Sequential Isolation of Essential Oils Repellent to the Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).

The push-pull approach using semiochemicals in pest control requires both an attractant and a repellent. Many previous studies have arbitrarily tested one or more known insect repellents or plant essential oils (EOs) hoping to find repellents of an insect pest. We used a comprehensive approach that synergistically tests in the field numerous natural volatiles from commercial EOs to identify repellents of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae), a worldwide pest of palms and date palms. Volatiles from 79 EOs in slow-release devices were divided into five groups and tested in traps with attractive pheromone compared to traps with pheromone alone. EO-treatment groups exhibiting repellency due to significant trap shutdown, were further subdivided into subgroups of four EOs each and tested further. Two groups of four EOs (cypress, desert wormwood, elemi, and Eucalyptus citriodora) and (niaouli, nutmeg, oregano, and orange sweet), or their corresponding mixtures of major volatiles, caused pheromone trap reductions of up to 92%. Further tests showed that seven of the eight EOs are similarly repellent as the corresponding subgroup. This systematic approach of successively testing sub-fractions of EOs in the field for trap shutdown should be useful to identify repellents of other insect pests of crops.

Absolute Configuration of the Spherical Mealybug Nipaecoccus viridis Sex Pheromone, γ-Necrodyl Isobutyrate: Chemoenzymatic Synthesis and Bioassays.

The spherical mealybug, Nipaecoccus viridis (Hemiptera: Pseudococcidae), is a major global pest causing feeding damage to leaves and fruits of citrus varieties, soybean, mango, pomegranate, and grapevines. Females of the mealybug release a sex pheromone that was identified recently as a mixture of γ-necrodyl isobutyrate and γ-necrodol. The identification required synthesis based on a natural source of trans-α-necrodol, of unknown chirality, obtained from essential oil of Spanish lavender, Lavandula luisieri. To determine the chirality of the sex pheromone, here, we synthesize the γ-necrodyl acetate enriched in (+)-(S)-enantiomer and separate the enantiomers using a lipase enzyme. We confirm that the natural components, both in the mealybug and in the lavender essential oil, consist of (-)-(R)-enantiomers. Bioassays conducted in the lab and field show that males are attracted to (-)-(R)-γ-necrodyl isobutyrate alone.

Biostable beta-amino acid PK/PBAN analogs: agonist and antagonist properties.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in a multifunctional array of important physiological processes in insects. PK/PBAN analogs incorporating beta-amino acids were synthesized and evaluated in a pheromonotropic assay in Heliothis peltigera, a melanotropic assay in Spodoptera littoralis, a pupariation assay in Neobellieria bullata, and a hindgut contractile assay in Leucophaea maderae. Two analogs (PK-betaA-1 and PK-betaA-4) demonstrate greatly enhanced resistance to the peptidases neprilysin and angiotensin converting enzyme that are shown to degrade the natural peptides. Despite the changes to the PK core, analog PK-betaA-4 represents a biostable, non-selective agonist in all four bioassays, essentially matching the potency of a natural PK in pupariation assay. Analog PK-betaA-2 is a potent agonist in the melanotropic assay, demonstrating full efficacy at 1pmol. In some cases, the structural changes imparted to the analogs modify the physiological responses. Analog PK-betaA-3 is a non-selective agonist in all four bioassays. The analog PK-betaA-1 shows greater selectivity than parent PK peptides; it is virtually inactive in the pupariation assay and represents a biostable antagonist in the pheromonotropic and melanotropic assays, without the significant agonism of the parent hexapeptide. These analogs provide new, and in some cases, biostable tools to endocrinologists studying similarities and differences in the mechanisms of the variety of PK/PBAN mediated physiological processes. They also may provide leads in the development of PK/PBAN-based, insect-specific pest management agents.

Inhibition of PK/PBAN-mediated functions in insects: discovery of selective and non-selective inhibitors.

The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in Heliothis peltigera female moths, cuticular melanization in Spodoptera littoralis larvae, pupariation in the fleshfly Neobellieria bullata and hindgut contraction in Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.

Backbone cyclic pheromone biosynthesis activating neuropeptide (PBAN) antagonists: inhibition of melanization in the moth Spodoptera littoralis (Insecta, Lepidoptera).

Antagonistic and agonistic activities of backbone cyclic (BBC) pheromone biosynthesis activating neuropeptide (PBAN) analogues were evaluated in an attempt to identify potent melanotropic antagonists, to gain an insight into their structure-activity relationship (SAR), and to discover molecules with selective and non-selective melanotropic and pheromonotropic properties. Eight potent melanotropic BBC antagonists and seven agonists were disclosed. SAR studies revealed that the structural requirements of the melanotropic and pheromonotropic agonists and antagonists are different. The cyclic structure of the BBC peptides was unimportant for antagonistic activity, and linearization retained their melanotropic and pheromonotropic antagonistic properties. Comparison of the antagonistic activities of the BBC and precyclic peptides with respect to both functions revealed eight selective antagonists (six that were selective melanotropic antagonists and two selective pheromonotropic antagonists) and four non-selective (melanotropic and pheromonotropic) antagonists. The selective melanotropic antagonists exhibited both, pure or mixed agonistic/antagonistic activities. The selective pheromonotropic compounds were pure antagonists. All non-selective compounds were pure antagonists. Comparison of the agonistic activities of the BBC peptides with respect to both functions revealed six selective melanotropic agonists and one non-selective agonistic compound. All compounds (whether selective or non-selective) exhibited pure agonistic activity. Discovery of the selective compounds hints at the possibility that the receptors that mediate the respective activities may have different properties.

PBAN selective antagonists: inhibition of PBAN induced cuticular melanization and sex pheromone biosynthesis in moths.

A D-Phe scan (sequential D-Phe replacement) library of linear peptides, synthesized on the basis of a slightly modified active sequence of PBAN (YFSPRL-amide) was employed to detect potential inhibitors of cuticular melanization in Spodoptera littoralis larvae and to compare their stimulatory and inhibitory melanization activity with their pheromonotropic agonistic and antagonistic activities. A quantitative melanotropic assay was used to monitor the extent of cuticular melanization elicited by Hez-PBAN1-33NH2 in S. littoralis larvae in the presence and absence of the D-Phe peptides. The data revealed the presence of two partial melanotropic antagonists, and disclosed the presence of selective pure melanotropic agonists and pure pheromonotropic antagonists indicating differences in the inhibitory and stimulatory patterns of the library with respect to both activities. The differences between the pheromonotropic and melanotropic inhibitory patterns of the peptides hints at the possibility that sex pheromone biosynthesis in the pheromone gland of Heliothis peltigera females and induction of cuticular melanization in S. littoralis may be mediated by different receptors (that may result either from presence of different receptor sub-types or may reflect species differences in receptor structure and/or properties) despite the fact that they are induced by the same peptide (PBAN1-33NH2).

Histochemical localization of the PBAN receptor in the pheromone gland of Heliothis peltigera.

The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH(2) and BpaArg(27)-PBAN28-33NH(2). Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar.

Bioavailability of backbone cyclic PK/PBAN neuropeptide antagonists--inhibition of sex pheromone biosynthesis elicited by the natural mechanism in Heliothis peltigera females.

The bioavailability (i.e. ability to penetrate the insect cuticle, to reach the target organ and to exert bioactivity) of two backbone cyclic (BBC) pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) antagonistic peptides was tested by applying them topically to Heliothis peltigera females and monitoring the resulting inhibition of sex pheromone production elicited by the natural (endogenous) mechanism during scotophase. Peptides were applied at various time points before the onset of scotophase, in aqueous or organic solvents, and pheromone content was examined at the 5th or 6th hour of scotophase. Both peptides penetrated the cuticle very efficiently and inhibited sex pheromone biosynthesis elicited by the natural mechanism for up to 8 or 9 h after application. The degree of inhibition differed between solvents: those applied in double-distilled water (DDW) were more active than those applied in dimethylsulfoxide (inhibition by 53-73% and 15-38%, respectively, for BBC-25, and 46-67% and 36-40%, respectively for BBC-28). Peptides applied in dimethylsulfoxide and hexane exhibited slightly more persistent inhibitory activity than those applied in DDW. The solvents themselves did not affect sex pheromone production. Multiple applications (at -2, 0, +2 and +4 h) resulted in almost complete (87%) inhibition of sex pheromone biosynthesis, compared with 52% inhibition following a single application. The present study is the first demonstration of the ability of topically applied PK/PBAN antagonists to inhibit sex pheromone biosynthesis elicited by the natural mechanism in female moths, and provides important information on the bioavailability of BBC peptides and the mechanism responsible for sex pheromone production in these insects.

Development of an immunoassay and a sol-gel-based immunoaffinity cleanup method for coplanar polychlorinated biphenyls from soil and sediment samples.

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL(-1)) with sensitivities in the range of 6-11 ng mL(-1). The Abs cross-reacted with PCB-126 and the heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF but not with PCB-169, PCB-118, Aroclor 1232, 1248, 1260 or the hexachlorodibenzofuran 2,3,4,6,7,8-HxCDF. The IgGs were also used to develop a sol-gel-based immunoaffinity purification (IAP) method for cleanup of PCB-126. Recovery efficiencies depended on the sol-gel formats; a 1:12 format resulted in the highest binding capacity. Net binding capacity ranged from 112 to 257 ng, and 90% of the analyte could be eluted with only 2 mL of ethanol. The method was also very efficient in purifying PCB-126 from spiked soil and sediment samples from contaminated sites; and eliminating matrix interferences to a degree that enabled analysis of the purified samples by ELISA. The approaches developed in the course of the study form a basis for the development of additional IAP methods for other PCBs, and their implementation in high-throughput screening programs for PCB in food, soil, and other environmental and biological samples.

A novel dihydroimidazoline, trans-Pro mimetic analog is a selective PK/PBAN agonist.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in the regulation of reproductive and developmental processes in a variety of insects. A transPro, type I beta-turn has been previously identified as important for the activity of PK/PBAN peptides. A PK/PBAN analog (PPK-Jo) incorporating a novel dihydroimidazole transPro mimetic motif was evaluated in four PK/PBAN bioassays (pheromonotropic, melanotropic, pupariation and hindgut myotropic). PPK-Jo proved to be a pure, selective melanotropic agonist in S. littoralis. The melanotropic receptor in S. littoralis demonstrates more tolerance to deviations from the ideal transPro structure than those of other PK/PBAN assays. The selective PK/PBAN agonist represents a new tool to better understand the endogenous mechanisms of these peptides and serves as a probe of the plasticity of PK/PBAN regulated systems and receptors. The dihydroimidazoline moiety is shown to function as a surrogate for a transPro in certain circumstances, and provides a novel scaffold with which to construct mimetic PK/PBAN analogs with enhanced selectivity and the potential to disrupt critical physiological processes in insect pests.

Bioavailability of insect neuropeptides: the PK/PBAN family as a case study.

The ability of unmodified linear peptides to penetrate the insect cuticle and exert bioactivity (e.g., stimulation of sex pheromone biosynthesis) was tested by topical application onto Heliothis peltigera moths of four insect neuropeptides (Nps) of the pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family: Helicoverpa zea PBAN (Hez-PBAN), Pseudaletia (Mythimna) separata pheromonotropin (PT), Leucophaea maderae PK (LPK) and Locusta migratoria myotropin (Lom-MT-II). The time kinetic of the peptides applied in double distilled water (DDW) or dimethylsulfoxide (DMSO) was tested and the activities of topically applied and injected peptides were compared. The results clearly indicated that all four peptides were highly potent but with differing activities in the two solvents: PBAN was most active in water, and PT in DMSO. The activity of PBAN in DDW lasted up to 8h post-application and its activity in this solvent showed a faster onset and a longer persistence than in DMSO. LPK and MT differed less in their kinetics between the two solvents. Topically applied PBAN at 1 nmol exhibited an equivalent or even significantly higher potency than the injected peptide at several different times post-treatment. Similar results were obtained with topically applied and injected LPK. The present results add important information on the bioavailability of unmodified linear peptides in moths, clearly indicate that linear hydrophilic peptides can penetrate the cuticle by contact application in aqueous solutions and in organic solvents very efficiently, reach their target organ and activate it.

PBAN receptor: employment of anti-receptor antibodies for its characterization and for development of a microplate binding assay.

This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists.

Bioavailability of beta-amino acid and C-terminally derived PK/PBAN analogs.

The ability of linear beta-amino acid substituted peptides (PK-betaA-1: Ac-YFT[beta(3)P]RLa; PK-betaA-2: Ac-Y[beta(3)homoF]TPRLa; PK-betaA-3: Ac-Y[beta(3)F]TPRLa; PK-betaA-4: Ac-[beta(3)F]FT[beta(3)P]RLa) and unsubstituted analogs (Ac-YFTPRLa and YFTPRLa) of the pyrokinin(PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family to penetrate the insect cuticle and exert biological activity (i.e., stimulate sex pheromone biosynthesis), was tested by topical application on Heliothis peltigera moths. The present results clearly indicate that small linear synthetic peptides can penetrate the cuticle very efficiently by contact application and activate their target organ. The time responses of the peptides applied in DDW and DMSO were tested and the activities of topically applied and injected peptides were compared. The results clearly indicate that PK-betaA-4 and PK-betaA-3 exhibited high bioavailability (ability to penetrate through the cuticle and exertion of bioactivity) with the latter showing longer persistence in both solvents than any other analog in the study; indicative that incorporation of a beta-amino acid at the Phe(2) position can enhance longevity in topical PK/PBAN analogs. PK-betaA-4 was significantly more active in DMSO than in DDW, and significantly more active than the parent peptide LPK in DMSO. PK-betaA-1 and PK-betaA-2 exhibited negligible activity. Interestingly, Ac-YFTPRLa was highly potent in both solvents; its activity in DDW did not differ from that of PK-betaA-4 and PK-betaA-3, and was higher than that of LPK. Even the unacylated peptide YFTPRLa was active in both solvents, at a similar level to LPK. Topically applied PK-betaA-4 and Ac-YFTPRLa exhibited significantly higher activity than the injected peptides. PK-betaA-3 and YFTPRLa were equally potent in both routes of administration.