Identification and field verification of an aggregation pheromone from the white-spotted flower chafer, Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae)

The white-spotted flower chafer (WSFC), Protaetia brevitarsis Lewis, is native to East Asia. Although their larvae are considered a potential resource insect for degrading plant residues, producing protein fodder, and processing to traditional medicine, adult WSFCs inflict damage to dozens of fruit and economic crops. The control of the WSFC still relies heavily on pesticides and the inefficient manual extraction of adults. Here, we report the identification and evaluation of the aggregation pheromone of WSFCs. From the headspace volatiles emitted from WSFC adults, anisole, 4-methylanisole, 2-heptanone and 2-nonanone were identified as WSFC-specific components. However, only anisole and 4-methylanisole elicited positive dose–response relationship in electroantennography tests, and only 4-methylanisole significantly attracted WSFCs of both sexes in olfactometer bioassays and field experiments. These results concluded that 4-methylanisole is the aggregation pheromone of WSFCs. Furthermore, we developed polyethylene vials as long-term dispensers of 4-methylanisole to attract and kill WSFCs. The polyethylene vial lures could effectively attracted WSFCs for more than four weeks. Pheromone-based lures can be developed as an environmentally friendly protocol for monitoring and controlling WSFC adults.

Identification of Components of the Aggregation Pheromone of the Guam Strain of Coconut Rhinoceros Beetle, Oryctes rhinoceros, and Determination of Stereochemistry

The coconut rhinoceros beetle, Oryctes rhinoceros (Linnaeus 1758) (Coleoptera: Scarabaeidae: Dynastinae) (CRB), is endemic to tropical Asia where it damages both coconut and oil palm. A new invasion by CRB occurred on Guam in 2007 and eradication attempts failed using commonly applied Oryctes rhinoceros nudivirus (OrNV) isolates. This and subsequent invasive outbreaks were found to have been caused by a previously unrecognized haplotype, CRB-G, which appeared to be tolerant to OrNV. The male-produced aggregation pheromone of the endemic, susceptible strain of O. rhinoceros (CRB-S) was previously identified as ethyl 4-methyloctanoate. Following reports from growers that commercial lures containing this compound were not attractive to CRB-G, the aim of this work was to identify the pheromone of CRB-G. Initial collections of volatiles from virgin male and female CRB-G adults from the Solomon Islands failed to show any male- or female-specific compounds as candidate pheromone components. Only after five months were significant quantities of ethyl 4-methyloctanoate and 4-methyloctanoic acid produced by males but not by females. No other male-specific compounds could be detected, in particular methyl 4-methyloctanoate, 4-methyl-1-octanol, or 4-methyl-1-octyl acetate, compounds identified in volatiles from some other species of Oryctes. Ethyl 4-methyloctanoate elicited a strong electroantennogram response from both male and female CRB-G, but these other compounds, including 4-methyloctanoic acid, did not. The enantiomers of ethyl 4-methyloctanoate and 4-methyloctanoic acid were conveniently prepared by enzymatic resolution of the commercially-available acid, and the enantiomers of the acid, but not the ester, could be separated by gas chromatography on an enantioselective cyclodextrin phase. Using this approach, both ethyl 4-methyloctanoate and 4-methyloctanoic acid produced by male CRB-G were shown to be exclusively the (R)-enantiomers whereas previous reports had suggested male O. rhinoceros produced the (S)-enantiomers. However, re-examination of the ester and acid produced by male CRB-S from Papua New Guinea showed that these were also the (R)-enantiomers. In field trapping experiments carried out in the Solomon Islands, both racemic and ethyl (R)-4-methyloctanoate were highly attractive to both male and female CRB-G beetles. The (S)-enantiomer and the corresponding acids were only weakly attractive. The addition of racemic 4-methyloctanoic acid to ethyl 4-methyloctanoate did significantly increase attractiveness, but the addition of (R)- or (S)-4-methyloctanoic acid to the corresponding ethyl esters did not. Possible reasons for the difference in assignment of configuration of the components of the CRB pheromone are discussed along with the practical implications of these results.

A larval aggregation pheromone as foraging cue for insectivorous birds

Although birds have traditionally been considered anosmic, increasing evidence indicates that olfaction plays an important role in the foraging behaviours of insectivorous birds. Recent studies have shown that birds can exploit herbivore-induced plant volatiles and sexual pheromones of adult insects to locate their prey. Many insectivorous birds prey on immature insects, providing relevant ecosystem services as pest regulators in natural and agricultural ecosystems. We asked whether birds could rely on chemical cues emitted by the immature stages of insects to prey on them. To address this question, we performed field experiments to evaluate if insectivorous birds can detect the aggregation pheromone produced by the larvae of the carpenter worm, Chilecomadia valdiviana . Groups of five artificial larvae were placed in branches of 72 adult trees in a remnant fragment of a sclerophyllous forest in central Chile. Each grouping of larvae contained a rubber septum loaded with either larval pheromone as treatment or solvent alone as control. We found that the number of larvae damaged by bird pecks was significantly higher in groups with dispensers containing the larval extract than in control groups. Our results show that birds can rely on immature insect-derived chemical cues used for larvae aggregation to prey on them.

Social communication activates the circadian gene Tctimeless in Tribolium castaneum

Chemical communication via pheromones is an integral component in insect behavior, particularly for mate searching and reproduction. Aggregation pheromones, that attract conspecifics of both sexes, are particularly common and have been identified for hundreds of species. These pheromones are among the most ecologically selective pest suppression agents. In this study, we identified an activating effect of the aggregation pheromone of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenibroidae) on a highly conserved circadian clock gene (Tctimeless). Tribolium castaneum is one of the most damaging cosmopolitan pest of flour and other stored food products. Its male produced aggregation pheromone, 4,8-dimethyldecanal (DMD), attracts both conspecific males and females and is used for pest management via monitoring and mating disruption. The Tctimeless gene is an essential component for daily expression patterns of the circadian clock and plays vital roles in eclosion, egg production, and embryonic development. In this study, we demonstrate that constant exposure to the species-specific aggregation pheromone led to Tctimeless up-regulation and a different pattern of rhythmic locomotive behavior. We propose that changing the well-adapted "alarm clock", using DMD is liable to reduce fitness and can be highly useful for pest management.

Synthesis of a synthetic analogue for the Sitophilus weevil aggregation pheromone and study on its hygienic and toxicological indexes

The work was initiated to study hygienic and toxicological indices of a synthetic analogue for the Sitophilus weevil aggregation pheromone. The toxicity testing of 5-hydroxy-4-methyl-3-heptanone demonstrated its extremely low toxicity for the warm-blood animals, as compared to the one of the typical pesticides. The average lethal dose of the product per orally administered to the white mice was established to be 4375.0 mg kg-1 LD16 and LD84 being 2225.0 mg kg-1 and 6550.0 mg kg-1, respectively. The average lethal dose for rabbits was 5900.0 mg kg-1 5-hydroxy-4-methyl-3-heptanone proved to have a mild skin and conjunctival irritant action, and equally mild functional cumulation. As to chronic toxicity, the acceptable daily dose of 4.3 mg/person/d was calculated and scientifically substantiated. The odor threshold was determined at the dose ranging from 0.35 to 0.7 mg l-1 with the practical limit ranging from 0.35 to 1.5 mg l-1, taste sensation threshold was found at the dose ranging from 1.0 to 3.0 mg l-1 with the practical limit ranging from 3.0 to 7.0 mg l-1

Aggregation Pheromone of the Guam Strain of Coconut Rhinoceros Beetle, Oryctes Rhinoceros, and Revision of Stereochemistry

The coconut rhinoceros beetle, Oryctes rhinoceros (Linnaeus 1758) (Coleoptera: Scarabaeidae: Dynastinae) (CRB), is endemic to tropical Asia where it damages both coconut and oil palm. A new invasion by CRB occurred on Guam in 2007 and eradication attempts failed using commonly applied O. rhinoceros nudivirus (OrNV) isolates. This and subsequent invasive outbreaks were found to have been caused by a previously unrecognized haplotype, CRB-G, which appeared to be tolerant to OrNV. The male-produced aggregation pheromone of the endemic, susceptible strain of O. rhinoceros (CRB-S) was previously identified as ethyl 4-methyloctanoate. There were anecdotal reports that the commercial lures containing this compound were not attractive to CRB-G and the aim of this work was to identify the pheromone of CRB-G. Initial collections of volatiles from virgin male and female CRB-G adults failed to show any male- or female-specific compounds as candidate pheromone components. Only after five months were significant quantities of ethyl 4-methyloctanoate and 4-methyloctanoic acid produced by males but not by females. No other male-specific compounds could be detected, in particular methyl 4-methyloctanoate, 4-methyl-1-octanol or 4-methyl-1-octyl acetate, compounds identified in volatiles from some other species of Oryctes. Ethyl 4-methyloctanoate elicited a strong electroantennogram response from both male and female CRB-G, but these other compounds, including 4-methyloctanoic acid, did not. The enantiomers of ethyl 4-methyloctanoate and 4-methyloctanoic acid were conveniently prepared by enzymatic resolution of the commercially-available acid, and the enantiomers of the acid, but not the ester, could be separated by gas chromatography on an enantiospecific cyclodextrin phase. Using this approach, both the ethyl 4-methyloctanoate and the 4-methyloctanoic acid produced by male CRB-G were shown to be exclusively the (R)-enantiomers whereas previous reports had suggested male CRB-S produced the (S)-enantiomers. However, re-examination of the ester and acid produced by male CRB-S showed that these were also the (R)-enantiomers. In field trapping experiments, both racemic and ethyl (R)-4-methyloctanoate were highly attractive to both male and female CRB-G. The (S)-enantiomer and the corresponding acids were only weakly attractive. Addition of racemic 4-methyloctanoic acid to ethyl 4-methyloctanoate did significantly increase attractiveness, but addition of (R)- or (S)-4-methyloctanoic acid to the corresponding ethyl esters did not. Reasons for the previous misassignment of the configuration of the components of the CRB-S pheromone are discussed along with the practical implications of these results.

Identification of male produced compounds in the bark beetle Polygraphus subopacus and establishment of (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol as an aggregation pheromone component

Bark beetles of the genus Polygraphus have recently been involved in large bark beetle outbreaks in central Sweden, together with the European spruce bark beetle Ips typographus. Three species of Polygraphus can be found in this region; Polygraphus poligraphus, Polygraphus punctifrons and Polygraphus subopacus. Efficient pheromone traps would facilitate further investigations of these species and their role in bark beetle outbreaks. Pheromone compounds have previously been identified in P. poligraphus and P. punctifrons, but not in P. subopacus. Thus, we allowed males and females of P. subopacus to bore in the bark of stem sections of Norway spruce (Picea abies) in the laboratory. Volatile organic compounds from boring insects were sampled with SPME and analysed with GC–MS and several male-specific compounds were observed. The male specific compounds were 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, 3-methyl-2-butenal, grandisol, fragranol, (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol, (E)-2-(3,3-dimethylcyclohexylidene)-ethanol, (Z)-2-(3,3-dimethylcyclohexylidene)-acetaldehyde, (E)-2-(3,3-dimethylcyclohexylidene)-acetaldehyde, geranial and γ-isogeraniol. (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol, [(Z)-DMCHE], was identified from GC–MS analysis to be the major male-specific compound while the (E)-isomer, [(E)-DMCHE], was found as a minor compound. These two compounds gave positive responses in EAG analyses with antennae from males and females of P. subopacus. Thus, (Z)- and (E)-DMCHE were used in a field experiment in central Sweden but only (Z)-DMCHE was found to be attractive to males and females of P. subopacus. Consequently, (Z)-DMCHE was established to be a component of P. subopacus aggregation pheromone.

COMPOUNDS FROM PEPPER FLOWERS AND FRUITS AS POTENTIAL ATTRACTANTS FOR THE CAPTURE OF PEPPER WEEVIL

Two field experiments were conducted to evaluate synthetic attractants derived from pepper flowers, flower buds and fruits, alone or in combination with the aggregation pheromone. The evaluation was carried out with the release and recapture of Anthonomus eugenii adults at different distances from the four cardinal points in separate trials. The volatility of the synthetic mixture and aggregation pheromone was determined by gas chromatographic analysis of the volatiles captured by dynamic headspace. The traps with synthetic mixture and essential oil captured insects at 10 m, while the aggregation pheromone trapped up to 60 m. The combination of synthetic mixture or essential oil with the aggregation pheromone did not increase the number of recaptures compared to the single pheromone. The synthetic mixture together with geranic acid recaptured adults up to 15 m, although they were not significantly different from the control. The exclusion of geranic acid from the aggregation pheromone significantly reduced the number of recaptured insects (P<0.05), while geranic acid alone failed to capture weevils. The results could be improved by increasing the concentrations of the compounds or by adding other compounds released during the reproductive stages of pepper. These results could guide future efforts for the development of tools based on synthetic plant volatiles for the monitoring of this pest.

Area wide management of white grub Holotrichia consanguinea Blachard through IPM modules in groundnut

Field experiments were conducted on groundnut for white grub Holotrichia consanguinea Blachard management using different IPM components in Gujarat during kharif 2018 and 2019. Two different eco-friendly modules were evaluated and compare with farmers practices. In Module I included on set of monsoon, spraying of chlorpyrifos 20 EC @ 0.04% on surrounding trees, seed treatment of chlorpyrifos 20 EC @ 20 ml/kg, Placement of aggregation pheromone on host tree (5 block/tree), Soil application of Beauveria bassiana @ 5 kg ha-1 (Min. 2 x 106 CFU/g) + castor cake (250 kg ha-1) before sowing, Application of B. bassiana @ 5 kg ha-1 in plant row with FYM (250 kg ha-1) after 30 days of germination. While Module II used Metarhizium anisopliae instead of B. bassiana. While in farmers practices included application of chlorpyriphos 10 G @ 10 kg ha-1 with urea at initiation of pest attack. Among the evaluated two ecofriendly module and farmers practices the lowest per cent plant mortality (3.03 %) and 0.56 grub/m2 was recorded in Module II. The highest pod and halum yield 2180 and 3942 kg ha-1, yield increase over control 31.00 and 39.37 %, avoidable loss 23.67 and 28.25% was recorded in module II. The maximum (32474 Rs/ha) net realization was found in the treatment of Module II. Looking to the NICBR, the highest (1:5.35) return was obtained with the treatment of Module II followed by Module I (1:2.93)