Pea leaf weevil, Sitona lineatus (L.) (Coleoptera: Curculionidae), adult feeding preference and larval development on pulse crop varieties in Montana.

BACKGROUND: Montana accounts for approximately 45% of US dry pea production and the pea leaf weevil (PLW; Sitona lineatus (L.)) is the most common insect pest in this region. After crop emergence adult PLW feed on the foliage to mature and subsequently mate, and the soil-dwelling larvae feed and develop on the nitrogen-fixing root nodules. Producers commonly apply prophylactic insecticide treatments to the seed at planting as well as one or two post-emergent insecticide sprays to control PLW damage. To develop alternative management strategies based on integrated pest management (IPM), this field study evaluated pulse crops grown in Montana for adult feeding preference and larval development. Ten different field pea varieties, along with two faba bean, lentil and chickpea varieties, were evaluated during the 2020 and 2021 field seasons at the Montana State University Arthur H. Post Agronomy Farm. RESULTS: Significant PLW pest pressure was observed within the research plots during both experimental years. Field pea and faba bean were preferred by the foliage feeding adult stage, with all but one variety averaging 39.2 to 86.3 average notches per plant. The pea variety Lifter was significantly preferred over all other comparisons, averaging 142.4 and 95.0 notches per plant in 2020 and 2021, respectively. Adult PLW feeding on lentil and chickpea was minimal, averaging 3.3 to 8.2 and 0.5 to 1.6 notches per plant, respectively. Numbers of larvae were highest on the roots of pea varieties, a known reproductive host, and almost nil on lentil and chickpea roots. Faba bean is also known as reproductive host, but, unexpectedly, larval populations were also low on the two faba bean varieties. CONCLUSIONS: The results from this study provide some limited evidence for alternative IPM strategies for field peas based on host plant tolerance or resistance within the range of varieties tested. Adult preference and larval development of PLW varied between the different pulse crops with field peas and faba beans being the most susceptible and lentils and chickpeas being the least susceptible. Host plant resistance against PLW could provide more sustainable IPM approaches in the future. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Alfalfa weevils (Coleoptera: Curculionidae) in the western United States are resistant to multiple type II pyrethroid insecticides.

The alfalfa weevil (Hypera postica Gyllenhal (Coleoptera: Curculionidae)), a key pest of alfalfa (Medicago sativa L. (Fabales: Fabacae)) across the US, has developed resistance to pyrethroids lambda-cyhalothrin and zeta-cypermethrin in at least 6 western US states. Unfortunately, 6 pyrethroid active ingredients represent most commercial insecticides registered for alfalfa weevil control in forage alfalfa systems. Thus, the loss of efficacy of this mode of action group due to multiple resistance represents a significant agricultural challenge because of a limited registered alternative mode of actions. To evaluate the extent and severity of resistance among pyrethroids around the United States, laboratory bioassays using larvae from Arizona, California, Montana, Oregon, Washington, and Wyoming, including both the Egyptian and western strains, were conducted. Results indicated that similar degrees of resistance among type II pyrethroids as determined by both laboratory bioassays and field trials exist. The LC50 values of alpha-cypermethrin, beta-cyfluthrin and zeta-cypermethrin produced significant correlations with the LC50 values of lambda-cyhalothrin. In contrast, resistance did not include type I pyrethroid, bifenthrin (registered for seed alfalfa production), whose LC50 values yielded a slope not significantly different from zero when correlated with lambda-cyhalothrin. Field trials conducted in Arizona, Montana, and Washington corroborated laboratory results, as commercial formulations with type II pyrethroid active ingredients failed to adequately control alfalfa weevils resistant to lambda-cyhalothrin. Integrated resistance management recommendations are discussed.

First Report of Alfalfa Weevil (Coleoptera: Curculionidae) Resistance to Lambda-Cyhalothrin in Montana.

Forage alfalfa (Medicago sativa L. [Fabales: Fabacae]) is a major agronomic crop grown nationally and Montana ranks highly in acres harvested. The alfalfa weevil (Hypera postica Gyllenhal [Coleoptera: Curculionidae]) is the primary defoliating pest that requires insecticide applications to prevent yield loss, particularly pyrethroid active ingredients (a.i.) that are both efficacious and cost-effective. Reports from commercial alfalfa producers in Big Horn County, MT, suggested local populations of alfalfa weevil had developed resistance to the pyrethroid a.i. lambda-cyhalothrin (type II pyrethroid). Chemical control is an important component of integrated pest management (IPM) of alfalfa weevil and the loss of pyrethroid a.i. as an effective tool would result in additional production costs. Two locations in southern Big Horn County and nine locations in four other Montana counties where resistance has not been reported were sampled and assayed for resistance to lambda-cyhalothrin. Populations from three counties were susceptible, the concentration causing 50% mortality (LC50) ranged from 0.02 to 0.10 µg/cm2. In contrast, populations from Big Horn County did not reach 50% mortality at the highest concentration of lambda-cyhalothrin tested (3.30 µg/cm2), indicating high levels of resistance have developed in these populations. A field trial in Big Horn County supported laboratory results of resistance; lambda-cyhalothrin at the highest label rate did not reduce alfalfa weevil populations. Additional bioassays suggest cross-resistance to zeta-cypermethrin (type II pyrethroid), but only partial cross-resistance to permethrin (type I pyrethroid).

Wireworm (Coleoptera: Elateridae) genomic analysis reveals putative cryptic species, population structure, and adaptation to pest control.

The larvae of click beetles (Coleoptera: Elateridae), known as "wireworms," are agricultural pests that pose a substantial economic threat worldwide. We produced one of the first wireworm genome assemblies (Limonius californicus), and investigated population structure and phylogenetic relationships of three species (L. californicus, L. infuscatus, L. canus) across the northwest US and southwest Canada using genome-wide markers (RADseq) and genome skimming. We found two species (L. californicus and L. infuscatus) are comprised of multiple genetically distinct groups that diverged in the Pleistocene but have no known distinguishing morphological characters, and therefore could be considered cryptic species complexes. We also found within-species population structure across relatively short geographic distances. Genome scans for selection provided preliminary evidence for signatures of adaptation associated with different pesticide treatments in an agricultural field trial for L. canus. We demonstrate that genomic tools can be a strong asset in developing effective wireworm control strategies.

Pheromone Odorant Receptor Responses Reveal the Presence of a Cryptic, Redundant Sex Pheromone Component in the European Corn Borer, Ostrinia nubilalis.

Two odorant receptors (ORs), OnubOR3 and OnubOR6, in the sex pheromone communication systems of E- and Z-strain European corn borers, Ostrinia nubilalis, were broadly receptive to analogs of their pheromone components. In addition to responding to their natural 14-carbon pheromone components, (Z)-11- and (E)-11-tetradecenyl acetates (Z11- and E11-14:OAc), these pheromone ORs responded to the longer-chain compounds, (Z)-11- and (E)-11-hexadecenyl acetate (Z11- and E11-16:OAc). Z11-16:OAc is a pheromone gland constituent of E-strain O. nubilalis females in Europe but has not previously been shown to have behavioral activity to males. Here, we demonstrate that Z11-16:OAc evokes high levels of upwind flight and source location in E-strain males when substituted for Z11-14:OAc (minor component) in the E-strain blend. Since Z11-16:OAc is found in the gland and has behavioral activity when Z11-14:OAc is missing, then it should be classified as a cryptic, redundant minor pheromone component in E-strain O. nubilalis. The opposite geometric isomer, E11-16:OAc, also functions in Z-strain O. nubilalis, substituting behaviorally for the E11-14:OAc minor component, but has not been found in Z-strain female glands. Single-sensillum recordings showed that sensory neurons of E- and Z-strain male antennae expressing OnubOR3 and OnubOR6 produced responses to these hexadecenyl acetates similar to those evoked by the natural (tetradecenyl acetate) pheromone components. We postulate that the wide responsiveness of these two ORs to the 16-carbon acetates could be a preadaptation for O. nubilalis to use these compounds as minor components in lieu of the respective 14-carbon acetates. Alternatively, the responsiveness of OnubOR3 to E11-16:OAc and OnubOR6 to Z11-16:OAc could represent a vestigial state of these receptors, with the 16-carbon acetates having previously acted as functional minor components in an ancestral blend.

Genome Sequence of the Wheat Stem Sawfly, Cephus cinctus, Representing an Early-Branching Lineage of the Hymenoptera, Illuminates Evolution of Hymenopteran Chemoreceptors.

The wheat stem sawfly, Cephus cinctus, is a major pest of wheat and key ecological player in the grasslands of western North America. It also represents the distinctive Cephoidea superfamily of sawflies (Symphyta) that appeared early during the hymenopteran radiation, but after three early-branching eusymphytan superfamilies that form the base of the order Hymenoptera. We present a high-quality draft genome assembly of 162 Mb in 1,976 scaffolds with a scaffold N50 of 622 kb. Automated gene annotation identified 11,210 protein-coding gene models and 1,307 noncoding RNA models. Thirteen percent of the assembly consists of ∼58,000 transposable elements partitioned equally between Class-I and Class-II elements. Orthology analysis reveals that 86% of Cephus proteins have identifiable orthologs in other insects. Phylogenomic analysis of conserved subsets of these proteins supports the placement of the Cephoidea between the Eusymphyta and the parasitic woodwasp superfamily Orussoidea. Manual annotation and phylogenetic analysis of families of odorant, gustatory, and ionotropic receptors, plus odorant-binding proteins, shows that Cephus has representatives for most conserved and expanded gene lineages in the Apocrita (wasps, ants, and bees). Cephus has also maintained several insect gene lineages that have been lost from the Apocrita, most prominently the carbon dioxide receptor subfamily. Furthermore, Cephus encodes a few small lineage-specific chemoreceptor gene family expansions that might be involved in adaptations to new grasses including wheat. These comparative analyses identify gene family members likely to have been present in the hymenopteran ancestor and provide a new perspective on the evolution of the chemosensory gene repertoire.

Genomic mechanisms of sympatric ecological and sexual divergence in a model agricultural pest, the European corn borer.

The European corn borer, Ostrinia nubilalis, is a model species for elucidating mechanisms underlying adaptively differentiated subpopulations in the face of reciprocal gene flow, and is a major pest of cultivated maize in North America and Eurasia. Strains are characterized by different pheromone communication systems in combination with voltinism strains that are adapted to distinct local climate and photoperiod through adjustments in diapause traits. However, only partial barriers to inter-strain hybridization exist in areas of sympatry. Recent research shows that genes governing important strain-specific isolating traits are disproportionately located on the Z-chromosome. Furthermore, co-adapted combinations of some of these genes are non-recombining due to location within a large chromosomal inversion, and assist in maintaining strain integrity despite hybridization.

Evaluation of Four Bait Traps for Sampling Wireworm (Coleoptera: Elateridae) Infesting Cereal Crops in Montana.

The basic principles of a reliable integrated pest management program include pest identification, monitoring, and distribution. Selecting the appropriate sampling protocol to monitor wireworm for research or applied entomology depends on the objective, including simply detecting the presence or absence of wireworm, surveying the composition of wireworm assemblages, or estimating spatial and temporal population densities. In this study, the efficacy of pitfall, stocking, pot, and canister traps baited with wheat and barley mixtures was evaluated for monitoring wireworm populations in four commercial cereal fields in Montana. Pitfall and stocking traps collected greater numbers of wireworm (1625 and 1575, respectively) followed by pot-type and canister-type traps (1173 and 725, respectively). The 5098 wireworm collected from four sites included seven species: Aeolus mellillus, Agriotes sp, Dalopius sp, Hypnoidus bicolor, Limonius californicus, Limonius infuscatus, and S. aeripennis.

Binding affinity of five PBPs to Ostrinia sex pheromones.

BACKGROUND: Pheromone binding proteins (PBPs) of male Lepidoptera function in chemical communication, mate attraction and recognition. Directional selection was previously predicted between PBP3 orthologs of Ostrinia furnacalis and Ostrinia nubilalis were interpreted as being involved in sexual isolation. RESULTS: In vitro assays show that recombinant male OfurPBP3 bound O. furnacalis sex pheromones, Z-12-tetradecenyl acetate (Z12-14:OAc) and E-12-tetradecenyl acetate (E12-14:OAc), as well as to ECB pheromones Z11- and E11-14:OAc. Recombinant OfurPBP4 and OfurPBP5 bound E11- and Z11-14:OAc with greater affinity compared to Z12- and E12-14:OAc, and OfurPBP4 incapable of binding with E12-14:OAc. In silico molecular docking predicted OfurPBP3 residues Phe12, Ile52, Leu94, Ile113 within a hydrophobic ligand-binding pocket and may participate in E12- and Z12-14:OAc binding. Independent site-directed mutagenesis experiments demonstrated that Ser12, Asn52, Arg94, and Asn113 residues variants caused an approximately 1.7- to 4.6-fold reduction in OfurPBP3 affinity for Z12- and E12-14:OAc, and a 2.7- to 8.4-fold decrease in affinity towards E11- and Z11-14:OAc. CONCLUSIONS: Five PBPs of O. furnacalis play important functions in Ostrinia pheromones binding. These four amino acids may play a role in binding of sex pheromone, but this study does not address questions regarding specific response between males of O. furnacalis and O. nubilalis. Additional studies are required determine the role, if any, PBPs play in the evolution of sex pheromone communication.

Efficacy of thiamethoxam and fipronil, applied alone and in combination, to control Limonius californicus and Hypnoidus bicolor (Coleoptera: Elateridae).

BACKGROUND: Wireworms, the larval stage of click beetles (family Elateridae), are significant soil pests of wheat and barley crops in the Pacific Northwest. At present, few pest management alternatives exist. For several decades, wireworms were effectively controlled by first-generation insecticides applied to the soil or as seed treatments. Currently used neonicotinoid insecticides protect crop seeds and germinating seedlings by temporary toxicity but limited mortality. As a result, field populations may increase, reaching levels too high for crop protection. In this study an investigation was made of the combination of two insecticides to achieve crop protection as well as insect mortality in wheat fields. RESULTS: Laboratory bioassays using wheat seed treated with fipronil at 1.0 and 5.0 g AI 100 kg(-1) seed resulted in 72-90% mortality of two wireworm species, Limonius californicus and Hypnoidus bicolor. At a rate of 39 g AI 100 kg(-1) seed, 8 times higher than the high rate of fipronil, thiamethoxam caused only 10-31% larval mortality in the bioassays, but did protect developing wheat stands from damage in field trials. Field plots planted with wheat seed treated with both fipronil (5.0 g AI 100 kg(-1) seed) and thiamethoxam (39.0 g AI 100 kg(-1) seed) had 83% fewer wireworms the following year compared with untreated check plots. No reduction in population was observed in plots treated with 39.0 g of thiamethoxam alone. CONCLUSIONS: Fipronil and thiamethoxam can be combined as a seed treatment to protect wheat crops from wireworm damage and reduce larval populations in the field.

DNA barcoding to improve the species-level management of wireworms (Coleoptera: Elateridae).

Economically important species of wireworms (Coleoptera: Elateridae) were successfully associated with adults using cytochrome oxidase I (COI) barcoding, proving the usefulness of this technique to associate life stages in taxonomically difficult pest groups. Previously unrecognized and morphologically difficult, even indistinguishable, pest larvae were shown to be identifiable using this technique. This is a critical step toward discovering effective species-based integrated pest management strategies for this resurgent pest group following the loss of Lindane seed treatments. Three new adult-larval associations were discovered for Hadromorphus callidus (Brown), Hemicrepidius carbonatus (LeConte) and Metanomus insidiosus (LeConte). Hypnoidus bicolor (Eschscholtz) was shown to comprise multiple divergent lineages at a level usually considered different species, indicating that the population structure of some pest species requires more investigation. The status of the prairie grain wireworm, Selatosomus destructor (Brown), as a full species or as a subspecies of Selatosomus aeripennis (Kirby) is called into question.

Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species.

Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.

Gene genealogies reveal differentiation at sex pheromone olfactory receptor loci in pheromone strains of the European corn borer, Ostrinia nubilalis.

Males of the E and Z strains of the European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) are attracted to different blends of the same pheromone components. The difference in male behavioral response is controlled by the sex-linked locus Resp. The two types of males have identical neuroanatomy but their physiological specificity is reversed, suggesting that variation at the periphery results in behavioral change. Differences in the olfactory receptors (ORs) could explain the strain-specific antennal response and blend preference. Gene genealogies can provide insights into the processes involved in speciation and allow delineation of genome regions that contribute to reproductive barriers. We used intronic DNA sequences from five OR-encoding genes to investigate whether they exhibit fixed differences between strains and therefore might contribute to reproductive isolation. Although two genealogies revealed shared polymorphism, molecular polymorphism at three genes revealed nearly fixed differences between strains. These three OR genes map to the sex chromosome, but our data indicate that the distance between Resp and the ORs is >20 cM, making it unlikely that variation in pheromone-sensitive OR genes is directly responsible for the difference in behavioral response. However, differences in male antennal response may have their origin in the selection of strain-specific alleles.

The application and performance of single nucleotide polymorphism markers for population genetic analyses of lepidoptera.

Microsatellite markers are difficult to apply within lepidopteran studies due to the lack of locus-specific PCR amplification and the high proportion of "null" alleles, such that erroneous estimations of population genetic parameters often result. Herein single nucleotide polymorphism (SNP) markers are developed from Ostrinia nubilalis (Lepidoptera: Crambidae) using next generation expressed sequence tag (EST) data. A total of 2742 SNPs were predicted within a reference assembly of 7414 EST contigs, and a subset of 763 were incorporated into 24 multiplex PCR reactions. To validate this pipeline, 5 European and North American sample sites were genotyped at 178 SNP loci, which indicated 84 (47.2%) were in Hardy-Weinberg equilibrium. Locus-by-locus F(ST), analysis of molecular variance, and STRUCTURE analyses indicate significant genetic differentiation may exist between European and North American O. nubilalis. The observed genetic diversity was significantly lower among European sites, which may result from genetic drift, natural selection, a genetic bottleneck, or ascertainment bias due to North American origin of EST sequence data. SNPs are an abundant source of mutation data for molecular genetic marker development in non-model species, with shared ancestral SNPs showing application within closely related species. These markers offer advantages over microsatellite markers for genetic and genomic analyses of Lepidoptera, but the source of mutation data may affect the estimation of population parameters and likely need to be considered in the interpretation of empirical data.

Asian corn borer pheromone binding protein 3, a candidate for evolving specificity to the 12-tetradecenyl acetate sex pheromone.

Most moth species in the genus Ostrinia use varying ratios of (Z)-11- and (E)-11-tetradecenyl acetate as their main sex pheromone components. The Asian corn borer is unique within the genus having evolved to use pheromone components with a shift in the location of the double bond, (Z)-12- and (E)-12-tetradecenyl acetate. We identified cDNAs representing five pheromone binding proteins (PBPs) and two sensory neuron membrane protein genes from an antennal transcriptome. The coding regions of the orthologous genes were cloned from the Asian corn borer and the (E) and (Z) sex pheromone races of the European corn borer. Their nucleotide sequences and transcript expression levels were analyzed to identify candidate genes from the Asian corn borer that may have evolved specificity to the 12-tetradecenyl acetate ligand. PBP2 and PBP3 transcripts were expressed at high male-biased levels. PBP3 had the most nonsynonymous nucleotide substitutions resulting in ten amino acid changes. Based on the predicted three-dimensional structure of PBP3, six of these ten amino acid changes occur in domains that may interact with the pheromone ligand. Future studies will determine whether PBP3 has evolved specificity to the Asian corn borer sex pheromone.

Iridovirus and microsporidian linked to honey bee colony decline.

BACKGROUND: In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses. METHODOLOGY/PRINCIPAL FINDINGS: We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006-2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone. CONCLUSIONS/SIGNIFICANCE: These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.

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 97ratio3 blend of Z11- and E11-tetradecenyl acetate whereas ECB(E) females produce an opposite 1ratio99 ratio of the Z and E isomers. Males of each race respond specifically to their conspecific female's blend. A closely related species, the Asian corn borer (ACB), O. furnacalis, uses a 3ratio2 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 (EC(50) = 0.86+/-0.27 microM) 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.

Neonate silkworm (Bombyx mori) larvae are attracted to mulberry (Morus alba) leaves with conspecific feeding damage.

The silkworm Bombyx mori is a molecular genetic model for the Lepidoptera. Its odorant receptor genes have been described, and preliminary studies suggest that several are expressed specifically in the larval caterpillar stage. This study was undertaken to identify olfactory behaviors specific to the larvae. A two-choice leaf disk bioassay with naive neonate larvae was used to evaluate the attractiveness of three types of mulberry leaf (Morus alba): newly flushed leaves from branch tips, mature leaves, and mature leaves with feeding damage caused by conspecific larvae. Mature leaves with feeding damage were the most attractive, newly flushed leaves were moderately favored, and undamaged mature leaves were the least attractive. Volatile odors collected from whole mulberry leaves by using solid-phase microextraction fibers were analyzed by gas chromatography-mass spectrometry. The volatile profile of newly flushed leaves and mature leaves damaged by conspecific larvae was more complex compared to undamaged mature leaves. By comparing the volatile makeup of each leaf type, a list of 22 candidate odors responsible for attracting the neonate larvae was generated; alpha-farnesene was particularly notable as a herbivore-induced volatile. These odors will be used in future in vitro studies to determine whether they activate larval-specific odorant receptors.

Molecular basis of female-specific odorant responses in Bombyx mori.

Males and females of many moth species exhibit important differences in sexual behaviours. Much research in this field has focused on the male-specific behaviour, electrophysiology and molecular biology of sex pheromone reception. Female-specific behaviours have been less well studied although, like male-specific behaviours, they could provide opportunities for intervention and management of lepidopteran pests. Previously, we identified genes encoding putative odorant receptors (ORs) from the genome of the silkworm, Bombyx mori, some of which have higher levels of steady-state transcript in the antennae of adult females compared with males. We have identified the full-length cDNA sequences of some of these ORs and described a novel OR that is part of a female-biased clade. Using expression in Sf9 cells and a calcium-imaging assay, we tested a range of compounds for their ability to activate the most highly female-biased ORs, BmOR19, BmOR30, BmOR45 and BmOR47. BmOR19 responds to linalool, while BmOR45 and BmOR47 respond to benzoic acid > 2-phenylethanol > benzaldehyde. No activating ligands were found for BmOR30. RNA in situ hybridisation experiments reveal that BmOR19 is expressed in female olfactory sensory neurons that are co-located in the same sensilla as a second ORN expressing BmOR45 and/or BmOR47. Taken together the activity and expression of these receptors is likely explanatory of the observed electrophysiology of long sensilla trichoidea of female B. mori, previously shown to each contain one terpene (BmOR19) and one benzoic acid (BmOR45, BmOR47) sensory neuron. Plant volatiles such as linalool, benzoic acid, 2-phenylethanol and benzaldehyde are oviposition cues for females of some moths. These compounds have also been found in male-produced pheromone blends extracted from the hair pencils of many noctuid species. Hair pencil structures have not previously been reported for B. mori, but we have found hair pencil-like structures in adult male B. mori that are absent in female moths. It is proposed that BmOR19, BmOR45 and BmOR47 account for some of the female-specific odorant responses in B. mori, such as oviposition and/or detection of an as yet unidentified male-produced sex pheromone.

A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid.

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.