Synthetic Co-Attractants of the Aggregation Pheromone of the Date Palm Root Borer Oryctes agamemnon.

Laboratory and field investigations to identify and evaluate plant co-attractants of the aggregation pheromone of the date palm pest Oryctes agamemnon are reported. Volatiles emitted by freshly cut palm core and palm core with feeding males, were collected, analyzed by gas chromatography coupled to mass spectrometry and evaluated in olfactometers alone or combined with synthetic pheromone. A collection of palm odor without male effluvia was attractive alone and enhanced attraction to synthetic pheromone in an olfactometer similar to that to a collection of palm odor emitted with feeding males and containing natural pheromone. Behavioral responses to collections of palm volatiles were correlated to the amount of volatiles material in them. Enhancement of the attractiveness of the pheromone was not correlated to chemicals specific to beetle feeding. The chemicals common to the active collections extracts were benzoate esters, mostly ethyl benzoate, anisole derivatives and sesquiterpenes. Blends of the most abundant components of the extracts were evaluated for enhancement of the attractiveness of pheromone (1 µg) in olfactometers at 1 or 10 µg doses. The mixtures were further evaluated by field trapping in Tunisia at 3-10 mg/day using reference (6 mg/day) or experimental pheromone formulations. A mixture of ethyl benzoate, 4-methylanisole and farnesol (1:1:1 w/w at 6.5 mg/day) enhanced captures in pheromone baited traps in 2014 and 2015 and this mixture was as active as the natural palm bait. The practical prospect of the result for the management for O. agamemnon, and other palm beetles is discussed.

Identification of the Aggregation Pheromone of the Date Palm Root Borer Oryctes agamemnon.

Laboratory and field investigations aimed to characterize the chemical communication system of the date palm pest Oryctes agamemnon. Live males or extracts of male effluvia attracted conspecifics in an olfactometer, whereas female effluvia attracted only males. Volatile emissions from adults feeding on sugarcane were sampled and analysed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Males emitted a blend of 1) ethyl 4-methyloctanoate, 2) 4-methyloctanoic acid, 3) 4-methyloctanyl acetate, and 4) 4-methyloctanol in variable ratio. Single sensillum recordings demonstrated that compounds 1, 2, and 3 are detected by specific olfactory receptor neurons. Olfactometric experiments showed that compounds 1 and 3 attract both sexes of O. agamemnon, but females are more attracted by compound 1 and males by compound 3. Compound 2 was more attractive for females, especially virgin ones. Field experiments confirmed that compound 1 and compound 2 attracted O. agamemnon of both sexes and showed synergy with palm odors. No clear activity of compound 3 was observed. A mix of compounds 1 and 2 with date palm core odor was significantly the most attractive, and captured more females than males. The male aggregation pheromone of O. agamemnon appears therefore to be based on a mixture in contrast to previously identified Oryctes pheromones. Our results provide the basis for developing mass trapping to control this pest.

Changes in odor background affect the locomotory response to pheromone in moths.

Many animals rely on chemical cues to recognize and locate a resource, and they must extract the relevant information from a complex and changing odor environment. For example, in moths, finding a mate is mediated by a sex pheromone, which is detected in a rich environment of volatile plant compounds. Here, we investigated the effects of a volatile plant background on the walking response of male Spodoptera littoralis to the female pheromone. Males were stimulated by combining pheromone with one of three plant compounds, and their walking paths were recorded with a locomotion compensator and analyzed. We found that the addition of certain volatile plant compounds disturbed the orientation toward the sex pheromone. The effect on locomotion was correlated with the capacity of the plant compound to antagonize pheromone detection by olfactory receptor neurons, suggesting a masking effect of the background over the pheromone signal. Moths were more sensitive to changes in background compared to a constant background, suggesting that a background odor also acts as a distracting stimulus. Our experiments show that the effects of odorant background on insect responses to chemical signals are complex and cannot be explained by a single mechanism.

The use of the sex pheromone as an evolutionary solution to food source selection in caterpillars.

Sex pheromones are released by adults of a species to elicit a sexual interaction with the other sex of the same species. Here we report an unexpected effect of a moth sex pheromone on the caterpillars of the same species. We demonstrate that larvae of the cotton leafworm Spodoptera littoralis are attracted by the moth sex pheromone and that this phenomenon is independent of sex determination. In addition, we show that the olfactory sensilla carried by the caterpillar antennae are sensitive to the pheromone and that the caterpillar sensilla express pheromone-binding proteins that are used by adult antennae to bind pheromone components. Finally, we demonstrate that the larvae are preferentially attracted to a food source when it contains the sex pheromone main component. A possible interpretation of these results is that the sex pheromone is used to promote food search in caterpillars, opening potential new routes for insect pest management.

Antimicrobial peptides keep insect endosymbionts under control.

Vertically transmitted endosymbionts persist for millions of years in invertebrates and play an important role in animal evolution. However, the functional basis underlying the maintenance of these long-term resident bacteria is unknown. We report that the weevil coleoptericin-A (ColA) antimicrobial peptide selectively targets endosymbionts within the bacteriocytes and regulates their growth through the inhibition of cell division. Silencing the colA gene with RNA interference resulted in a decrease in size of the giant filamentous endosymbionts, which escaped from the bacteriocytes and spread into insect tissues. Although this family of peptides is commonly linked with microbe clearance, this work shows that endosymbiosis benefits from ColA, suggesting that long-term host-symbiont coevolution might have shaped immune effectors for symbiont maintenance.

Evaluation and modeling of synergy to pheromone and plant kairomone in American palm weevil.

BACKGROUND: Many behavioral responses to odors are synergistic, particularly in insects. In beetles, synergy often involves a pheromone and a plant odor, and pest management relies on them for the use of combined lures. To investigate olfactory synergy mechanisms, we need to distinguish synergistic effects from additive ones, when all components of the mixture are active. RESULTS: As versatile tools and procedures were not available, we developed a bioassay, and a mathematical model to evaluate synergy between aggregation pheromone (P) and host plant odors (kairomone: K) in the American palm weevil, a pest insect showing enhanced responses to P+K mixtures. Responses to synthetic P and natural K were obtained using a 4-arm olfactometer coupled to a controlled volatile delivery system. We showed that: (1) Response thresholds were ca. 10 and 100 pg/s respectively for P and K. (2) Both stimuli induced similar maximum response. (3) Increasing the dose decreased the response for P to the point of repellence and maintained a maximum response for K. (4) P and K were synergistic over a 100-fold range of doses with experimental responses to P+K mixtures greater than the ones predicted assuming additive effects. Responses close to maximum were associated with the mixture amounts below the response threshold for both P and K. CONCLUSION: These results confirm the role of olfactory synergy in optimizing active host-plant localization by phytophagous insects. Our evaluation procedure can be generalized to test synergistic or inhibitory integrated responses of various odor mixtures for various insects.

Pulsed odors from maize or spinach elicit orientation in European corn borer neonate larvae.

Lepidoptera larvae are capable of orienting towards or away from plants by using odors as cues but whether this attraction is innate or secondarily acquired remains unknown. We tested the hypothesis that European corn borer (ECB) neonate larvae express an innate attraction towards odors released from maize, and avoidance towards odors from spinach. Neonate larvae were placed on a locomotion compensator within a constant stream of humidified air that was loaded intermittently with airborne odors drawn from potted plants. The odor stream was delivered continuously or pulsed (1 to 10 sec pulses) at 40 ml/min. ECB larvae oriented toward maize odors pulsed at 2 to 6 sec but walked away from maize odors delivered at lower frequencies (9 and 10 sec pulses or to continuous ones). They consistently walked away from spinach odors, irrespective of the pulsing regime except at 1 sec pulses that did not elicit orientation. We further explored odor intensity on orientation towards maize odors by adjusting the odor stream intensity. At higher intensity (60 ml/min), the direction reversal started at the 6 sec half period, while at lower intensity (20 ml/min), it showed up only for the continuous stimulus. ECB larvae exhibit a striking ability to lock on to a direction, which they maintained despite gaps of up to 10 sec in the odor stream. Our results demonstrate that ECB neonate larvae express innate orientation preferences towards natural odors from plants. These reactions correlate well with the biological value of these plants for ECB: maize generally is accepted by ECB larvae and adults, while spinach represents a poor host because it produces (non-volatile) phytoecdysteroids that are toxic and deterrent.

Plant terpenes affect intensity and temporal parameters of pheromone detection in a moth.

In moths, the components of the female pheromone blend are detected in the male antennae by pheromone olfactory receptor neurons (Ph-ORNs) expressing narrowly tuned olfactory receptors. Responses to sex pheromones have generally been thought to be independent from the odorant background. However, interactions between pheromone components and plant volatiles have been reported at behavioral and detection levels. To document the mechanisms of such interactions, we analyzed Ph-ORN responses of Spodoptera littoralis to the main pheromone component, Z9E11-14:Ac, in the presence of 4 monoterpenes. To mimic natural contexts in which plant odors and pheromone emanate from different sources, the 2 stimuli were presented with different temporal patterns and from independent sources. Linalool reversibly reduced the firing response to Z9E11-14:Ac and produced an off effect. Geraniol and geranyl and linalyl acetates reduced the responses to Z9E11-14:Ac with a longer time course. Pulses of linalool over prolonged pheromone stimulation resulted in a discontinuous firing activity. Pulses of pheromone were better separated over a background of linalool, compared with odorless air. The data confirm that plant compounds may modulate the intensity and the temporal coding by Ph-ORNs of pheromone information. This modulation might positively affect mate location at high pheromone density especially nearby a pheromone source.

An antennal circadian clock and circadian rhythms in peripheral pheromone reception in the moth Spodoptera littoralis.

Circadian rhythms are observed in mating behaviors in moths: females emit sex pheromones and males are attracted by these pheromones in rhythmic fashions. In the moth Spodoptera littoralis, we demonstrated the occurrence of a circadian oscillator in the antenna, the peripheral olfactory organ. We identified different clock genes, period (per), cryptochrome1 (cry1) and cryptochrome2 (cry2), in this organ. Using quantitative real-time PCR (qPCR), we found that their corresponding transcripts cycled circadianly in the antenna as well as in the brain. Electroantennogram (EAG) recordings over 24 h demonstrated for the first time a circadian rhythm in antennal responses of a moth to sex pheromone. qPCR showed that out of one pheromone-binding protein (PBP), one olfactory receptor (OR), and one odorant-degrading enzyme (ODE), all putatively involved in the pheromone reception, only the ODE transcript presented a circadian rhythm that may be related to rhythms in olfactory signal resolution. Peripheral or central circadian clock control of olfaction is then discussed in light of recent data.

Oryctes monoceros trapping with synthetic pheromone and palm material in Ivory Coast.

Oryctes monoceros is the most serious pest in coconut plantations, causing up to 40% damage in tropical Africa, especially in Ivory Coast. With a view to reducing pest populations by olfactory trapping, field trials were carried out to assess the efficiency of a synthetic aggregation pheromone: ethyl 4-methyloctanoate (1), 4-methyloctanoic acid (2), a related volatile produced by males, and decaying palm material, either oil palm empty fruit bunches (EFB) or pieces of coconut wood (CW) of various ages. Vertical polyvinyl chloride tube traps (2 x 0.16 m with two openings in the upper half), embedded in the soil, were more efficient than 30-L pail traps 1.5 m above ground. EFB, which were inactive alone, synergized captures with synthetic pheromone. CW was more effective than EFB in comparative trials. Compound 2 did not catch any beetles when assessed with EFB, and reduced catches by 1 + EFB when tested at >10% with the pheromone. Trapping over 6 mo in 2002 and 2003 in a 19-ha coconut plot inside a 4,000-ha oil palm estate reduced damage from 3.8% in 2001 to 0.5% in 2002, then to 0.2% in 2003. Damage was 0.0% in 2004 with routine trapping using 32 traps, which caught 3369 beetles in 9 mo. The results are discussed in relation to other Dynastid palm pests and coconut protection in Ivory Coast.

Interactions between acetoin, a plant volatile, and pheromone in Rhynchophorus palmarum: behavioral and olfactory neuron responses.

Aggregation of Rhynchophorus palmarum weevils on host plants is mediated by a male pheromone (rhynchophorol: R) and host-plant volatiles (PVs) acting in synergy. Synthetic PV blends synergizing pheromone contain acetoin (A) and ethyl acetate (EtAc). R, A, and EtAc are detected by specialized olfactory receptor neurons (ORNs). In addition, particular types of ORNs are tuned to both A and R. To specify the role played by acetoin in pheromone perception, we recorded the responses of ORNs to 100 ng of A or R presented either separately or mixed. Behavioral responses to R, A, and EtAc were studied in a four-armed olfactometer and by field trapping. We screened 59 R-, A-, and AR-tuned ORNs by recording specific responses to odors presented either separately or mixed. Stimulations by blends elicited complex response profiles from the three ORN types: some gave synergistic responses, others were inhibited, and the remainder responded as though both odors were detected independently. Several gave either a weak or no response to a first stimulation by R, but responded clearly to a second stimulation after an intercalary stimulation by A. In the olfactometer, both sexes were more attracted to a blend of A + R (1 + 0.01 ng/sec) than to pure compounds, whereas EtAc did not enhance response to R. Pheromone-baited traps (1 mg/day) containing PV blends (650 mg/day) based on an ethanol/EtAc blend (1:1), plus either 5 or 10% A, or a more complex reference blend, or sugarcane (natural pheromone synergist), caught similar numbers of weevils and about twice as many insects as a control ethanol/EtAc blend. Traps with only pheromone caught about 10 times fewer insects. Behavioral results support the role of acetoin as a pheromone synergist for R. palmarum, and electrophysiological data provide evidence of modulation of peripheral sensory responses to pheromone by acetoin. Sexual dimorphism was observed neither at the ORN nor at the behavioral levels.

Male aggregation pheromone of date palm fruit stalk borer Oryctes elegans.

Laboratory and field investigations were carried out to characterize the chemical communication system of the date palm fruit stalk borer, Oryctes elegans, and to develop pheromone-based trapping in Eastern Iran. Adults of both sexes feeding on date palm pieces attracted conspecifics, whereas date palm alone was minimally attractive. Males were twice as attractive as females. More beetles were captured at the palm crown than at ground level. Odors from adults feeding on sugarcane were sampled and analyzed by gas chromatography and mass spectrometry. Whereas females did not emit sex specific volatiles, males emitted a blend of 4-methyloctanoic acid (1: major component) and ethyl 4-methyloctanoate (2), occasionally mixed with minor components: 4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), 4-methyloctanol (5), and nonanyl acetate (6). Electroantennography and field trapping experiments demonstrated that compound 1 is an essential component of the male aggregation pheromone of O. elegans. It was barely attractive by itself but synergistic with fresh date palm odor. It attracted many more beetles than any of compounds 2-6. The addition of one or several of compounds 2-6 to 1 did not improve trap captures. During the course of 2 years, we captured 4000 beetles, with a weekly average of 6.3 beetles/trap, and were able to monitor the seasonal flight of O. elegans. Our results provide the basis for developing mass trapping for control of this pest.

Structure and function of the antennal sensilla of the palm weevil Rhynchophorus palmarum (Coleoptera, Curculionidae).

The distribution, fine structure and function of the sensilla present on the antennal club of Rhynchophorus palmarum were studied. No sex dimorphism was observed. Scanning and transmission electron microscopy showed five types of hair-like structures, four of which were evenly distributed on the antennal club. Two types of hair (IV and V) showed wall pores, a characteristic of olfactory sensilla. The antenna numbers 11,190 +/- 3040 type IV and 7360 +/- 1500 type V hairs. Using single sensillum recording, we identified 17 types of olfactory receptor neurons (ORNs) on the basis of their responses to pheromone and host plant odors, triggering synergic behavioral responses. We characterized highly specific and sensitive ORNs tuned to the aggregation pheromone (18% ORNs; 0.01-1 ng response threshold) and to host plant odors such as ethyl acetate, ethanol, acetoin and guaiacol (10% ORNs; 1-10 ng response threshold). Eleven percent of the ORNs were more generalist, responding to several odors with low sensitivity. Nine percent of the ORNs showed a complex pattern of responses, being co-activated by the pheromone and plant odors. This suggests an interaction at the sensory neuron level between pheromone and plant odors, triggering synergic behavioral responses.

Activity of male pheromone of Melanesian rhinoceros beetle Scapanes australis.

Laboratory and field investigations were carried out to investigate the nature and role of the male pheromone emitted by the Dynast beetle Scapanes australis and to develop a mass trapping technique against this major coconut pest in Papua New Guinea. We report the biological data obtained from natural and synthetic pheromone, previously described as an 84:12:4 (w/w) mixture of 2-butanol (1), 3-hydoxy-2-butanone (2), and 2,3-butanediol (3). EAG recordings from natural and synthetic pheromone and a pitfall olfactometer were poorly informative. In contrast, extensive field trapping trials with various synthetic pheromone mixtures and doses showed that 1 and 2 (formulated in polyethylene sachets in 90:5 v/v ratio) were necessary and sufficient for optimum long-range attraction. Beetles were captured in traps baited with racemic 1 plus 2, with or without a stereoisomer mixture of 3 (2.5- to 2500-mg/day doses). Plant pieces, either sugarcane or coconut, enhanced captures by the synthetic pheromone, which was active alone. Traps with the pheromone caught both sexes in a 3:2 female-male ratio. A pheromone-based mass trapping led to the capture of 2173 beetles in 14 traps surrounding 40 ha of a cocoa-coconut plantation. The captures followed a log-linear decrease during the 125-week trapping program. The role of the male pheromone and its potential for crop protection are discussed.