Development and characterization of microsatellite markers for population genetics of the cocoa pod borer Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae).

The cocoa pod borer (CPB) Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae) is one of the major constraints for cocoa production in South East Asia. In addition to cultural and chemical control methods, autocidal control tactics such as the Sterile Insect Technique (SIT) could be an efficient addition to the currently control strategy, however SIT implementation will depend on the population genetics of the targeted pest. The aim of the present work was to search for suitable microsatellite loci in the genome of CPB that is partially sequenced. Twelve microsatellites were initially selected and used to analyze moths collected from Indonesia, Malaysia, and the Philippines. A quality control verification process was carried out and seven microsatellites found to be suitable and efficient to distinguish differences between CPB populations from different locations. The selected microsatellites were also tested against a closely related species, i.e. the lychee fruit borer Conopomorpha sinensis (LFB) from Vietnam and eight loci were found to be suitable. The availability of these novel microsatellite loci will provide useful tools for the analysis of the population genetics and gene flow of these pests, to select suitable CPB strains to implement the SIT.

Development of a Diet Production System for Conopomorpha cramerella (Lepidoptera: Gracillariidae), a Major Cocoa Production Pest in Southeast Asia and the Pacific Islands.

The development of artificial diets for the cocoa pod borer Conopomorpha cramerella, a major pest of cocoa plants, has undergone significant advancements. In this study, we present the success rates of two diet formulations, MM1 and MM4, which have been progressively improved. Nutritional composition analysis revealed that the MM1 diet differed from the natural host, cocoa pods, in several aspects, including protein, carbohydrate, and vitamin C content. To address these differences, modifications were made to the diet compositions, leading to the MM4 diet version. These modifications resulted in improved diet quality and reduced contamination, leading to enhanced success rates in all stages of C. cramerella development. Larval development, pupation success rates, and adult emergence rates were significantly higher in the MM4 diet compared with the MM1 diet. Moreover, the duration of larval development and pupal stage decreased, while adult longevity increased with the MM4 diet. The overall development success of diet-reared insects from egg to adult was comparable with that of insects reared on cocoa pods. However, the cocoon formation, body length and fresh weight of the adults reared on the artificial diets were lower than those reared on cocoa pods. This diet formulation provides a promising approach for laboratory rearing of C. cramerella and opens avenues for further research and mass-rearing initiatives to mitigate the impact of this pest on cocoa production.

Development of a Kairomone-Based Attractant as a Monitoring Tool for the Cocoa Pod Borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae).

The cocoa pod borer (CPB), Conopomorpha cramerella, is a major economic pest of cocoa, Theobroma cacao, in Southeast Asia. CPB monitoring programs currently use a costly synthetic pheromone lure attractive to males. Field trapping experiments demonstrating an effective plant-based alternative are presented in this study. Five lychee-based products were compared for their attractiveness to CPB males. The organic lychee flavor extract (OLFE), the most attractive product, captured significantly more CPB as a 1 mL vial formulation than unbaited traps, while being competitive with the commercial pheromone lures. Additional experiments show that a 20 mL membrane OLFE lure was most effective, attracting significantly more CPB than the pheromone. When the kairomone and pheromone lures were combined, no additive or synergistic effects were observed. Concentrating the OLFE product (OLFEc) using a rotary evaporator increased the lure attractiveness to field longevity for up to 28 weeks; in contrast, pheromone lures were effective for approximately 4 weeks. The 20 mL concentrated OLFE membrane lures should provide a cheaper and more efficient monitoring tool for CPB than the current commercial pheromone lures.

Sex Pheromone of Cocoa Pod Borer, Conopomorpha cramerella: Field Activity Evaluation of Pheromone Formulations in an Indonesia Plantation.

The previously identified female sex pheromone of cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae), was re-evaluated for male attraction using six different pheromone formulations in Indonesian cocoa plantations. In a dose-response experiment, the 0.1 mg pheromone load was significantly more attractive to male CPB than the lower doses tested. Additionally, during the first four-week trapping period, USDA (Beltsville, MD, USA) lures containing 0.1 mg of synthetic pheromone blend exhibited significantly better attraction than the commercial lure obtained from Alpha Scents, Inc. (Canby, OR, USA) with the same pheromone load (0.1 mg). Although the 1.0 mg lure did not show any higher attraction than the 0.1 mg lure during the first month, it was significantly attractive for CPB males with the same weekly average capture efficacy for the whole twenty-seven weeks in field conditions in 2018. A long-life pheromone lure can be particularly useful in monitoring large-scale cocoa farms.

Mealybug (Hemiptera: Pseudococcidae) Species Associated with Cacao Mild Mosaic Virus and Evidence of Virus Acquisition.

Theobroma cacao is affected by viruses on every continent where the crop is cultivated, with the most well-known ones belonging to the Badnavirus genus. One of these, cacao mild mosaic virus (CaMMV), is present in the Americas, and is transmitted by several species of Pseudococcidae (mealybugs). To determine which species are associated with virus-affected cacao plants in North America, and to assess their potential as vectors, mealybugs (n = 166) were collected from infected trees in Florida, and identified using COI, ITS2, and 28S markers. The species present were Pseudococcus jackbeardsleyi (38%; n = 63), Maconellicoccus hirsutus (34.3%; n = 57), Pseudococcus comstocki (15.7%; n = 26), and Ferrisia virgata (12%; n = 20). Virus acquisition was assessed by testing mealybug DNA (0.8 ng) using a nested PCR that amplified a 500 bp fragment of the movement protein-coat protein region of CaMMV. Virus sequences were obtained from 34.6 to 43.1% of the insects tested; however, acquisition did not differ among species, X2 (3, N = 166) = 0.56, p < 0.91. This study identified two new mealybug species, P. jackbeardsleyi and M. hirsutus, as potential vectors of CaMMV. This information is essential for understanding the infection cycle of CaMMV and developing effective management strategies.

TLC-Based Bioassay to Isolate Kairomones from Tea Tree Essential Oil That Attract Male Mediterranean Fruit Flies, Ceratitis capitata (Wiedemann).

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) poses a major threat to fruit and vegetable production in the United States and throughout the world. New attractants and detection methods could improve control strategies for this invasive pest. In this study, we developed a method that combined thin-layer chromatography (TLC) of tea tree essential oil (TTO) (Melaleuca alternifolia) with short-range bioassays to isolate attractive kairomones for male C. capitata. After development, the TLC chromatogram indicated that TTO separated into five major spots, designated as zones 1 to 5. When the TLC plate was exposed to flies, zones 1 and 3 were strongly attractive to male C. capitata. To confirm activity, the developed TLC plate was cut into five zones which were then tested in short-range bioassays. Again, flies were observed to aggregate around zones 1 and 3, which corresponded with Rf values of 0.93 and 0.59. In addition, zones 1 to 5 were separated using preparative-TLC, and olfactory responses to volatile emissions from the five fractions were quantified by electroantennography (EAG). Highest amplitude EAG responses were recorded with fractions 1 and 3, further supporting the bioactivity of these samples. In conclusion, a TLC-based bioassay system can provide an effective, rapid screening protocol for initial isolation of insect kairomones from complex mixtures such as essential oils or plant extracts. Further analysis of TTO fractions 1 and 3 is needed to identify the specific constituents attractive to male C. capitata.

Laboratory Evaluation of Natural and Synthetic Aromatic Compounds as Potential Attractants for Male Mediterranean fruit Fly, Ceratitis capitata.

Ceratitis capitata, the Mediterranean fruit fly, is one of the most serious agricultural pests worldwide responsible for significant reduction in fruit and vegetable yields. Eradication is expensive and often not feasible. Current control methods include the application of conventional insecticides, leading to pesticide resistance and unwanted environmental effects. The aim of this study was to identify potential new attractants for incorporation into more environmentally sound management programs for C. capitata. In initial binary choice bioassays against control, a series of naturally occurring plant and fungal aromatic compounds and their related analogs were screened, identifying phenyllactic acid (7), estragole (24), o-eugenol (21), and 2-allylphenol (23) as promising attractants for male C. capitata. Subsequent binary choice tests evaluated five semisynthetic derivatives prepared from 2-allylphenol, but none of these were as attractive as 2-allylphenol. In binary choice bioassays with the four most attractive compounds, males were more attracted to o-eugenol (21) than to estragole (24), 2-allylphenol (23), or phenyllactic acid (7). In addition, electroantennography (EAG) was used to quantify antennal olfactory responses to the individual compounds (1-29), and the strongest EAG responses were elicited by 1-allyl-4-(trifluoromethyl)benzene (11), estragole (24), 4-allyltoluene (14), trans-anethole (9), o-eugenol (21), and 2-allylphenol (23). The compounds evaluated in the current investigation provide insight into chemical structure-function relationships and help direct future efforts in the development of improved attractants for the detection and control of invasive C. capitata.

Chemosystematics Using Cuticular Compounds: A Powerful Tool to Separate Species in Mediterranean Dung Beetles (Coleoptera: Geotrupidae).

The use of chemical characters to infer a phylogeny is known to be promising to ascertain phylogenetic relationships in controversial groups. Dung beetle classifications containing the Geotrupidae family, based on morphological characters and genes, are debated with respect to the subfamilies, such as the Bolboceratids. In our study, we used different approaches to generate and compare the Geotrupidae phylogenies based on genetics and chemotaxonomy. Cuticular compounds were analyzed for 12 species of Mediterranean dung beetles to build a chemical phylogeny. In addition, mitochondrial and nuclear marker concatenation have been used to elaborate the molecular phylogeny. Using the cuticular compound continuous data, our results showed that each species was associated with a specific chemical pattern and that all individuals belonging to the same species displayed a similar chemical blend. The most distant species was Bolbelasmus gallicus, with an evident distinction from the other species due to several compounds. The maximum parsimony tree showed that all genera belonging to a Geotrupidae subfamily were grouped in the same clade, with B. gallicus species isolated in another clade, similar to the chemotaxonomy grouping. A strong positive correlation between chemotaxonomy and genetic phylogeny has been demonstrated, underlying a genetic basis for cuticular hydrocarbon variations. Our results are congruent with previous studies using morphological or genetic data. Our results also showed that only 10 compounds can be used to distinguish at least six species of dung beetle and that chemotaxonomy could become a useful and affordable tool to determine phylogenetic relationships in insects.

Attraction of Ceratitis capitata (Diptera: Tephritidae) Sterile Males to Essential Oils: The Importance of Linalool.

Small cage and wind tunnel bioassays were used to understand the role of volatile chemicals found in ginger root oil and other essential oils in the attraction of sterile male Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Small cage bioassays found that both a 10 component blend (geraniol, linalool, ß-myrcene, limonene, α-pinene, ß-pinene, ß-caryophyllene, terpinen-4-ol, α-terpineol, and α-humulene) and a 4 component subset of that blend (geraniol, linalool, ß-myrcene and limonene) were more attractive than paired mineral oil controls. Both blends were equally attractive as ginger root oil and each other. Deletion studies, which tested all 3 component blends, found decreased attraction when linalool was deleted from the 4 component blend. Linalool alone attracted an equal percentage of flies as the 4 component blend, confirming that this chemical was responsible primarily for attraction to ginger root oil. Wind tunnel bioassays confirmed previous studies that panel traps baited with ginger root oil captured more flies than traps baited with manuka oil. Addition of linalool to manuka oil resulted in capture equal to ginger root oil, and addition of linalool to ginger root oil resulted in capture of more flies than ginger root alone. The results of this study will allow a better understanding of the role of individual plant-based chemicals in the attraction of male C. capitata.

Cubeb Oil Lures: Terpenoid Emissions, Trapping Efficacy, and Longevity for Attraction of Redbay Ambrosia Beetle (Coleoptera: Curculionidae: Scolytinae).

Redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is an exotic wood borer and the primary vector of Raffaelea lauricola, a symbiotic fungus that causes laurel wilt. This lethal disease has decimated native redbay [Persea borbonia (L.) Sprengel] and swampbay [Persea palustris (Rafinesque) Sargent] throughout southeastern U.S. forests, and currently threatens avocado (Persea americana Miller) in Florida. To curtail the spread of laurel wilt, effective attractants are needed for early detection of the vector. Phoebe oil lures were the best known attractant for X. glabratus, but they are no longer available. The current detection system uses manuka oil lures, but previous research indicated that manuka lures have a short field life in Florida. Recently, cubeb oil was identified as a new attractant for X. glabratus, and cubeb bubble lures are now available commercially. This study compared trapping efficacy and field longevity of cubeb and manuka lures with phoebe lures that had been in storage since 2010 over a 12-wk period in south Florida. In addition, terpenoid emissions were quantified from cubeb and manuka lures aged outdoors for 12 wk. Captures were comparable with all three lures for 3 wk, but by 4 wk, captures with manuka were significantly less. Equivalent captures were obtained with cubeb and phoebe lures for 7 wk, but captures with cubeb were significantly greater from 8 to 12 wk. Our results indicate that cubeb bubble lures are the most effective tool currently available for detection of X. glabratus, with a field life of 3 months due to extended low release of attractive sesquiterpenes, primarily α-copaene and α-cubebene.

North American Lauraceae: terpenoid emissions, relative attraction and boring preferences of redbay ambrosia beetle, Xyleborus glabratus (coleoptera: curculionidae: scolytinae).

The invasive redbay ambrosia beetle, Xyleborus glabratus, is the primary vector of Raffaelea lauricola, a symbiotic fungus and the etiologic agent of laurel wilt. This lethal disease has caused severe mortality of redbay (Persea borbonia) and swampbay (P. palustris) trees in the southeastern USA, threatens avocado (P. americana) production in Florida, and has potential to impact additional New World species. To date, all North American hosts of X. glabratus and suscepts of laurel wilt are members of the family Lauraceae. This comparative study combined field tests and laboratory bioassays to evaluate attraction and boring preferences of female X. glabratus using freshly-cut bolts from nine species of Lauraceae: avocado (one cultivar of each botanical race), redbay, swampbay, silkbay (Persea humilis), California bay laurel (Umbellularia californica), sassafras (Sassafras albidum), northern spicebush (Lindera benzoin), camphor tree (Cinnamomum camphora), and lancewood (Nectandra coriacea). In addition, volatile collections and gas chromatography-mass spectroscopy (GC-MS) were conducted to quantify terpenoid emissions from test bolts, and electroantennography (EAG) was performed to measure olfactory responses of X. glabratus to terpenoids identified by GC-MS. Significant differences were observed among treatments in both field and laboratory tests. Silkbay and camphor tree attracted the highest numbers of the beetle in the field, and lancewood and spicebush the lowest, whereas boring activity was greatest on silkbay, bay laurel, swampbay, and redbay, and lowest on lancewood, spicebush, and camphor tree. The Guatemalan cultivar of avocado was more attractive than those of the other races, but boring response among the three was equivalent. The results suggest that camphor tree may contain a chemical deterrent to boring, and that different cues are associated with host location and host acceptance. Emissions of α-cubebene, α-copaene, α-humulene, and calamenene were positively correlated with attraction, and EAG analyses confirmed chemoreception of terpenoids by antennal receptors of X. glabratus.

Terpenoid variations within and among half-sibling avocado trees, Persea americana Mill. (Lauraceae).

Chemical analyses were conducted to determine the qualitative and quantitative differences in monoterpenes and sesquiterpenes in plant material from avocado trees, Persea americana Mill. (Lauraceae). The initial study analyzed plant material sampled from the trunk to the leaves through different branch diameters to quantify proximo-distal spatial differences within a tree. All trees were seedlings initiated from a single maternal tree. Two-way analysis of variance was conducted on 34 chemicals that comprised at least 3% of the total chemical content of at least one tree and/or location within a tree. There were significant interactions between genotype and location sampled for most chemicals. Parentage analysis using microsatellite molecular markers (SSR's) determined that the four trees had three fathers and that they represented two full-siblings and two half-sibling trees. Descriptive discriminant analysis found that both genotype and location within a tree could be separated based on chemical content, and that the chemical content from full-siblings tended to be more similar than chemical content from half-siblings. To further explore the relationship between genetic background and chemical content, samples were analyzed from leaf material from 20 trees that included two sets of full-sibling seedling trees, the maternal tree and the surviving paternal tree. Descriptive discriminant analysis found good separation between the two full-sibling groups, and that the separation was associated with chemistry of the parental trees. Six groups of chemicals were identified that explained the variation among the trees. We discuss the results in relation to the discrimination process used by wood-boring insects for site-selection on host trees, for tree selection among potential host trees, and the potential use of terpenoid chemical content in chemotaxonomy of avocado trees.

Temporal analysis of sesquiterpene emissions from manuka and phoebe oil lures and efficacy for attraction of Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae).

Redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is an exotic wood-borer that vectors the fungal agent (Raffaelea lauricola) responsible for laurel wilt. Laurel wilt has had severe impact on forest ecosystems in the southeastern United States, killing a large proportion of native Persea trees, particularly redbay (P. borbonia) and swampbay (P. palustris), and currently poses an economic threat to avocado (P. americana) in Florida. To control the spread of this lethal disease, effective attractants are needed for early detection of the vector. Two 12-wk field tests were conducted in Florida to evaluate efficacy and longevity of manuka and phoebe oil lures, and to relate captures of X. glabratus to release rates of putative sesquiterpene attractants. Two trap types were also evaluated, Lindgren funnel traps and sticky panel traps. To document lure emissions over time, a separate set of lures was aged outdoors for 12 wk and sampled periodically to quantify volatile sesquiterpenes using super-Q adsorbant and gas chromatography-mass spectroscopy analysis. Phoebe lures captured significantly more X. glabratus than manuka lures, and sticky traps captured more beetles than funnel traps. Phoebe lures captured X. glabratus for 10-12 wk, but field life of manuka lures was 2-3 wk. Emissions of alpha-copaene, alpha-humulene, and cadinene were consistently higher from phoebe lures, particularly during the 2-3 wk window when manuka lures lost efficacy, suggesting that these sesquiterpenes are primary kairomones used by host-seeking females. Results indicate that the current monitoring system is suboptimal for early detection of X. glabratus because of rapid depletion of sesquiterpenes from manuka lures.

Xyleborus glabratus, X. affinis, and X. ferrugineus (Coleoptera: Curculionidae: Scolytinae): electroantennogram responses to host-based attractants and temporal patterns in host-seeking flight.

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is an exotic wood-boring insect that vectors the mycopathogen responsible for laurel wilt, a lethal vascular disease of trees in the Lauraceae, including avocado (Persea americana Mill.). Effective semiochemical-based detection and control programs for X. glabratus will require an understanding of the chemical ecology and host-seeking behaviors of this new invasive pest. This study 1) presents an electroantennography (EAG) method developed for assessment of olfactory responses in ambrosia beetles; 2) uses that new method to quantify EAG responses of X. glabratus, X. affinis, and X. ferrugineus to volatiles from three host-based attractants: manuka oil (essential oil extract from Leptospermum scoparium Forst. & Forst.), phoebe oil (extract from Phoebe porosa Mex.), and wood from silkbay (Persea humilis Nash); and 3) documents temporal differences in host-seeking flight of the sympatric Xyleborus species. Field observations revealed that X. glabratus engages in flight several hours earlier than X. affinis and X. ferrugineus, providing a window for selective capture of the target pest species. In EAG analyses with X. glabratus, antennal response to phoebe oil was equivalent to response to host Persea wood, but EAG response elicited with manuka oil was significantly less. In comparative studies, EAG response of X. glabratus was significantly higher than response of either X. affinis or X. ferrugineus to all three host-based substrates. Future research will use this EAG method to measure olfactory responses to synthetic terpenoids, facilitating identification of the specific kairomones used by X. glabratus for host location.

Attraction of the redbay ambrosia beetle, Xyleborus glabratus, to avocado, lychee, and essential oil Lures.

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff, is an exotic wood-boring insect that vectors the mycopathogen responsible for laurel wilt, a lethal vascular disease of trees in the Lauraceae. High mortality has occurred in native Persea species in the southeastern U.S., and the vector-pathogen complex poses an imminent threat to the production of commercial avocado, P. americana, in south Florida. There is a critical need for effective attractants to detect, monitor, and control this invasive pest. This study combined field tests and laboratory bioassays to evaluate the response of female X. glabratus to host-based volatiles from wood of avocado (cultivars of West Indian, Guatemalan, and Mexican races); from wood of lychee (Litchi chinensis, a presumed non-host that is high in the sesquiterpene α-copaene, a putative attractant); and to commercial lures containing manuka and phoebe oils, two reported attractive baits. Volatile collections and GC-MS analyses were performed to quantify the sesquiterpene content of test substrates. In the field, traps baited with lychee wood captured more beetles than those with wood from avocado cultivars; traps baited with phoebe oil lures captured more beetles than those with manuka oil lures (the current monitoring tool). In field and laboratory tests, X. glabratus did not show a preference among avocado races in either attraction or host acceptance (initiation of boring). In choice tests, lychee was more attractive than avocado initially, but a higher percentage of beetles bored into avocado, suggesting that lychee emits more powerful olfactory/visual cues, but that avocado contains more of the secondary cues necessary for host recognition. Emissions of α-copaene, ß-caryophyllene, and α-humulene were correlated with field captures, and lychee wood may be a source of additional semiochemicals for X. glabratus.

Attraction and electroantennogram responses of male Mediterranean fruit fly to volatile chemicals from Persea, Litchi and Ficus wood.

Trimedlure is the most effective male-targeted lure for the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). A similar response is elicited by plant substances that contain α-copaene, a naturally-occurring sesquiterpene. α-Copaene is a complex, highly-volatile, widely-distributed plant compound, and male C. capitata respond to material from both hosts (e.g., Litchi chinensis) and non-hosts (e.g., Ficus benjamina) that contain α-copaene. Avocado, Persea americana, recently was found to contain varying amounts of α-copaene in the bark and underlying cambial tissue. Short-range attraction bioassays and electroantennography (EAG) were used to quantify responses of sterile male C. capitata to samples of rasped wood from four avocado genotypes, L. chinensis, and F. benjamina. Gas chromatography-mass spectral (GC-MS) analysis was used to identify and quantify the major sesquiterpenes. Attraction and EAG amplitude were correlated, with L. chinensis eliciting the highest and F. benjamina the lowest responses. Responses to the avocado genotypes were intermediate, but varied among the four types. GC-MS identified 13 sesquiterpenes, including α-copaene, from all samples. Amounts of α-copaene in volatile collections from samples (3 g) ranged from 11.8 µg in L. chinensis to 0.09 µg in F. benjamina, which correlated with short-range attraction and EAG response. α-Copaene ranged from 8.0 to 0.8 µg in the avocado genotypes, but attraction and EAG responses were not correlated with the amount of α-copaene. Differences in enantiomeric structure of the α-copaene in the different genotypes and/or presence of additional sesquiterpenes may be responsible for the variation in male response. EAG responses were correlated with the amount of several other sesquiterpenes including α-humulene, and this compound elicited a strong antennal response when tested alone.

Effect of 20-hydroxyecdysone on cannibalism, sexual behavior, and contact sex pheromone in the solitary female spider, Tegenaria atrica.

We investigated the effect of exogenously administered 20-hydroxyecdysone (20E) on sexual behavior and on contact sex signals of adult females of the spider Tegenaria atrica. In untreated or control females cannibalism occurred in 36-47% of encounters, whereas no cannibalism at all was observed with 20E-treated females. The frequency of sexual receptivity was low (42%) in untreated or control females and high (100%) in 20E-treated females. Variability of female sexual behavior could be related to quantitative changes in web and cuticular compounds. We showed that 20E altered the production of polar contact sex pheromones on web and cuticle: methylesters and fatty acids are produced in higher amounts in 20E-treated females. Bioassays demonstrated clearly that methylesters and fatty acids of webs and cuticles were involved in attraction of males. Moreover, bioassays with synthetic substances showed that four fatty acids (myristic, pentadecanoic, hexadecanoic, and oleic acids) and one methylester (methyl stearate) are particularly effective in eliciting sexual behavior in males. In T. atrica, 20E inhibits cannibalism during sexual activity and changes sex pheromone production.