecological components, and most importantly, identifies and supports the development of suppression strategies for X. fastidiosa aimed at reducing vector populations through habitat manipulation.

Prevalence and natural host range of Homalodisca coagulata virus-1 (HoCV-1).

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 approximately 95 genome equivalents. A survey employing this assay in conjunction with GWSS samples collected throughout the United States including California, Hawaii, Florida Georgia, and the 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 lifestage or sex. Examination of alternate leafhopper species further demonstrated that, although infection is not ubiquitous to all sharpshooter genera, HoCV-1 is not limited to Homalodisca vitripennis (=H. coagulata).

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae).

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.

Molecular analysis of capsid protein of Homalodisca coagulata Virus-1, a new leafhopper-infecting virus from the glassy-winged sharpshooter, Homalodisca coagulata.

A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5(th) instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of beta-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly alpha-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

Iridovirus in the root weevil Diaprepes abbreviatus.

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 transmission electron microscopy. 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 strategies based on induced epizootics. This is the first report of a virus infecting D. abbreviatus.

Iridovirus infection of cell cultures from the Diaprepes root weevil, Diaprepes abbreviatus.

We here report 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 degrees 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.

Aphid biology: expressed genes from alate Toxoptera citricida, the brown citrus aphid.

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 < or = -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.

Development of a continuous whitefly cell line [Homoptera: Aleryrodidae: Bemisia tabaci (Gennadius)] for the study of begomovirus.

The whitefly Bemisia tabaci (Gennadius) is a widely distributed 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 degrees C. Continuous cell cultures were established and have since undergone 92 passages in 25-cm(2) flasks. Cell doubling time is approximately 3 days and the cells have been successfully revived after 1 year after storage at -80 degrees 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.

Discovering new insect viruses: whitefly iridovirus (Homoptera: Aleyrodidae: Bemisia tabaci).

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 polymerase chain 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.

Location of Geminiviruses in the Whitefly Bemisia tabaci (Homoptera: Aleyrodidae).

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, which is discussed.

Pharmacist career ladder with clinical privilege categories.

The development of a career ladder program for staff pharmacists at a Department of Veterans Affairs (VA) medical center is recounted. Center policy required the establishment of clinical privileges for all pharmacists with direct patient contact and specified three VA privilege categories with increasing degrees of autonomy. The pharmacy department supported the need for all pharmacists to incorporate clinical activities into their daily practice but faced several problems, including inadequate instruction, insufficient incentives, fragmentation of clinical services, and subjectivity of measures of competence. In response, a pharmacy credentialing committee created a career ladder with three levels based on the established system of clinical privileges. Level A integrated basic clinical pharmacy knowledge with dispensing activities. Level B increased the number of clinical skills required and allowed the pharmacist to act as a therapeutic consultant. Level C incorporated the skills necessary for specialty practice. Instructors were designated for each clinical service area, readings and sample problems were assigned, and staff development presentations were improved. Objective tests of skills were designed. Combining the three levels on the career ladder with the three categories of clinical privileges formed a matrix of nine options for advancement. Pharmacists applying for advancement must master all requisite skills and submit relevant documentation. Each level carries a pay increase of 2%. A total of 53% of the staff pharmacists have participated in the program, which has had a favorable impact on staff retention. By combining nationally established categories of clinical privileges with an institution-specific career ladder, a pharmacy department helped ensure the consistency of services and promote the development of clinical practitioners.