Behavioral Evidence for Olfactory-Based Location of Honeybee Colonies by the Scarab Oplostomus haroldi.

The Afro-tropical scarab Oplostomus haroldi (Witte) is a pest of honeybees in East Africa with little information available on its chemical ecology. Recently, we identified a female-produced contact sex pheromone, (Z)-9-pentacosene, from the cuticular lipids that attracted males. Here, we investigated the kairomonal basis of host location in O. haroldi. We used coupled gas chromatography/electroantennographic detection (GC/EAD) and GC/mass spectrometry to identify antennally-active compounds from volatiles collected from honeybee colonies. Antennae of both sexes of the beetle consistently detected seven components, which were identified as 3-hydroxy-2-butanone, 2,3-butanediol, butyl acetate, isopentyl acetate, butyl butyrate, hexyl acetate, and methyl benzoate. In olfactometer bioassays, both sexes responded to the full seven-component synthetic blend over solvent controls, but chose honeybee colony odors over the blend. These findings suggest that the seven compounds are components of a kairomone from honeybee colonies used by O. haroldi.

Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense.

Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. As the first described plant peptide signal, systemin regulates antiherbivore defenses in the Solanaceae, but in other plant families, peptides with analogous activity have remained elusive. In the current study, we demonstrate that a member of the maize (Zea mays) plant elicitor peptide (Pep) family, ZmPep3, regulates responses against herbivores. Consistent with being a signal, expression of the ZmPROPEP3 precursor gene is rapidly induced by Spodoptera exigua oral secretions. At concentrations starting at 5 pmol per leaf, ZmPep3 stimulates production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivory defense. These include proteinase inhibitors and biosynthetic enzymes for production of volatile terpenes and benzoxazinoids. In accordance with gene expression data, plants treated with ZmPep3 emit volatiles similar to those from plants subjected to herbivory. ZmPep3-treated plants also exhibit induced accumulation of the benzoxazinoid phytoalexin 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside. Direct and indirect defenses induced by ZmPep3 contribute to resistance against S. exigua through significant reduction of larval growth and attraction of Cotesia marginiventris parasitoids. ZmPep3 activity is specific to Poaceous species; however, peptides derived from PROPEP orthologs identified in Solanaceous and Fabaceous plants also induce herbivory-associated volatiles in their respective species. These studies demonstrate that Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing functional homologs of systemin outside of the Solanaceae.

Conservation and modification of genetic and physiological toolkits underpinning diapause in bumble bee queens.

Diapause is the key adaptation allowing insects to survive unfavourable conditions and inhabit an array of environments. Physiological changes during diapause are largely conserved across species and are hypothesized to be regulated by a conserved suite of genes (a 'toolkit'). Furthermore, it is hypothesized that in social insects, this toolkit was co-opted to mediate caste differentiation between long-lived, reproductive, diapause-capable queens and short-lived, sterile workers. Using Bombus terrestris queens, we examined the physiological and transcriptomic changes associated with diapause and CO2 treatment, which causes queens to bypass diapause. We performed comparative analyses with genes previously identified to be associated with diapause in the Dipteran Sarcophaga crassipalpis and with caste differentiation in bumble bees. As in Diptera, diapause in bumble bees is associated with physiological and transcriptional changes related to nutrient storage, stress resistance and core metabolic pathways. There is a significant overlap, both at the level of transcript and gene ontology, between the genetic mechanisms mediating diapause in B. terrestris and S. crassipalpis, reaffirming the existence of a conserved insect diapause genetic toolkit. However, a substantial proportion (10%) of the differentially regulated transcripts in diapausing queens have no clear orthologs in other species, and key players regulating diapause in Diptera (juvenile hormone and vitellogenin) appear to have distinct functions in bumble bees. We also found a substantial overlap between genes related to caste determination and diapause in bumble bees. Thus, our studies demonstrate an intriguing interplay between pathways underpinning adaptation to environmental extremes and the evolution of sociality in insects.

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance.

Maize (Zea mays) production, which is of global agro-economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought-induced phytoalexins is positively correlated with the root-to-shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.

Sex-specific mating pheromones in the nematode Panagrellus redivivus.

Nematodes use an extensive chemical language based on glycosides of the dideoxysugar ascarylose for developmental regulation (dauer formation), male sex attraction, aggregation, and dispersal. However, no examples of a female- or hermaphrodite-specific sex attractant have been identified to date. In this study, we investigated the pheromone system of the gonochoristic sour paste nematode Panagrellus redivivus, which produces sex-specific attractants of the opposite sex. Activity-guided fractionation of the P. redivivus exometabolome revealed that males are strongly attracted to ascr#1 (also known as daumone), an ascaroside previously identified from Caenorhabditis elegans hermaphrodites. Female P. redivivus are repelled by high concentrations of ascr#1 but are specifically attracted to a previously unknown ascaroside that we named dhas#18, a dihydroxy derivative of the known ascr#18 and an ascaroside that features extensive functionalization of the lipid-derived side chain. Targeted profiling of the P. redivivus exometabolome revealed several additional ascarosides that did not induce strong chemotaxis. We show that P. redivivus females, but not males, produce the male-attracting ascr#1, whereas males, but not females, produce the female-attracting dhas#18. These results show that ascaroside biosynthesis in P. redivivus is highly sex-specific. Furthermore, the extensive side chain functionalization in dhas#18, which is reminiscent of polyketide-derived natural products, indicates unanticipated biosynthetic capabilities in nematodes.

Mi-1-Mediated Nematode Resistance in Tomatoes is Broken by Short-Term Heat Stress but Recovers Over Time.

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, 'Amelia', which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of 'Amelia' but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in 'Amelia' did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.

Effects of elevated [CO2 ] on maize defence against mycotoxigenic Fusarium verticillioides.

Maize is by quantity the most important C4 cereal crop; however, future climate changes are expected to increase maize susceptibility to mycotoxigenic fungal pathogens and reduce productivity. While rising atmospheric [CO2 ] is a driving force behind the warmer temperatures and drought, which aggravate fungal disease and mycotoxin accumulation, our understanding of how elevated [CO2 ] will effect maize defences against such pathogens is limited. Here we report that elevated [CO2 ] increases maize susceptibility to Fusarium verticillioides proliferation, while mycotoxin levels are unaltered. Fumonisin production is not proportional to the increase in F. verticillioides biomass, and the amount of fumonisin produced per unit pathogen is reduced at elevated [CO2 ]. Following F. verticillioides stalk inoculation, the accumulation of sugars, free fatty acids, lipoxygenase (LOX) transcripts, phytohormones and downstream phytoalexins is dampened in maize grown at elevated [CO2 ]. The attenuation of maize 13-LOXs and jasmonic acid production correlates with reduced terpenoid phytoalexins and increased susceptibility. Furthermore, the attenuated induction of 9-LOXs, which have been suggested to stimulate mycotoxin biosynthesis, is consistent with reduced fumonisin per unit fungal biomass at elevated [CO2 ]. Our findings suggest that elevated [CO2 ] will compromise maize LOX-dependent signalling, which will influence the interactions between maize and mycotoxigenic fungi.

Plant volatiles influence the African weaver ant-cashew tree mutualism.

Plant volatiles influence virtually all forms of ant-plant symbioses. However, little is known about their role in the mutualistic relationship between the African weaver ant and the cashew tree. In this study, we tested the hypothesis that cashew tree volatiles from plant parts most vulnerable to herbivory viz. inflorescence, leaves, and fruits, are attractive to weaver ants. Using behavioral assays, we show that these volatiles attract weaver ants but without significant difference in preference for any of the odors. These same plant parts are associated with extra floral nectaries (EFNs') and therefore we evaluated the possibility that the ants associate the volatiles with food rewards. We found that perception of the odors was followed by a searching response that led the ants to non-volatile sugar rewards. More importantly, we observed that weaver ants spent significantly more time around the odor when it was paired to a reward. Chemical analysis of volatiles showed that the plant parts shared similarities in chemical composition, dominated by monoterpenes and sesquiterpenes. Additionally, we evaluated the attractiveness of a synthetic blend of three ocimene isomers ((E)-ß-ocimene, (Z)-ß-ocimene and allo-ocimene) identified in cashew leaf odor and shown to constitute a candidate kairomone for the cashew pest Pseudotheraptus wayi. We found that the attractiveness of the blend was dose dependent, and the response of the ants was not significantly different to that established with the crude volatiles from plant tissues. These results present new and interesting possibilities for improving weaver ant performance in cashew pest management.

Identification of methyl farnesoate from the hemolymph of insects.

Methyl farnesoate, [methyl (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienoate (1)] has not been thought be present in the hemolymph of insects, although it is the immediate biosynthetic precursor of the circulating insect hormone juvenile hormone III (methyl (2E,6E)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate) (2). Compound 1 was identified from the hemolymph obtained from five orders of insects. Identification of 1 from the American bird grasshopper was facilitated using both electron impact and chemical-ionization GC-MS, GC-FTIR, and 2D NMR techniques. The identifications from other insects were made using GC-MS, and the amounts of all were quantified using LIM-CI-GC-MS. The ratios of 1 and 2 varied in these insects during different developmental stages. The present results underscore the need for further studies on methyl farnesoate (1) as a circulating hormone in insects.

A blend of small molecules regulates both mating and development in Caenorhabditis elegans.

In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the 'ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.

Examining the role of foraging and malvolio in host-finding behavior in the honey bee parasite, Varroa destructor (Anderson & Trueman).

When a female varroa mite, Varroa destructor (Anderson & Trueman), invades a honey bee brood cell, the physiology rapidly changes from feeding phoretic to reproductive. Changes in foraging and malvolio transcript levels in the brain have been associated with modulated intra-specific food searching behaviors in insects and other invertebrates. Transcription profiles for both genes were examined during and immediately following brood cell invasion to assess their role as potential control elements. Vdfor and Vdmvl transcripts were found in all organs of varroa mites with the highest Vdfor transcript levels in ovary-lyrate organs and the highest Vdmvl in Malpighian tubules. Changes in transcript levels of Vdfor and Vdmvl in synganglia were not associated with the cell invasion process, remaining comparable between early reproductive mites (collected from the pre-capping brood cells) and phoretic mites. However, Vdfor and Vdmvl transcript levels were lowered by 37 and 53%, respectively, in synganglia from reproductive mites compared to early reproductive mites, but not significantly different to levels in synganglia from phoretic mites. On the other hand, in whole body preparations the Vdfor and Vdmvl had significantly higher levels of transcript in reproductive mites compared to phoretic and early reproductive, mainly due to the presence of both transcripts accumulating in the eggs carried by the ovipositing mite. Varroa mites are a critical component for honey bee population decline and finding varroa mite genes associated with brood cell invasion, reproduction, ion balance and other physiological processes will facilitate development of novel control avenues for this honey bee parasite.

An amino acid substitution inhibits specialist herbivore production of an antagonist effector and recovers insect-induced plant defenses.

Plants respond to insect herbivory through the production of biochemicals that function as either direct defenses or indirect defenses via the attraction of natural enemies. While attack by closely related insect pests can result in distinctive levels of induced plant defenses, precise biochemical mechanisms responsible for differing responses remain largely unknown. Cowpea (Vigna unguiculata) responds to Fall armyworm (Spodoptera frugiperda) herbivory through the detection of fragments of chloroplastic ATP synthase γ-subunit proteins, termed inceptin-related peptides, present in larval oral secretions (OS). In contrast to generalists like Fall armyworm, OS of the legume-specializing velvetbean caterpillar (VBC; Anticarsia gemmatalis) do not elicit ethylene production and demonstrate significantly lower induced volatile emission in direct herbivory comparisons. Unlike all other Lepidoptera OS examined, which preferentially contain inceptin (Vu-In; +ICDINGVCVDA-), VBC OS contain predominantly a C-terminal truncated peptide, Vu-In(-A) (+ICDINGVCVD-). Vu-In(-A) is both inactive and functions as a potent naturally occurring antagonist of Vu-In-induced responses. To block antagonist production, amino acid substitutions at the C terminus were screened for differences in VBC gut proteolysis. A valine-substituted peptide (Vu-In(ΔV); +ICDINGVCVDV-) retaining full elicitor activity was found to accumulate in VBC OS. Compared with the native polypeptide, VBC that previously ingested 500 pmol of the valine-modified chloroplastic ATP synthase γ-subunit precursor elicited significantly stronger plant responses in herbivory assays. We demonstrate that a specialist herbivore minimizes the activation of defenses by converting an elicitor into an antagonist effector and identify an amino acid substitution that recovers these induced plant defenses to a level observed with generalist herbivores.

Sesquiterpene action, and morphogenetic signaling through the ortholog of retinoid X receptor, in higher Diptera.

Morphogenetic signaling by small terpenoid hormones is a common feature of both vertebrate and invertebrate development. Most attention on insect developmental signaling by small terpenoids has focused on signaling by juvenile hormone through bHLH-PAS proteins (e.g., the MET protein), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a series of endocrine and pharmacological studies on pupariation in cyclorrhaphous Diptera have remained persistently refractory to explanation with the above two-axis model. Recently, the terpenoid compound methyl farnesoate has been physicochemically demonstrated to exist in circulation at physiological concentrations, in several mecopterid orders, including Diptera. In addition, it has also been recently demonstrated that the receptor to which methyl farnesoate binds with nanomolar affinity (ultraspiracle, an ortholog of retinoid X receptor) requires a functioning ligand binding pocket to sustain the morphogenetic transition to puparium formation. This review evaluates endocrine and pharmacological evidence for developmental pathways reached by methyl farnesoate action, and assesses the participation of the retinoid X receptor ligand pocket in signal transduction to those developmental endpoints.

Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize.

Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 µg g⁻¹ fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles ß-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze ß-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.

Chemical communication in the honey bee scarab pest Oplostomus haroldi: role of (Z)-9-pentacosene.

Oplostomus haroldi Witte belongs to a unique genus of afro-tropical scarabs that have associations with honey bee colonies, from which they derive vital nutrients. Although the attributes of the honey bee nest impose barriers to communication among nest invaders, this beetle still is able to detect conspecific mates for reproduction. Here, we show, through behavioral studies, that cuticular lipids serve as mate discrimination cues in this beetle. We observed five steps during mating: arrestment, alignment, mounting, and copulation, and a post-copulatory stage, lasting ~40-70 % of the total mating duration, that suggested mate guarding. Chemical analysis identified the same nine straight-chain alkanes (C(23)-C(31)), six methyl-branched alkanes (6), and five mono-unsaturated alkenes in the cuticular lipids of both sexes. Methyl alkanes constituted the major component (46 %) of male cuticular lipids, while mono-unsaturated alkenes were most abundant (53 %) in females. (Z)-9-Pentacosene was twice as abundant in females than in males, and ~20 fold more concentrated in beetles than in worker bees. In mating assays, (Z)-9-pentacosene elicited arrestment, alignment, and mounting, but not copulation, by male beetles. These results represent the first evidence of a contact sex pheromone in a scarab beetle. Such contact pheromones may be an essential, cryptic mechanism for arthropods associated with eusocial insects.

Phytohormone-based activity mapping of insect herbivore-produced elicitors.

In response to insect attack, many plants exhibit dynamic biochemical changes, resulting in the induced production of direct and indirect defenses. Elicitors present in herbivore oral secretions are believed to positively regulate many inducible plant defenses; however, little is known about the specificity of elicitor recognition in plants. To investigate the phylogenic distribution of elicitor activity, we tested representatives from three different elicitor classes on the time course of defense-related phytohormone production, including ethylene (E), jasmonic acid (JA), and salicylic acid, in a range of plant species spanning angiosperm diversity. All families examined responded to at least one elicitor class with significant increases in E and JA production within 1 to 2 h after treatment, yet elicitation activity among species was highly idiosyncratic. The fatty-acid amino acid conjugate volicitin exhibited the widest range of phytohormone and volatile inducing activity, which spanned maize (Zea mays), soybean (Glycine max), and eggplant (Solanum melongena). In contrast, the activity of inceptin-related peptides, originally described in cowpea (Vigna unguiculata), was limited even within the Fabaceae. Similarly, caeliferin A16:0, a disulfooxy fatty acid from grasshoppers, was the only elicitor with demonstrable activity in Arabidopsis thaliana. Although precise mechanisms remain unknown, the unpredictable nature of elicitor activity between plant species supports the existence of specific receptor-ligand interactions mediating recognition. Despite the lack of an ideal plant model for studying the action of numerous elicitors, E and JA exist as highly conserved and readily quantifiable markers for future discoveries in this field.

Evidence for potential of managing some African fruit fly species (Diptera: Tephritidae) using the mango fruit fly host-marking pheromone.

We investigated conspecific and heterospecific oviposition host discrimination among four economically important fruit fly pests of mango in Africa (Ceratitis capitata, Wiedemann; C. fasciventris, Bezzi; C. rosa, Karsch, and C. cosyra, Walker) with regard to host-marking behavior and fecal matter aqueous solutions. The objective of the study was to get insight into the potential of managing these pests using the host-marking technique. Observations were done on mango slices marked by the flies and treated with aqueous solutions of fecal matter of the flies, respectively. In both host-marking and fecal matter experiments, C. cosyra, which is the most destructive species of the four on mango, was exceptional. It only discriminated against hosts treated with its fecal matter but with lower sensitivity while C. capitata and C.fasciventris discriminated against hosts marked by it or treated with its fecal matter and with higher sensitivity. Our results provide evidence for potential of managing some of the major fruit fly species infesting mango in Africa using the host-marking pheromone of the mango fruit fly, C. cosyra.

Density-related volatile emissions and responses in the red flour beetle, Tribolium castaneum.

Intraspecific attraction depends both on the cues provided by the attracting individual and the response of the attracted individual. These attracting cues are related not only to current conditions, but also are a reflection of individual and population life history. These relationships were examined by placing red flour beetle, Tribolium castaneum (Herbst.), adults in flasks at increasing densities and monitoring the changes in volatile chemical emission over time. Only certain chemicals were quantified: methyl benzoquinone, ethyl benzoquinone and 4,8-dimethyldecanal, all of which are known to impact the biology of T. castaneum. The flasks were used as sources for both quantification of the chemicals and for bioassays. Additional bioassays were conducted with synthetic 4,8-dimethyldecanal, a known aggregation pheromone component, to evaluate attraction with respect to population density. Tribolium castaneum density affected both the release of volatile chemicals and the responses of conspecifics to those chemicals. The results indicated that while there were important effects of beetle density on chemical emission and response, none of the chemicals evaluated emerged as promising synergists to the current aggregation pheromone 4,8-dimethyldecanal. The benzoquinones released in response to stress and density acted as anti-aggregation pheromones along with their accepted defensive function.

Influence of a juvenile hormone analog and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) sexual success.

Juvenile hormone levels and adult diet have important effects on the attractiveness and competitiveness of male Anastrepha suspensa (Loew) (Caribbean fruit fly). Because the success of the sterile insect technique requires the release of males that can compete in the wild, these effects are of crucial importance. Laboratory and field cage experiments were conducted to compare male sexual performance on a lifetime basis and daily basis when submitted to four different treatments: (M+P+) application of the juvenile hormone analog, methoprene (M) and sugar and hydrolyzed yeast as adult food; (M+P-) application of M and sugar as adult food; (M-P+) no application of M and sugar and hydrolyzed yeast as adult food; and (M-P-) no application of M and sugar as adult food. On a daily basis, M+P+ males always performed better sexually, and 10% of these individuals were able to mate three consecutive times in the same day. However, the copula duration decreased with the increased number of matings on same day. In addition, M caused earlier maturation. On a lifetime basis, M+P+ males had significantly greater sexual success than other flies. The substantial improvement in male sexual performance because of the hormone application, protein supply, and interaction of hormone and protein has the potential of producing more efficacious sterile males.

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect.

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.