Diel rhythm of volatile emissions from males and females of the olive fruit fly Bactrocera oleae using PTR-ToF and GC-MS.

The olive fruit fly Bactrocera oleae, is the major key pest of olive groves worldwide. As an odor-driven species, its intraspecific communication has been thoroughly investigated, yielding a combination of spiroacetals, esters and hydrocarbons. However, its management with pheromone is still restricted to olean, the major pheromone component. Given the crucial role of circadian rhythm and pheromone blends in mediating flies reproductive behavior compared to single compounds, B. oleae headspace chemical profile was carefully examined, through the combination of Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToF) and Gas Chromatography coupled with Mass Spectrometry (GC-MS). This novel approach aimed at continuously investigating the temporal scale of volatilome profile of B. oleae individuals, as well as the determination of new candidate sex-borne compounds (particularly those emitted in traces or having low molecular weight), that may be relevant to the fly's chemical communication and were unreported due to limitations of frequently used analytical techniques. Our results describe the dynamics and diversity of B. oleae chemical profile, highlighting the emission of 90 compounds, with clear diel rhythm of release, of known pheromone components of B. oleae (e.g., olean, alpha-pinene and muscalure) and new candidates. In contrast to ammonia, acetaldehyde and muscalure, which were highly emitted during the afternoon by males and mixed groups, olean was mostly released by mature females and mixed groups, with a peak of emission during early-morning and afternoon. This emission of olean around dawn is reported for the first time, suggesting early-morning mating activity in B. oleae. Furthermore, esters, such as methyl tetradecanoate, which had been earlier identified as a pheromone for B. oleae, did not exhibit any discernible release patterns. These findings are the first to demonstrate the emission of chemicals, which are only produced when males and females are close to one another, with an emission peak during the afternoon (mating period), and that may have aphrodisiac properties for B. oleae males. These results emphasize the relevance of compounds with distinct diel rhythm and address their potential function as intraspecific messengers, according to their source and timing of release.

Bioactive Male-Produced Volatiles from Anastrepha obliqua and their Role in Attraction of Conspecific Females.

The study of insect semiochemicals, especially pheromones, is of fundamental importance for the development of strategies for controlling agricultural pests. In this study, volatile compounds involved in the communication between males and females of the fruit fly, Anastrepha obliqua (Diptera: Tephritidae), for mating purposes were characterized to develop attractant formulations for females of this species. Extracts containing volatile compounds released by males of A. obliqua were obtained by the dynamic headspace technique and analyzed by gas chromatography coupled with an electroantennographic detector (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). Twenty-one volatile compounds were identified in the aeration extracts of males. Five of them caused EAD responses from the antennae of females: 1-heptanol, linalool, (Z)-3-nonen-1-ol, (E,Z)-3,6-nonadien-1-ol, and (Z,E)-α-farnesene. Six synthetic mixtures of these compounds, including the five-component blend and all possible four-component blends, were formulated in a biopolymer and used in behavioral bioassays conducted in the laboratory arena with conspecific virgin females. One blend of 1-heptanol, linalool, (Z)-3-nonen-1-ol, and (Z,E)-α-farnesene attracted more females than the collection of volatiles from virgin males used as control. The other mixtures were as attractive to A. obliqua females as the control treatment. This study indicates potential for use of these compounds in monitoring and control strategies for this pest.

OBP2 in the Midlegs of the Male Bactrocera dorsalis Is Involved in the Perception of the Female-Biased Sex Pheromone 4-Allyl-2,6-dimethoxyphenol.

Short-range semiochemicals convey individual-specific information, which is important for final successful courtship and copulation. In this study, an electroantennography (EAG) instrument was used to determine the legs of male Bactrocera dorsalis that might participate in the perception of 4-allyl-2,6-dimethoxyphenol (4-DMP), a female-biased cuticular hydrocarbon (CHC) component. By performing comparative RNA-seq analysis, nine differentially expressed OBPs between 4-DMP-stimulated (M4-DMP) and unstimulated (M) male flies were screened out. Among the four downregulated OBPs, BdorOBP2 exhibited the strongest binding to 4-DMP than BdorOBP4, BdorOBP19a, and BdorOBP56h. Functional analyses confirmed that the reduction in transcript abundance of BdorOBP2 led to a significant decrease in behavioral responses of male flies to 4-DMP. In silico simulation revealed dramatic changes in the key residues and conformation between the two complexes, implying that BdorOBP2 might activate different receptors after binding to 4-DMP or methyl eugenol (ME). The results of this study suggest that BdorOBP2 mediates behavioral responses to 4-DMP and could be a promising molecular target for strategies of pest control.

Sampling technique biases in the analysis of fruit fly volatiles: a case study of Queensland fruit fly.

Diverse methods have been used to sample insect semiochemicals. Sampling methods can differ in efficiency and affinity and this can introduce significant biases when interpreting biological patterns. We compare common methods used to sample tephritid fruit fly rectal gland volatiles ('pheromones'), focusing on Queensland fruit fly, Bactrocera tryoni. Solvents of different polarity, n-hexane, dichloromethane and ethanol, were compared using intact and crushed glands. Polydimethylsiloxane, polydimethylsiloxane/divinylbenzene and polyacrylate were compared as adsorbents for solid phase microextraction. Tenax-GR and Porapak Q were compared as adsorbents for dynamic headspace sampling. Along with compounds previously reported for B. tryoni, we detected five previously unreported compounds in males, and three in females. Dichloromethane extracted more amides while there was no significant difference between the three solvents in extraction of spiroacetals except for (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane for which n-hexane extracted higher amount than both dichloromethane and ethanol. Ethanol failed to contain many of the more volatile compounds. Crushed rectal gland samples provided higher concentrations of extracted compounds than intact rectal gland samples, but no compounds were missed in intact samples. Of solid phase microextraction fibers, polyacrylate had low affinity for spiroacetals, ethyl isobutyrate and ethyl-2-methylbutanoate. Polydimethylsiloxane was more efficient for spiroacetals while type of fiber did not affect the amounts of amides and esters. In dynamic headspace sampling, Porapak was more efficient for ethyl isobutyrate and spiroacetals, while Tenax was more efficient for other esters and amides, and sampling time was a critical factor. Biases that can be introduced by sampling methods are important considerations when collecting and interpreting insect semiochemical profiles.

Cuticular Chemistry of the Queensland Fruit Fly Bactrocera tryoni (Froggatt).

The cuticular layer of the insect exoskeleton contains diverse compounds that serve important biological functions, including the maintenance of homeostasis by protecting against water loss, protection from injury, pathogens and insecticides, and communication. Bactrocera tryoni (Froggatt) is the most destructive pest of fruit production in Australia, yet there are no published accounts of this species' cuticular chemistry. We here provide a comprehensive description of B. tryoni cuticular chemistry. We used gas chromatography-mass spectrometry to identify and characterize compounds in hexane extracts of B. tryoni adults reared from larvae in naturally infested fruits. The compounds found included spiroacetals, aliphatic amides, saturated/unsaturated and methyl branched C12 to C20 chain esters and C29 to C33 normal and methyl-branched alkanes. The spiroacetals and esters were found to be specific to mature females, while the amides were found in both sexes. Normal and methyl-branched alkanes were qualitatively the same in all age and sex groups but some of the alkanes differed in amounts (as estimated from internal standard-normalized peak areas) between mature males and females, as well as between mature and immature flies. This study provides essential foundations for studies investigating the functions of cuticular chemistry in this economically important species.

Attraction and Electrophysiological Response to Identified Rectal Gland Volatiles in Bactrocera frauenfeldi (Schiner).

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi.

Amorphous calcium phosphate in the pupal cuticle of Bactrocera dorsalis Hendel (Diptera: Tephritidae): A new discovery for reconsidering the mineralization of the insect cuticle.

It is now widely accepted that Hexapoda emerged from Crustacea. Compared to the ubiquitous calcified exoskeleton in crustaceans, a mineralized cuticle in insects is extremely rare. Catecholamine-driven protein cross-links play a leading role in the sclerotization of insect cuticle. In this study, mineralization was discovered in the pupal cuticle of Bactrocera dorsalis (Diptera: Tephritidae), a common pest of fruit farms. We mainly profiled the features of mineralized pupal cuticles from B. dorsalis and its white mutant B. dorsaliswh and unmineralized cuticle from Musca domestica using high-resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with structural analysis involving infrared (IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and synchrotron X-ray absorption near-edge structure (XANES) spectroscopy. We also compared the thermodynamic and mechanical properties of different pupal cuticles. The results showed that the pupal cuticles of B. dorsalis contain a phase of stable amorphous calcium phosphate (ACP) with a high level of magnesium, which is mainly distributed in the exocuticle and assists in the formation of a graded, stiffened cuticle structure. Unexpectedly, this ACP possesses a very low Ca/P ratio and has a composition similar to that of CaHPO4·2H2O. The degree of mineralization in the pupal cuticle of B. dorsaliswh (approximately 22 wt%) is significantly greater than that of wild-type B. dorsalis (approximately 12 wt%), which indicates that there may be a connection between the biomineralization and tyrosine-mediated tanning pathways. These findings provide new evidence for the mineralization of the insect cuticle, which may shed new light on the evolutionary mechanism underlying the divergence of cuticle sclerotization between insects and crustaceans.

Improving the Monitoring of the Walnut Husk Fly (Diptera: Tephritidae) Using Male-Produced Lactones.

It is important to monitor fruit flies (Diptera: Tephritidae) efficiently to implement sustainable means of control. Attractants are often used to increase the efficiency of sticky traps deployed in orchards to monitor Lepidopterans, but remains to be developed to monitor fruit flies. Rhagoletis completa Cresson (Diptera: Tephritidae) is an invasive species in the walnut orchards of Europe, and is commonly monitored with yellow sticky traps. In this study, we collected the volatile compounds released by male and female R. completa, and identified two lactones released exclusively by males. We then formulated both lactones in long-lasting volatile dispensers, and we quantified their release rate over a 26-d period. Finally, during the entire period when female flies are present in the field, we compared the efficiency of the conventional monitoring method using unbaited yellow sticky traps with yellow sticky traps associated with a dispenser releasing both male-produced lactones. These assays were conducted in 54 walnut orchards in France, in 2017. The number of fruit flies caught with sticky traps associated with lactones dispensers was increased by up to 10 times each week. Lactone-baited traps also allowed earlier detection in the season. These field results are promising for R. completa monitoring. A complete chiral identification of these lactones should be performed along with a clarification of their role in the sexual communication of R. completa.

Genome-wide identification of chitinase and chitin deacetylase gene families in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Chitinases (Chts) and chitin deacetylases (CDAs) are important enzymes required for chitin metabolism in insects. In this study, 12 Cht-related genes (including seven Cht genes and five imaginal disc growth factor genes) and 6 CDA genes (encoding seven proteins) were identified in Bactrocera dorsalis using genome-wide searching and transcript profiling. Based on the conserved sequences and phylogenetic relationships, 12 Cht-related proteins were clustered into eight groups (group I-V and VII-IX). Further domain architecture analysis showed that all contained at least one chitinase catalytic domain, however, only four (BdCht5, BdCht7, BdCht8 and BdCht10) possessed chitin-binding domains. The subsequent phylogenetic analysis revealed that seven CDAs were clustered into five groups (group I-V), and all had one chitin deacetylase catalytic domain. However, only six exhibited chitin-binding domains. Finally, the development- and tissue-specific expression profiling showed that transcript levels of the 12 Cht-related genes and 6 CDA genes varied considerably among eggs, larvae, pupae and adults, as well as among different tissues of larvae and adults. Our findings illustrate the structural differences and expression patterns of Cht and CDA genes in B. dorsalis, and provide important information for the development of new pest control strategies based on these vital enzymes.

Epicuticular chemistry reinforces the new taxonomic classification of the Bactrocera dorsalis species complex (Diptera: Tephritidae, Dacinae).

Bactrocera invadens Drew, Tsuruta & White, Bactrocera papayae Drew & Hancock, and Bactrocera philippinensis Drew & Hancock, key pest species within the Bactrocera dorsalis species complex, have been recently synonymized under the name Bactrocera dorsalis (Hendel). The closely related Bactrocera carambolae Drew & Hancock remains as a discrete taxonomic entity. Although the synonymizations have been accepted by most researchers, debate about the species limits remains. Because of the economic importance of this group of taxa, any new information available to support or deny the synonymizations is valuable. We investigated the chemical epicuticle composition of males and females of B. dorsalis, B. invadens, B. papayae, B. philippinensis, and B. carambolae by means of one- and two-dimensional gas chromatography-mass spectrometry, followed by multiple factor analyses and principal component analysis. Clear segregation of complex cuticule profiles of both B. carambolae sexes from B. dorsalis (Hendel) was observed. In addition to cuticular hydrocarbons, abundant complex mixtures of sex-specific oxygenated lipids (three fatty acids and 22 fatty acid esters) with so far unknown function were identified in epicuticle extracts from females of all species. The data obtained supports both taxonomic synonymization of B. invadens, B. papayae, and B. philippinensis with B. dorsalis, as well as the exclusion of B. carambolae from B. dorsalis.

Identification of the Ubiquitous Antioxidant Tripeptide Glutathione as a Fruit Fly Semiochemical.

Many insects mark their oviposition sites with a host marking pheromone (HMP) to deter other females from overexploiting these sites. Previous studies have identified and used HMPs to manage certain fruit fly species; however, few are known for African indigenous fruit flies. The HMP of the African fruit fly, Ceratitis cosyra, was identified as the ubiquitous plant and animal antioxidant tripeptide, glutathione (GSH). GSH was isolated from the aqueous extract of adult female fecal matter and characterized by LC-QTOF-MS. GSH level increased with increasing age of female fecal matter, with highest concentration detected from 2-week-old adult females. Additionally, GSH levels were 5-10-times higher in fecal matter than in the ovipositor or hemolymph extracts of females. In bioassays, synthetic GSH reduced oviposition responses in conspecifics of C. cosyra and the heterospecific species C. rosa, C. fasciventris, C. capitata, and Zeugodacus cucurbitae. These results represent the first report of a ubiquitous antioxidant as a semiochemical in insects and its potential use in fruit fly management.

Electrospun Micro/Nanofibers as Controlled Release Systems for Pheromones of Bactrocera oleae and Prays oleae.

New systems for the controlled release of 1,7-dioxaspiro[5.5]undecane and (Z)-7-tetradecenal, the sex pheromones of olive fruit fly, Bactrocera oleae, and olive moth, Prays oleae, respectively, were developed utilizing electrospun micro/nanofiber matrices from inexpensive, biodegradable polymers, namely polycaprolactone, cellulose acetate and polyhydroxybutyrate. The incorporation of the pheromones in 5, 10 and 20% w/w in the electrospinning polymer blends allowed for the production of fiber mats with variable loading levels and release rates, ensuring however in all cases the release of pheromones for more than 16 weeks. Laboratory bioassays and field trapping tests showed that the fiber mats obtained from electrospinning of polyhydroxybutyrate solution containing 5% w/w 1,7-dioxaspiro[5.5]undecane and polycaprolactone solution containing 5% w/w (Z)-7-tetradecenal were almost twice as effective in attracting B. oleae and P. oleae males, respectively, in comparison to the positive controls used.

Sex Pheromone of Anastrepha striata.

The guava fruit fly, Anastrepha striata, is a pest of several cultivated species of Myrtaceae in the American tropics and subtropics. During calling, A. striata males release numerous volatiles. This study was conducted to identify which of the male volatiles function as the A. striata sex pheromone and to investigate the effects of age and time of day on the emission of pheromone components. Analysis of the volatiles from males collected by solid phase microextraction using gas chromatography coupled to electroantennographic detection (GC-EAD) showed that three volatile compounds elicited repeatable responses from the antennae of females. The EAD-active compounds were identified by GC/mass spectrometry as ethyl hexanoate, linalool, and ethyl octanoate. In two-choice tests using Multilure traps placed in field cages, traps baited with live males, ethyl hexanoate, or the three-component blend captured more females than unbaited traps. However, there was no difference in catches when traps baited with live males were compared against traps baited with ethyl hexanoate. Although traps baited with the three-component blend caught more females than traps baited with live males, the difference was not significant. Analyses of pheromonal components released by A. striata males 8 to 26 days old showed that there was an effect of age on pheromone production and also a significant effect of time of day on pheromone emission. Release of the volatile compounds occurred from 14.00 to 18.00 hr, although traces of linalool were detected from 08.00 hr. Peak emission of pheromone compounds occurred at 14.00 hr.

Comparative proteomic analysis of Bactrocera dorsalis (Hendel) in response to thermal stress.

Temperature is one of the most important environmental variables affecting growth, reproduction and distribution of insects. The rise of comparative proteomics provides a powerful tool to explore the response in proteins to thermal stress. As an important worldwide pest, the oriental fruit fly Bactrocera dorsalis causes severe economic losses to crops. To understand the response of B. dorsalis to thermal stress, we performed a comparative proteome analysis of this insect after exposure to extreme low and high temperatures using two-dimensional electrophoresis. Among the separated proteins, 51 diverse protein spots were present differently in response to extreme temperatures. Using tandem mass spectrometry sequencing analysis 39 proteins were successfully identified, which included 13 oxidoreductases, 10 binding proteins, 5 transferases, and 2 each of lyases, isomerases, ligases, and developmental proteins. Subsequently, the expression of these protein transcripts was studied by RT-qPCR to validate the proteomic results. In conclusion, this study provides a first look into the thermal stress response of B. dorsalis at the protein level, and thus it paves the way for further functional studies in the physiological mechanism related to thermal stress.

Conserved metallomics in two insect families evolving separately for a hundred million years.

Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.

Resolution of three cryptic agricultural pests (Ceratitis fasciventris, C. anonae, C. rosa, Diptera: Tephritidae) using cuticular hydrocarbon profiling.

Discrimination of particular species within the species complexes of tephritid fruit flies is a very challenging task. In this fruit-fly family, several complexes of cryptic species have been reported, including the African cryptic species complex (FAR complex). Cuticular hydrocarbons (CHCs) appear to be an excellent tool for chemotaxonomical discrimination of these cryptic species. In the present study, CHC profiles have been used to discriminate among three important agricultural pests from the FAR complex, Ceratitis fasciventris, Ceratitis anonae and Ceratitis rosa. Hexane body surface extracts of mature males and females were analyzed by two-dimensional gas chromatography with mass spectrometric detection and differences in CHC profiles between species and sexes tested through multivariate statistics and compared with species identification by means of microsatellite markers. Quantitative as well as qualitative CHC profile differences between sexes and species are reported. The CHC profiles consisted of a mixture of linear, internally methyl-branched and mono-, di- and tri-unsaturated alkanes. Twelve compounds were pinpointed as potential chemotaxonomical markers. The present study shows that presence or absence of particular CHCs might be used in the chemical diagnosis of the FAR complex. Moreover, our results represent an important first step in the development of a useful chemotaxonomic tool for cryptic species identification of these important agricultural pests.

Oxidative carbon-carbon bond cleavage is a key step in spiroacetal biosynthesis in the fruit fly Bactrocera cacuminata.

The early steps of spiroacetal biosynthesis in the fruit fly Bactrocera cacuminata (Solanum fly) have been investigated using a series of deuterium-labeled, oxygenated fatty acid like compounds. These potential spiroacetal precursors were administered to male flies, and their volatile emissions were analyzed for specific deuterium incorporation by GC/MS. This has allowed the order of early oxidative events in the biosynthetic pathway to be determined. Together with the already well-established later steps, the results of these in vivo investigations have allowed essentially the complete delineation of the spiroacetal biosynthetic pathway, beginning from products of primary metabolism. A fatty acid equivalent undergoes a series of enzyme-mediated oxidations leading to a trioxygenated fatty acid like species that includes a vicinal diol. This moiety then undergoes enzyme-mediated oxidative carbon-carbon bond cleavage as the key step to generate the C9 unit of the final spiroacetal. This is the first time such an oxidative transformation has been reported in insects. A final hydroxylation step is followed by spontaneous spiro-cyclization. This distinct pathway adds further to the complexity and diversity of biosynthetic pathways to spiroacetals.

Seasonal accumulation of acetylated triacylglycerols by a freeze-tolerant insect.

Most animals store energy as long-chain triacylglycerols (lcTAGs). Trace amounts of acetylated triacylglycerols (acTAGs) have been reported in animals, but are not accumulated, likely because they have lower energy density than lcTAGs. Here we report that acTAGs comprise 36% of the neutral lipid pool of overwintering prepupae of the goldenrod gall fly, Eurosta solidaginis, while only 17% of the neutral lipid pool is made up of typical lcTAGs. These high concentrations of acTAGs, present only during winter, appear to be synthesized by E. solidaginis and are not found in other freeze-tolerant insects, nor in the plant host. The mixture of acTAGs found in E. solidaginis has a significantly lower melting point than equivalent lcTAGs, and thus remains liquid at temperatures at which E. solidaginis is frozen in the field, and depresses the melting point of aqueous solutions in a manner unusual for neutral lipids. We note that accumulation of acTAGs coincides with preparation for overwintering and the seasonal acquisition of freeze tolerance. This is the first observation of accumulation of acTAGs by an animal, and the first evidence of dynamic interconversion between acTAGs and lcTAGs during development and in response to stress.

Computational reverse chemical ecology: virtual screening and predicting behaviorally active semiochemicals for Bactrocera dorsalis.

BACKGROUND: Semiochemical is a generic term used for a chemical substance that influences the behaviour of an organism. It is a common term used in the field of chemical ecology to encompass pheromones, allomones, kairomones, attractants and repellents. Insects have mastered the art of using semiochemicals as communication signals and rely on them to find mates, host or habitat. This dependency of insects on semiochemicals has allowed chemical ecologists to develop environment friendly pest management strategies. However, discovering semiochemicals is a laborious process that involves a plethora of behavioural and analytical techniques, making it expansively time consuming. Recently, reverse chemical ecology approach using odorant binding proteins (OBPs) as target for elucidating behaviourally active compounds is gaining eminence. In this scenario, we describe a "computational reverse chemical ecology" approach for rapid screening of potential semiochemicals. RESULTS: We illustrate the high prediction accuracy of our computational method. We screened 25 semiochemicals for their binding potential to a GOBP of B. dorsalis using molecular docking (in silico) and molecular dynamics. Parallely, compounds were subjected to fluorescent quenching assays (Experimental). The correlation between in silico and experimental data were significant (r2 = 0.9408; P < 0.0001). Further, predicted compounds were subjected to behavioral bioassays and were found to be highly attractive to insects. CONCLUSIONS: The present study provides a unique methodology for rapid screening and predicting behaviorally active semiochemicals. This methodology may be developed as a viable approach for prospecting active semiochemicals for pest control, which otherwise is a laborious process.

Characterization of olfactory sensilla of the olive fly: behavioral and electrophysiological responses to volatile organic compounds from the host plant and bacterial filtrate.

The responses of olive fly (Bactrocera oleae) antennal and palpal olfactory receptors to odors emitted by Pseudomonas putida bacterial filtrate and to volatiles from a host plant were evaluated using electrophysiological and behavioral bioassays. Morphological identification of olfactory receptors was also performed. The third antennal segment (flagellum) bears four types of multiporous sensilla: trichoid, short basiconica, clavate and grooved. Maxillary palps have mechanosensory bristles and multiporous basiconica sensilla. In wind-tunnel bioassays, olive fly responses to volatiles emitted by bacterial filtrate were higher than those to culture medium. Bacterial filtrate was more attractive than ammonium carbonate or a mixture of ethyl acetate and acetic acid in ethanol. GC-MS of bacterial filtrate identified some of the chemicals produced by bacterial activity, including methyl thiolacetate, ammonia, 2-pentanone, 2-heptanone, ethyl tiglate and methyl thiocyanate. Electrophysiological investigations proved that antennal sensilla are responsive to bacterial filtrate odor, methyl thiolacetate, olive leaves and olives, as well as to α-pinene, while acetic acid elicited an inhibitory response. Electropalpgrams recorded a specific response to bacterial filtrate by mated males and females, as well as a dose-dependent response relationship to methyl thiolacetate by mated females. The identification of new active volatile compounds in the semiochemical system of the olive fly is promising for the development of innovative control strategies in area-wide management.