Stephen F. Garczynski

Sex Pheromone Receptor Specificity in the European Corn Borer Moth, Ostrinia nubilalis

Background The European corn borer (ECB), Ostrinia nubilalis (Hubner), exists as two separate sex pheromone races. ECB(Z) females produce a 97∶3 blend of Z11- and E11-tetradecenyl acetate whereas ECB(E) females produce an opposite 1∶99 ratio of the Z and E isomers. Males of each race respond specifically to their conspecific females blend. A closely related species, the Asian corn borer (ACB), O. furnacalis, uses a 3∶2 blend of Z12- and E12-tetradecenyl acetate, and is believed to have evolved from an ECB-like ancestor. To further knowledge of the molecular mechanisms of pheromone detection and its evolution among closely related species we identified and characterized sex pheromone receptors from ECB(Z). Methodology Homology-dependent (degenerate PCR primers designed to conserved amino acid motifs) and homology-independent (pyrophosphate sequencing of antennal cDNA) approaches were used to identify candidate sex pheromone transcripts. Expression in male and female antennae was assayed by quantitative real-time PCR. Two-electrode voltage clamp electrophysiology was used to functionally characterize candidate receptors expressed in Xenopus oocytes. Conclusion We characterized five sex pheromone receptors, OnOrs1 and 3–6. Their transcripts were 14–100 times more abundant in male compared to female antennae. OnOr6 was highly selective for Z11-tetradecenyl acetate (EC50 = 0.86±0.27 µM) and was at least three orders of magnitude less responsive to E11-tetradecenyl acetate. Surprisingly, OnOr1, 3 and 5 responded to all four pheromones tested (Z11- and E11-tetradecenyl acetate, and Z12- and E12-tetradecenyl acetate) and to Z9-tetradecenyl acetate, a behavioral antagonist. OnOr1 was selective for E12-tetradecenyl acetate based on an efficacy that was at least 5-fold greater compared to the other four components. This combination of specifically- and broadly-responsive pheromone receptors corresponds to published results of sensory neuron activity in vivo. Receptors broadly-responsive to a class of pheromone components may provide a mechanism for variation in the male moth response that enables population level shifts in pheromone blend use.

Association of “Candidatus Liberibacter solanacearum” with the Psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe

ABSTRACT 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.”

Moth Sex Pheromone Receptors and Deceitful Parapheromones

The insects olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.

Characterization of three transcripts encoding small heat shock proteins expressed in the codling moth, Cydia pomonella (Lepidoptera: Tortricidae)

Abstract  Codling moth is a major pest of apples and pears worldwide. Increasing knowledge of how this insect responds to environmental stress will improve field and post‐harvest control measures used against it. The small heat shock proteins (sHsps) play a major role in cellular responses to environmental stressors. A degenerate oligonucleotide primer, designed against the conserved α‐crystallin domain, was used in 3′ rapid amplification of complementary DNA (cDNA) ends reactions to amplify transcripts encoding sHsps expressed in the codling moth cell line, Cp169, subjected to heat shock. Three full‐length cDNAs were cloned from Cp169 cells that contained open reading frames encoding sHsps. The cDNA for CpHsp19.8 was 795 bp encoding 177 amino acids. The cDNA for CpHsp19.9 was 749 bp encoding 175 amino acids. The cDNA for CpHsp22.2 was 737 bp encoding 192 amino acids. Analysis of the protein sequences of the three CpHsps indicated the presence of 83 amino acids with homology to the α‐crystallin domain. For each of the CpHsps, the α‐crystallin domain was surrounded by divergent N‐ and C‐terminal regions, consistent with the conserved structural features of sHsps. Real‐time polymerase chain reaction, used to determine the expression patterns of each of the sHsps in different developmental stages of codling moth revealed the presence of transcripts in all stages tested. Consistent with characteristics of other sHsps, expression of CpHsp transcripts were greatly enhanced when insects were subjected to heat shock. The results of this research can be used as a guide to study the roles of sHsps in codling moth control using various post‐harvest treatments.

Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth

The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8 341 expressed sequence tags was generated from Roche 454 GS‐FLX sequencing of eight tissue‐specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698 nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT‐PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1 289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily.

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.

Identification and initial characterization of the 3′ end of gene transcripts encoding putative members of the pheromone receptor subfamily in Lepidoptera

Abstract  Semiochemicals, including pheromones and kairomones, used in pest management programs reduce the need for chemical insecticides, and understanding their interactions with their membrane receptors may help make them more effective in the field. Identification of odorant receptors in the Lepidoptera has mainly been achieved using bioinformatics to search DNA sequences generated by genome or expressed sequence tag (EST) sequencing projects. This study reports a rapid method to identify members of the pheromone receptor subfamily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of known lepidopteran pheromone receptors, and the primers were used in a 3′ rapid amplification of complementary DNA (cDNA) ends procedure. Polymerase chain reaction products generated from seven different lepidopteran species were TA cloned and sequenced. The cDNA sequences of 25 transcripts were determined to encode potential members of the pheromone receptor subfamily. These cDNAs ranged from 238 to 642 bp and encoded 49–54 amino acids of the carboxyl terminus. Analysis of the 3′ untranslated region reveals that most of the transcripts contain multiple polyadenylation signal sequences, and in the case of Manduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3′ untranslated region was also useful in determining unique receptors encoded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of pheromone receptor identification in EST sequencing projects, or can be used as a stand‐alone method in conjunction with 5′ rapid amplification of cDNA ends procedures.

Development of Monoclonal Antibodies to Pear Psylla (Hemiptera: Psyllidae) and Evaluation of Field Predation by Two Key Predators

Abstract The pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), and related psyllids are important pests of pear (Pyrus spp.) worldwide. Many of these pests are thought to be partially controlled by predatory insects. To improve our understanding of the predator species that attack pear psylla, we developed monoclonal antibodies (MAbs) against this pest for predator gut content studies. Mice were immunized with homogenates of nymphal, adult, and egg stages of pear psylla. A mouse immunized with nymph homogenate showed high activity against all three antigen types and was used for MAb development. From 952 hybridomas screened, 35 showed good activity to pear psylla and low activity against nontarget arthropods. Four MAbs were retained: two from immunoglobulin M (IgM)-secreting hybridomas, both with high activity against all stages of psylla except young eggs, and two immunoglobin G-secreting hybridomas, both with high activity against psylla eggs and gravid adult females. Using one of the IgM-MAbs, pear psylla remains were detected in the predatory bugs Anthocoris tomentosus Péricart (Heteroptera: Anthocoridae) and Deraeocoris brevis (Uhler) (Heteroptera: Miridae) in laboratory feeding trials. Digestion half lives typically exceeded 24 h and were dependent on meal size and predator life stage. Gut content analysis of 970 field-collected D. brevis and Anthocoris spp. showed that the proportion which fed on psylla averaged 59% and that percentage closely tracked the density of pear psylla nymphs during three seasons. The utility of these antibodies for the study of trophic interactions and habitat management in relation to biological control of pear psylla is discussed.

Wolbachia Infection Differs Among Divergent Mitochondrial Haplotypes of Bactericera cockerelli (Hemiptera: Triozidae)

ABSTRACT Four mitochondrial (cytrochrome oxidase I) haplotypes of the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), have been identified in North America: western, central, northwestern, and southwestern. A recent study found that females of the northwestern haplotype mated by males of the western or central haplotypes failed to produce viable eggs. Our goal was to determine whether these patterns in reproductive incompatibility are associated with differences among haplotypes in the presence of cytoplasmic incompatibility-inducing bacteria, Wolbachia or Cardinium. Polymerase chain reaction (PCR) revealed that psyllids of the western and central haplotypes were both simultaneously infected with two strains of Wolbachia, but Wolbachia was not detected in psyllids of the northwestern haplotype. PCR using archived DNA from field-collected psyllids confirmed patterns in Wolbachia infection among the western, central, and northwestern haplotypes, and also indicated that Wolbachia was not detectable in psyllids of the southwestern haplotype, which were not available for the mating studies. Cardinium was not detected in psyllids regardless of haplotype. These results provide evidence that differences in Wolbachia infection may be the cause of cytoplasmic incompatibilities among sympatric yet biologically distinct populations of B. cockerelli that have highly divergent mitochondrial haplotypes. This knowledge will improve the interpretation of studies to assess interactions and biological differences among B. cockerelli haplotypes.

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 β‐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.