Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae).

The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss, Buchloe dactyloides (Nuttall) due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland morphology and the associated salivary complex of this insect. Whole and sectioned B. occiduus salivary glands were visualized using light and scanning electron microscopy to determine overall structure and cell types of the salivary glands and their individual lobes. Microscopy revealed a pair of trilobed principal glands and a pair of tubular accessory glands of differing cellular types. To link structure with function, the salivary gland proteome was characterized using liquid chromatography tandem mass spectrometry. The salivary proteome analysis resulted in B. occiduus sequences matching 228 nonhomologous protein sequences of the pea aphid, Acyrthosiphon pisum (Harris), with many specific to the proteins present in the salivary proteome of A. pisum. A number of sequences were assigned the molecular function of hydrolase and oxido-reductase activity, with one specific protein sequence revealing a peroxidase-like function. This is the first study to characterize the salivary proteome of B. occiduus and the first of any species in the family Blissidae.

Transfer of Escherichia coli O157:H7 to spinach by house flies, Musca domestica (Diptera: Muscidae).

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.

An analysis of the genomic variability of the phytopathogenic mollicute Spiroplasma kunkelii.

Corn stunt disease has become a factor limiting maize production in some areas of the Americas in recent years. Although resistant maize genotypes have been developed in the past, this resistance has been unstable over time or in some geographical locations. To better understand disease components that could affect the stability of host resistance, we assessed the genome variability of the etiologic agent, Spiroplasma kunkelii. Isolates were obtained from a number of areas, and characterized molecularly by amplification of several regions of the spiroplasma chromosome and sequencing of specific gene fragments. The degree of polymorphism between isolates of different geographic origins was low, and the level of genomic variability was similar within isolates of different countries. Polymorphism among isolates was found in viral insertions and in the sequence of Skarp, a gene that encodes a membrane protein implicated in attachment to insect cells. The results suggest that the genome composition of this species is highly conserved among isolates. Hence, it is unlikely that the instability of maize resistance is due to generation of new pathotypes of S. kunkelii. Instead, other components of this complex pathosystem could account for the breakdown of resistance.

Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis.

Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN™ Tnp transposome™ system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 10(9) spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon's presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN™ Tnp transposome™ system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.

Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum.

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorum-targeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.

Identification of Volatiles From Plants Infested With Honeydew-Producing Insects, and Attraction of House Flies (Diptera: Muscidae) to These Volatiles.

House flies (Musca domestica L.) are mechanical vectors of food-borne pathogens including Salmonella spp., Escherichia coli O157:H7, and Shigella spp., resulting in increased risk of diarrheal disease in areas where flies are abundant. Movement of house flies into food crops may be increased by the presence of honeydew-producing insects feeding on these crops. Using gas chromatography-electroantennogram detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS), volatile odors that elicited house fly antennal response were identified from naval orange (Osbeck) (Sapindales: Rutaceae) and Marsh grapefruit (Macfad.) (Sapindales: Rutaceae) leaves infested with whitefly (Hemiptera: Aleyrodidae) and from whole faba (L.) (Fabales: Fabaceae) bean plants infested with aphids (Hemiptera: Aphididae). Volatiles identified included benzaldehyde, butyl hexanoate, ß-caryophyllene, Δ3-carene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenyl acetate, myrcene, limonene, linalool, and naphthalene. This was followed by semifield bioassays of volatile blends and individual volatiles to determine house fly attraction to these volatiles. Although fly capture rates in the semifield setting were low, benzaldehyde and (Z)-3-hexenyl acetate were consistently attractive to house flies as individual compounds and as components of volatile blends.

Waveforms From Stylet Probing of the Mosquito Aedes aegypti (Diptera: Culicidae) Measured by AC-DC Electropenetrography.

Electropenetrography (EPG) has been used for many years to visualize unseen stylet probing behaviors of plant-feeding piercing-sucking insects, primarily hemipterans. Yet, EPG has not been extensively used with blood-feeding insects. In this study, an AC-DC electropenetrograph with variable input resistors (Ri), i.e., amplifier sensitivities, was used to construct a waveform library for the mosquito arbovirus vector, Aedes aegypti (Linneaus), while feeding on human hands. EPG waveforms representing feeding activities were: 1) electrically characterized, 2) defined by visual observation of biological activities, 3) analyzed for differences in appearance by Ri level and type of applied signal (AC or DC), and 4) quantified. Electrical origins of waveforms were identified from five different Ri levels and AC versus DC. Mosquitoes produced short stylet probes ('bites') that typically contained five waveform families. Behaviors occurred in the following order: surface salivation (waveform family J), stylet penetration through the outer skin (K), penetration of deeper tissues and location of blood vessels/pathway activities (L), active ingestion with engorgement (M), and an unknown behavior that terminated the probe (N). Only K, L, and M were performed by every insect. A kinetogram of conditional probabilities for waveform performance demonstrated plasticity among individuals in L and M, which were alternated. Now that EPG waveforms for mosquito feeding have been defined, EPG can be used as a tool for improved biological understanding of mosquito-borne diseases.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals.

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.

Single-Target and Multiplex Discrimination of Whiteflies (Hemiptera: Aleyrodidae) Bemisia tabaci and Trialeurodes vaporariorum With Modified Priming Oligonucleotide Thermodynamics.

The whitefly species Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) are worldwide agricultural pests and virus vectors. Bemisia tabaci, in particular, is often transported internationally via trade routes leading to potential introductions of exotic whiteflies or plant viruses. Quick identification of agriculturally important whiteflies can facilitate interventions that prevent these cross-border introductions. Polymerase chain reaction (PCR) primers were designed to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) sequence of members of the B. tabaci complex, MEAM1, MED, and NW, and T. vaporariorum. Primers incorporated an A/T-rich overhang sequence at the 5' terminus (5' flap) to test for increased primer sensitivity and assay efficiency. Single-target and multiplex endpoint PCR assays with the eight primer sets were performed using genomic DNA template extracted from individual adult whiteflies. Resultant PCR amplicons obtained for B. tabaci MEAM1, MED, and NW, and T. vaporariorum primers with the 5' flap were 559-, 717-, 353-, and 258-bp, respectively, and without the 5' flap were 550-, 712-, 329-, and 252-bp in length, respectively. In single-target and multiplex reactions, specific amplification was achieved using both the unmodified and 5' flap-modified primers. Sequencing and phylogenetic analysis confirmed primer-target amplification specificity. Using these primer sets in single-target or multiplex PCR allows for quick discrimination and specific identification of B. tabaci complex members and T. vaporariorum, and the addition of 5'A/T-rich overhang sequences increases the sensitivity and amplification of some primer sets.

House Fly (Musca domestica L.) Attraction to Insect Honeydew.

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.

Effect of Insect Exclusion on the Incidence of Yellow Vine Disease and of the Associated Bacterium in Squash.

Yellow vine (YV) of cucurbits, associated with a phloem-limited bacterium, causes rapid wilting and death in affected plants. In a previous study, experimental insecticide-treated plots had a lower incidence of YV than untreated plots, suggesting that insects were involved in the transmission of the bacterium. In the study reported here, we compared the incidence of YV and polymerase chain reaction (PCR) detection of the YV bacterium in noncovered squash plants (Cucurbita pepo var. melopepo) with plants covered with fine-mesh fabric secured in such a way that insects were excluded. Rows of squash were covered with row mesh cover that was stretched over hoops and anchored in the soil. The row cover was removed after 40 or 50 days, at which time all plants were sampled destructively by harvesting the crown and root. In the first experiment, 3% of the noncovered plants had foliar symptoms, 7% were positive with the use of Dienes' stain, and 25% were positive when analyzed by PCR with specific primers. No covered plants were positive by any detection method, and no plants in the second experiment had foliar symptoms or tested positive with Dienes' stain. However, 20% of noncovered and 0% of covered plants were PCR positive. These data support the hypothesis that insects were involved in the transmission of the bacterium.

Characterization of putative membrane protein genes of the 'Candidatus Phytoplasma asteris', chrysanthemum yellows isolate.

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches' broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the 'Candidatus Phytoplasma asteris'. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of 'Ca. Phytoplasma asteris' from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.