Neuropeptides and peptide hormones identified in codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

The codling moth, Cydia pomonella, is a worldwide pest of pome fruits. Neuropeptides regulate most physiological functions in insects and represent new targets for the development of control agents. The only neuropeptides reported from the codling moth to date are the allatostatin A family peptides. To identify other neuropeptides and peptide hormones from codling moth, we analyzed head transcriptomes, identified 50 transcripts, and predicted 120 prepropeptides for the codling moth neuropeptides and peptide hormones. All transcripts were amplified, and these sequences were verified. One of the notable findings in this study is that diapause hormones (DHs) reported from Tortricid moths, including the codling moth, do not have the WFGPRL sequence in C-terminal ends in the pban genes. The C-terminal motif is critical to characterize insect DH peptides, and always conserved in pban/dh genes in Lepidoptera and many insect orders. Interestingly, the WFGPRL sequence was produced only from the capa gene in the codling moth. The allatostatin A-family encoding transcript predicted nine peptides, seven of which, as expected, are identical to those previously isolated from the moth. We also identified new codling moth orthologs of insect neuropeptides including CCHamides, allatostatin CC, RYamides, and natalisins. The information provided in this study will benefit future codling moth investigations using peptidoproteomics to determine peptide presence and functions.

Relative Abundance of Carsonella ruddii (Gamma Proteobacterium) in Females and Males of Cacopsylla pyricola (Hemiptera: Psyllidae) and Bactericera cockerelli (Hemiptera: Triozidae).

Carsonella ruddii (Gamma Proteobacterium) is an obligate bacterial endosymbiont of psyllids that produces essential amino acids that are lacking in the insect's diet. Accurate estimations of Carsonella populations are important to studies of Carsonella-psyllid interactions and to developing ways to target Carsonella for control of psyllid pests including pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) and potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). We used two methods, namely fluorescence in situ hybridization and quantitative polymerase chain reaction (qPCR), to estimate relative abundance of Carsonella in bacteriocytes and whole bodies of psyllids, respectively. Using these two methods, we compared Carsonella populations between female and male insects. Estimations using fluorescence in situ hybridization indicated that Carsonella was more abundant in bacteriocytes of female C. pyricola than in those of males, but Carsonella abundance in bacteriocytes did not differ between sexes of B. cockerelli. Analyses by qPCR using whole-body specimens indicated Carsonella was more abundant in females than in males of both psyllids. Neither fluorescence in situ hybridization nor qPCR indicated that Carsonella populations differed in abundance among adults of different ages (0-3 wk after adult eclosion). Using fluorescence in situ hybridization, Carsonella was observed in ovarioles of newly emerged females and formed an aggregation in the posterior end of mature oocytes. Results of our study indicate that female psyllids harbor greater populations of Carsonella than do males and that sex should be controlled for in studies which require estimations of Carsonella populations.

Cloning and characterization of an mRNA encoding an insulin receptor from the horned scarab beetle Onthophagus nigriventris (Coleoptera: Scarabaeidae).

The insulin signaling pathway is the primary signaling pathway coupling growth with nutritional condition in all animals. Sensitivity to circulating levels of insulin has been shown to regulate the growth of specific traits in a dose-dependent manner in response to environmental conditions in a diversity of insect species. Alternative phenotypes in insects manifest in a variety of morphologies such as the sexually dimorphic and male dimorphic horned beetles. Large males of the sexually dimorphic dung beetle Onthophagus nigriventris develop a thoracic horn up to twice the length of the body whereas small males and females never develop this horn. The regulation of this dimorphism is known to be nutrition dependent for males. We focused on the insulin signaling pathway as a potential regulator of this dimorphism. We sequenced a full-length gene transcript encoding the O. nigriventris insulin receptor (OnInR), which is the receptor for circulating insulin and insulin-like peptides in animals. We show that the predicted OnInR protein is similar in overall amino acid identity to other insulin receptors (InRs) and is most closely related phylogenetically to insect InRs. Expression of the OnInR transcript was found during development of imaginal tissues in both males and females. However, expression of OnInR in the region where a horn would grow of small males and female was significantly higher than in the horn tissues of large males at the end of growth. This variation in OnInR expression between sexes and morphs indicates a role for the InR in polymorphic horn development.

Chemosensory Receptor Expression in the Abdomen Tip of the Female Codling Moth, Cydia pomonella L. (Lepidoptera: Tortricidae).

In insects, the chemical senses influence most vital behaviors, including mate seeking and egg laying; these sensory modalities are predominantly governed by odorant receptors (ORs), ionotropic receptors (IRs), and gustatory receptors (GRs). The codling moth, Cydia pomonella, is a global pest of apple, pear, and walnut, and semiochemically based management strategies limit the economic impacts of this species. The previous report of expression of a candidate pheromone-responsive OR in female codling moth ovipositor and pheromone glands raises further questions about the chemosensory capacity of these organs. With an RNA-sequencing approach, we examined chemoreceptors' expression in the female codling moth abdomen tip, sampling tissues from mated and unmated females and pupae. We report 37 ORs, 22 GRs, and 18 IRs expressed in our transcriptome showing overlap with receptors expressed in adult antennae as well as non-antennal candidate receptors. A quantitative PCR approach was also taken to assess the effect of mating on OR expression in adult female moths, revealing a few genes to be upregulated or downregulating after mating. These results provide a better understanding of the chemosensory role of codling moth female abdomen tip organs in female-specific behaviors. Future research will determine the function of specific receptors to augment current semiochemical-based strategies for codling moth management.

Identification of three new 'Candidatus Liberibacter solanacearum' haplotypes in four psyllid species (Hemiptera: Psylloidea).

Eleven haplotypes of the bacterium, 'Candidatus Liberibacter solanacearum', have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for 'Ca. L. solanacearum' by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the 'Ca. L. solanacearum' 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of 'Ca. L. solanacearum' haplotype F was not detected in this study.

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti.

Ingestion of vertebrate blood is essential for egg maturation and transmission of disease-causing parasites by female mosquitoes. Prior studies with the yellow fever mosquito, Aedes aegypti, indicated blood feeding stimulates egg production by triggering the release of hormones from medial neurosecretory cells in the mosquito brain. The ability of bovine insulin to stimulate a similar response further suggested this trigger is an endogenous insulin-like peptide (ILP). A. aegypti encodes eight predicted ILPs. Here, we report that synthetic ILP3 dose-dependently stimulated yolk uptake by oocytes and ecdysteroid production by the ovaries at lower concentrations than bovine insulin. ILP3 also exhibited metabolic activity by elevating carbohydrate and lipid storage. Binding studies using ovary membranes indicated that ILP3 had an IC(50) value of 5.9 nM that was poorly competed by bovine insulin. Autoradiography and immunoblotting studies suggested that ILP3 binds the mosquito insulin receptor (MIR), whereas loss-of-function experiments showed that ILP3 activity requires MIR expression. Overall, our results identify ILP3 as a critical regulator of egg production by A. aegypti.

Neuropeptidomics of the mosquito Aedes aegypti.

Neuropeptidomic data were collected on the mosquito Ae. aegypti, which is considered the most tractable mosquito species for physiological and endocrine studies. The data were solely obtained by direct mass spectrometric profiling, including tandem fragmentation, of selected tissues from single specimens, which yielded a largely complete accounting of the putative bioactive neuropeptides; truncated neuropeptides with low abundance were not counted as mature peptides. Differential processing within the CNS was detected for the CAPA-precursor, and differential post-translational processing (pyroglutamate formation) was detected for AST-C and CAPA-PVK-2. For the first time in insects, we succeeded in the direct mass spectrometric profiling of midgut tissue which yielded a comprehensive and immediate overview of the peptides involved in the endocrine system of the gut. Head peptides which were earlier identified as the most abundant RFamides of Ae. aegypti, were not detected in any part of the CNS or midgut. This study provides a framework for future investigations on mosquito endocrinology and neurobiology. Given the high sequence similarity of neuropeptide precursors identified in other medically important mosquitoes, conclusions regarding the peptidome of Ae. aegypti likely are applicable to these mosquitoes.

Association of "Candidatus Liberibacter solanacearum" with the psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe.

The psyllid Trioza apicalis Förster (Hemiptera: Triozidae) is a serious pest of carrots, Daucus carota L., in Europe. Carrots exhibiting symptoms of psyllid damage were observed in commercial fields in southern Finland in 2008. Symptoms in affected plants included leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots. Mechanisms by which T. apicalis induces symptoms in plants are not understood, and no plant pathogens have yet been associated with this insect. Given recent association of liberibacter with several crops affected by psyllids, an investigation on whether this bacterium is associated with T. apicalis was conducted. Polymerase chain reaction (PCR) primer pairs OA2/OI2c and LsoF/OI2c, specific for 16S rRNA gene from "Candidatus Liberibacter solanacearum," generated amplicons of 1,168 bp and 1,173 bp, respectively, from DNA extracted from field-collected psyllids (61 and 36.6%, respectively), laboratory-reared psyllids (70 and 33.3%, respectively), field-collected petioles from symptomatic carrots (80 and 55%, respectively), and laboratory-grown carrots (100% for both primer pairs). In contrast, no PCR products were detected in DNA extracted from insect-free plants. The DNA sequences of amplicons of the genes encoding liberibacter 16S rRNA from psyllids and carrots were identical. DNA of the 16S rRNA gene sequences determined from carrots and psyllids were 99.9% identical to analogous sequences of "Ca. L. solanacearum" amplified from several solanaceous crops and the psyllid Bactericera cockerelli (Sulc), a vector of this bacterium. This is the first report of a plant pathogen associated with T. apicalis and the second known psyllid species associated with "Ca. L. solanacearum".

Alternative Splicing of the CpomOR53 Gene Produces Three Transcripts Expressed in Codling Moth (Lepidoptera: Tortricidae) Antennae.

Incorporation of semiochemicals into codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), IPM programs has drastically reduced the amount of chemical insecticides needed to control this orchard pest. Odorant receptors are key sensors in the detection of semiochemicals and trigger downstream signaling events leading to behavioral responses. For codling moth, 58 odorant receptors have been identified in antennal transcriptomes, a few of which have been characterized for ligand activation. From the codling moth antennal transcriptome, a single transcript encoding CpomOR53 was annotated but re-evaluation suggests two or more variants of this receptor may be present and it is hypothesized that they are produced by alternative splicing. In this study, the complete open reading frame of CpomOR53 was amplified from codling moth male and female antennal RNAs, with three distinct transcripts detected. Characterization of these transcripts indicate that they are produced by alternative splicing of the CpomOR53 gene. The membrane topology for each of the CpomOR53 variants shows that alternative spliced products altered the length of intracellular loop two of the predicted proteins. The effects of these alterations were not determined but will be addressed in future studies determining the ligand(s) that activate each CpomOR53 transcript variant.

Characterization and expression of the short neuropeptide F receptor in the African malaria mosquito, Anopheles gambiae.

A short neuropeptide F (sNPF) precursor and a sNPF receptor (sNPFR) were characterized for the mosquito, Anopheles gambiae. The sNPFR was expressed in CHO-K1 cells, and it exhibited high affinity binding, IC(50) approximately 3-5 nM, for specific sNPFs. sNPF1 potently inhibited forskolin-stimulated cAMP production by transfected cells, suggesting sNPFR acts via G(i/o). Transcripts for sNPF and sNPFR were present in all body regions of larvae, pupae, and adults, and immunoblots for sNPFR confirmed this distribution in females. Membranes from female heads and thoraces exhibited prototypical high affinity binding for radiolabeled sNPF, indicating sNPFR is a bona fide endogenous receptor.

CRISPR/Cas9 Editing of the Codling Moth (Lepidoptera: Tortricidae) CpomOR1 Gene Affects Egg Production and Viability.

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major pest of pome fruit worldwide. Incorporation of semiochemicals, including the main sex pheromone (codlemone), into codling moth IPM programs has drastically reduced the amount of chemical insecticides needed to control this orchard pest. Odorant receptors located in sensory neuron membranes in the antennae are key sensors in the detection of semiochemicals and trigger downstream signaling events leading to a behavioral response. CpomOR1 is an odorant receptor belonging to the pheromone receptor subfamily in codling moth, and is a prime candidate for being a codlemone receptor based on its high expression levels in male antennae. In this study, the CpomOR1 gene was targeted using CRISPR/Cas9 genome editing to knockdown functional OR1 protein production to determine physiological function(s). By injecting early stage eggs, mutations were successfully introduced, including both deletions and insertions. When attempting to create stable populations of codling moth through mating of males with females containing mutations of the CpomOR1 gene, it was found that fecundity and fertility were affected, with edited females producing nonviable eggs. The role of CpomOR1 in fecundity and fertility in codling moth is unknown and will be the focus of future studies.

Bacterial Endosymbionts of the Psyllid Cacopsylla pyricola (Hemiptera: Psyllidae) in the Pacific Northwestern United States.

Insects often have facultative associations with bacterial endosymbionts, which can alter the insects' susceptibility to parasitism, pathogens, plant defenses, and certain classes of insecticides. We collected pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), from pear orchards in Washington and Oregon, and surveyed them for the presence of bacterial endosymbionts. Adult psyllids were collected on multiple dates to allow us to assay specimens of both the summer ("summerform") and the overwintering ("winterform") morphotypes. Two endosymbionts, Arsenophonus and Phytoplasma pyri, were detected in psyllids of both morphotypes in both states. A separate survey revealed similar associations present in psyllids collected in 1987. Arsenophonus was present in 80-100% of psyllids in all growing regions. A slightly lower proportion of summerform than winterform psyllids harbored the bacterium. Arsenophonus was present in the bacteriomes and developing oocytes of most psyllids, indicating that this endosymbiont is transovarially transmitted. This bacterium was also observed in the salivary glands and midguts of some psyllids. Phytoplasma pyri was present in a greater proportion of pear psylla from orchards near Yakima, WA, than from other regions, and was present in a higher proportion of winterforms than summerforms. We did not detect Wolbachia, Profftella, or Liberibacter europaeus, which are associated with other psyllid pests, including other species of Cacopsylla. Our study is the first to survey North American populations of C. pyricola for endosymbionts, and provides a foundation for further research on how bacterial associations may influence the ecology and management of this pest.

"Candidatus Liberibacter solanacearum" Associated With the Psyllid, Bactericera maculipennis (Hemiptera: Triozidae).

The psyllid Bactericera maculipennis (Crawford) (Hemiptera: Triozidae) often cohabits field bindweed (Convolvulus arvensis, Solanales: Convolvulaceae) and other plants with the congeneric psyllid, Bactericera cockerelli (Sulc), in the Pacific Northwestern United States. Bactericera cockerelli is a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease of potato (Solanales: Solanaceae). Because B. maculipennis and B. cockerelli both naturally occur on certain plants, we surveyed B. maculipennis adults collected from Washington and Idaho for presence of "Ca. L. solanacearum" to determine whether this psyllid also harbors this pathogen. Liberibacter was present in 30% of field-collected B. maculipennis and in 100% of colony-reared psyllids. Sequences of 16S rDNA and microsatellite markers revealed that "Ca. L. solanacearum" from B. maculipennis was closely related to Liberibacter haplotype B from B. cockerelli. Results of laboratory assays demonstrated that Liberibacter can be transmitted between B. cockerelli and B. maculipennis on plants within the Convolvulaceae. Potato plants challenged with Liberibacter-infected B. maculipennis did not become infected, apparently because potato is not a suitable host for the psyllid. We therefore conclude that B. maculipennis is not a direct threat to potato production, despite its association with Liberibacter. We are the first to report that "Ca. L. solanacearum" is associated with a psyllid other than B. cockerelli in North America. Results of our study demonstrate the importance of understanding the complete ecology of psyllids-including interactions with other psyllids on non-crop hosts-in predicting what crops or regions are potentially susceptible to the spread of Liberibacter.

Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity.

Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids were clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs.

Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 ß-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

Gut Content Analysis of a Phloem-Feeding Insect, Bactericera cockerelli (Hemiptera: Triozidae).

Potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a key pest of potato (Solanum tuberosum L., Solanales: Solanaceae) and a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease. In addition to its presence on cultivated crops, the psyllid regularly occurs on numerous uncultivated annual and perennial species within the Solanaceae. A better understanding of landscape-level ecology of B. cockerelli would substantially improve our ability to predict which potato fields are most likely to be colonized by infected psyllids. We developed three PCR-based methods of gut content analysis to identify what plant species B. cockerelli had previously fed upon. These methods included-1) sequencing PCR amplicons of regions of plant-derived internal transcribed spacer (ITS) or the chloroplast trnL gene from psyllids, 2) high-resolution melting analysis of ITS or trnL real-time PCR products, and 3) restriction enzyme digestion of trnL PCR product. Each method was used to test whether we could identify psyllids that had been reared continuously on potato versus psyllids reared continuously on the perennial nightshade, Solanum dulcamara. All three methods of gut content analysis correctly identified psyllids from potato and psyllids from S. dulcamara Our study is the first to demonstrate that plant DNA can be detected in a phloem-feeding insect. Gut content analysis, in combination with other landscape ecology approaches, could help elucidate patterns in landscape-level movements and host plant associations of B. cockerelli.

The chemosensory receptors of codling moth Cydia pomonella-expression in larvae and adults.

Olfaction and gustation play critical roles in the life history of insects, mediating vital behaviors such as food, mate and host seeking. Chemosensory receptor proteins, including odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) function to interface the insect with its chemical environment. Codling moth, Cydia pomonella, is a worldwide pest of apple, pear and walnut, and behavior-modifying semiochemicals are used for environmentally safe control. We produced an Illumina-based transcriptome from antennae of males and females as well as neonate head tissue, affording a qualitative and quantitative analysis of the codling moth chemosensory receptor repertoire. We identified 58 ORs, 20 GRs and 21 IRs, and provide a revised nomenclature that is consistent with homologous sequences in related species. Importantly, we have identified several OR transcripts displaying sex-biased expression in adults, as well as larval-enriched transcripts. Our analyses have expanded annotations of the chemosensory receptor gene families, and provide first-time transcript abundance estimates for codling moth. The results presented here provide a strong foundation for future work on codling moth behavioral physiology and ecology at the molecular level, and may lead to the development of more precise biorational control strategies.

Characterization of neuropeptide F and its receptor from the African malaria mosquito, Anopheles gambiae.

The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50 approximately 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF>Aea-NPF>Drm-NPF>Drm-NPF8-36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito.

Phenotypic screen for RNAi effects in the codling moth Cydia pomonella.

RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate the induction of RNAi in the codling moth, Cydia pomonella, a major lepidopteran pest of apple, pear, and walnut. Prior to a knockdown screen, fluorescently labeled small interfering RNA (siRNA) and double-stranded RNA (dsRNA) derived from green fluorescent protein (GFP) coding sequence were delivered to the surface of artificial diet to which neonate larvae were introduced and subsequently examined for the distribution of fluorescence in their tissues. Fluorescence was highly concentrated in the midgut but its presence in other tissues was equivocal. Next, dsRNAs were made for C. pomonella genes orthologous to those that have well defined deleterious phenotypes in Drosophila melanogaster. A screen was conducted using dsRNAs encoding cullin-1 (Cpcul1), maleless (Cpmle), musashi (Cpmsi), a homeobox gene (CpHbx), and pumilio (Cppum). The dsRNAs designed from these target genes were administered to neonate larvae by delivery to the surface of the growth medium. None of the dsRNA treatments affected larval viability, however Cpcul1-dsRNA had a significant effect on larval growth, with the average length of larvae about 3mm, compared to about 4mm in the control groups. Measurement of Cpcul1 transcript levels by quantitative real-time PCR (qRT-PCR) revealed a dose-dependent RNAi effect in response to increasing amount of Cpcul1-dsRNA. Despite their reduced size, Cpcul1-dsRNA-treated larvae molted normally and matured to adulthood in a manner similar to controls. In an additional experiment, Cpcul1-siRNA was found to induce similar stunting effect as that induced by Cpcul1-dsRNA.

Horizontal Transmission of "Candidatus Liberibacter solanacearum" by Bactericera cockerelli (Hemiptera: Triozidae) on Convolvulus and Ipomoea (Solanales: Convolvulaceae).

"Candidatus Liberibacter solanacearum" (Proteobacteria) is an important pathogen of solanaceous crops (Solanales: Solanaceae) in North America and New Zealand, and is the putative causal agent of zebra chip disease of potato. This phloem-limited pathogen is transmitted to potato and other solanaceous plants by the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). While some plants in the Convolvulaceae (Solanales) are also known hosts for B. cockerelli, previous efforts to detect Liberibacter in Convolvulaceae have been unsuccessful. Moreover, studies to determine whether Liberibacter can be acquired from these plants by B. cockerelli are lacking. The goal of this study was to determine whether horizontal transmission of Liberibacter occurs among potato psyllids on two species of Convolvulaceae, sweet potato (Ipomoea batatas) and field bindweed (Convolvulus arvensis), which grows abundantly in potato growing regions of the United States. Results indicated that uninfected psyllids acquired Liberibacter from both I. batatas and C. arvensis if infected psyllids were present on plants concurrently with the uninfected psyllids. Uninfected psyllids did not acquire Liberibacter from plants if the infected psyllids were removed from the plants before the uninfected psyllids were allowed access. In contrast with previous reports, PCR did detect the presence of Liberibacter DNA in some plants. However, visible amplicons were faint and did not correspond with acquisition of the pathogen by uninfected psyllids. None of the plants exhibited disease symptoms. Results indicate that horizontal transmission of Liberibacter among potato psyllids can occur on Convolvulaceae, and that the association between Liberibacter and Convolvulaceae merits additional attention.