Cuticular Hydrocarbon Biosynthesis Research Articles

Insulin signaling in female Drosophila links diet and sexual attractiveness.

Appropriate sexual selection or individual sexual attractiveness is closely associated with the reproductive success of a species. Here, we report that young male flies exhibit innate courtship preference for female flies that are raised on higher-yeast diets and that have greater body weight and fecundity, but reduced locomotor activity and shortened lifespan. Male flies discriminate among females that have been fed diets that contain 3 different yeast concentrations-1, 5, and 20% yeast- via gustatory, but not visual or olfactory, perception. Female flies that are raised on higher-yeast diets exhibit elevated expression levels of Drosophila insulin-like peptides (di lps), and we demonstrate that hypomorphic mutations of di lp2, 3, 5 or foxo, as well as oenocyte-specific gene disruption of the insulin receptor, all abolish this male courtship preference for high yeast-fed females. Moreover, our data demonstrate that disrupted di lp signaling can alter the expression profile of some cuticular hydrocarbons (CHCs) in female flies, and that genetic inhibition of an enzyme involved in the biosynthesis of CHCs in oenocytes, elongase F, also eliminates the male courtship preference. Together, our findings provide mechanistic insights that link female reproductive potential to sexual attractiveness, thereby encouraging adaptive mating and optimal reproductive success.-Lin, W.-S., Yeh, S.-R., Fan, S.-Z., Chen, L.-Y., Yen, J.-H., Fu, T.-F., Wu, M.-S., Wang, P.-Y. Insulin signaling in female Drosophila links diet and sexual attractiveness.

Regulation of cuticular hydrocarbon profile maturation by Drosophila tanning hormone, bursicon, and its interaction with desaturase activity

Shortly after emergence the exoskeleton (cuticle) of adult insects is rapidly expanded, hardened (sclerotized), and pigmented (melanized). In parallel with this process, the oenocytes, which are large polyploid cells located below the abdominal epidermis, secrete onto the cuticle a cocktail of cuticular hydrocarbons (CHs) and waxes. These improve the waterproofing of the cuticle, and also provide important chemosensory and pheromonal cues linked with gender, age, and species differentiation. The hardening and pigmentation of the new cuticle are controlled by the neurohormone, bursicon, and its receptor, encoded by the DLGR2 receptor, rickets (rk); by contrast, little is known about the timecourse of changes in CH profile and about the role of bursicon in this process. Here we show in Drosophila that rk function is also required for the normal maturation of the fly's CH profile, with flies mutant for rk function showing dramatically elevated levels of CHs. Interestingly, this effect is mostly abrogated by mutations in the Δ9 desaturase encoded by the desaturase1 gene, which introduces a first double bond into elongated fatty-acid chains, suggesting that desaturase1 acts downstream of rk. In addition, flies mutant for rk showed changes in the absolute and relative levels of specific 7-monoenes (in males) and 7,11-dienes (in females). The fact that these differences in CH amounts were obtained using extractions of very different durations suggests that the particular CH profile of flies mutant for rk is not simply due to their unsclerotized cuticle but that bursicon may be involved in the process of CH biosynthesis itself.

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.

Polymorphisms in a desaturase 2 ortholog associate with cuticular hydrocarbon and male mating success variation in a natural population of Drosophila serrata.

Elucidating the nature of genetic variation underlying both sexually selected traits and the fitness components of sexual selection is essential to understanding the broader consequences of sexual selection as an evolutionary process. To date, there have been relatively few attempts to connect the genetic variance in sexually selected traits with segregating DNA sequence polymorphisms. We set out to address this in a well-characterized sexual selection system--the cuticular hydrocarbons (CHCs) of Drosophila serrata--using an indirect association study design that allowed simultaneous estimation of the genetic variance in CHCs, sexual fitness and single nucleotide polymorphism (SNP) effects in an outbred population. We cloned and sequenced an ortholog of the D. melanogaster desaturase 2 gene, previously shown to affect CHC biosynthesis in D. melanogaster, and associated 36 SNPs with minor allele frequencies > 0.02 with variance in CHCs and sexual fitness. Three SNPs had significant multivariate associations with CHC phenotype (q-value < 0.05). At these loci, minor alleles had multivariate effects on CHCs that were weakly associated with the multivariate direction of sexual selection operating on these traits. Two of these SNPs had pleiotropic associations with male mating success, suggesting these variants may underlie responses to sexual selection due to this locus. There were 15 significant male mating success associations (q-value < 0.1), and interestingly, we detected a nonrandom pattern in the relationship between allele frequency and direction of effect on male mating success. The minor-frequency allele usually reduced male mating success, suggesting a positive association between male mating success and total fitness at this locus.

The effect of dietary fatty acids on the cuticular hydrocarbon phenotype of an herbivorous insect and consequences for mate recognition.

The cuticular hydrocarbon (CHC) profile of the mustard leaf beetle Phaedon cochleariae is known to mediate mate recognition and is dependent on food plant species; beetles previously were shown to prefer mates that fed on the same plant species and which have a similar CHC pattern. In order to elucidate whether the pattern of ingested fatty acids affects the CHC pattern of P. cochleariae adults, we fed beetles: (a) with two different host plant species differing in fatty acid profile; and (b) artificial diets differing mainly in their composition of mono-, di-, and triunsaturated fatty acids. Analyses of the beetles' CHCs revealed that ingestion of different fatty acid blends results in quantitative effects on the beetle's straight-chain and methyl-branched CHCs. Interestingly, CHC patterns of males and females were affected differently by ingestion of fatty acids. In contrast to the effect on mating caused by feeding on different host plant species, beetles that were fed with different artificial diets, leading to different beetle CHC profiles, did not exhibit mating preference. We suggest that the occurrence of CHC-dependent assortative mating in P. cochleariae does not depend on the dietary fatty acids offered to the beetles in this study, but on other food constituents that affect CHC biosynthesis.

Exoskeleton formation in Apis mellifera: Cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development

Cuticular hydrocarbons (CHCs) are abundant in the superficial cuticular layer (envelope) of insects where they play roles as structural, anti-desiccation and semiochemical compounds. Many studies have investigated the CHC composition in the adult insects. However, studies on the profiles of these compounds during cuticle formation and differentiation are scarce and restrict to specific stages of a few insect species. We characterized the CHCs developmental profiles in the honeybee workers during an entire molting cycle (from pupal-to-adult ecdyses) and in mature adults (forager bees). Gas chromatography/mass spectrometry (GC/MS) analysis revealed remarkable differences in the relative quantities of CHCs, thus discriminating pupae, developing and newly-ecdysed adults, and foragers from each other. In parallel, the honeybee genome database was searched for predicted gene models using known amino acid sequences of insect enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases) as queries in BLASTP analysis. The expression levels of six desaturase genes and ten elongase genes potentially involved in CHC biosynthesis were determined by reverse transcription and real time polymerase chain reaction (RT-qPCR) in the developing integument (cuticle and subjacent epidermis). Aiming to predict roles for these genes in CHC biosynthesis, the developmental profiles of CHCs and desaturase/elongase transcript levels were evaluated using Spearman correlation coefficient. This analysis pointed to differential roles for these gene products in the biosynthesis of certain CHC classes. Based on the assumption that homologous proteins may share a similar function, phylogenetic trees were reconstructed as an additional strategy to predict functions and evolutionary relationships of the honeybee desaturases and elongases. Together, these approaches highlighted the molecular complexity underlying the formation of the lesser known layer of the cuticular exoskeleton, the envelope.

Effects of immunostimulation on social behavior, chemical communication and genome-wide gene expression in honey bee workers (Apis mellifera)

BackgroundSocial insects, such as honey bees, use molecular, physiological and behavioral responses to combat pathogens and parasites. The honey bee genome contains all of the canonical insect immune response pathways, and several studies have demonstrated that pathogens can activate expression of immune effectors. Honey bees also use behavioral responses, termed social immunity, to collectively defend their hives from pathogens and parasites. These responses include hygienic behavior (where workers remove diseased brood) and allo-grooming (where workers remove ectoparasites from nestmates). We have previously demonstrated that immunostimulation causes changes in the cuticular hydrocarbon profiles of workers, which results in altered worker-worker social interactions. Thus, cuticular hydrocarbons may enable workers to identify sick nestmates, and adjust their behavior in response. Here, we test the specificity of behavioral, chemical and genomic responses to immunostimulation by challenging workers with a panel of different immune stimulants (saline, Sephadex beads and Gram-negative bacteria E. coli).ResultsWhile only bacteria-injected bees elicited altered behavioral responses from healthy nestmates compared to controls, all treatments resulted in significant changes in cuticular hydrocarbon profiles. Immunostimulation caused significant changes in expression of hundreds of genes, the majority of which have not been identified as members of the canonical immune response pathways. Furthermore, several new candidate genes that may play a role in cuticular hydrocarbon biosynthesis were identified. Effects of immune challenge expression of several genes involved in immune response, cuticular hydrocarbon biosynthesis, and the Notch signaling pathway were confirmed using quantitative real-time PCR. Finally, we identified common genes regulated by pathogen challenge in honey bees and other insects.ConclusionsThese results demonstrate that honey bee genomic responses to immunostimulation are substantially broader than the previously identified canonical immune response pathways, and may mediate the behavioral changes associated with social immunity by orchestrating changes in chemical signaling. These studies lay the groundwork for future research into the genomic responses of honey bees to native honey bee parasites and pathogens.

Genetics of cuticular hydrocarbon differences between males of the parasitoid wasps Nasonia giraulti and Nasonia vitripennis

Many insects rely on cuticular hydrocarbons (CHCs) as major recognition signals between individuals. Previous research on the genetics of CHCs has focused on Drosophila in which the roles of three desaturases and one elongase were highlighted. Comparable studies in other insect taxa have not been conducted so far. Here, we explore the genetics of CHCs in hybrids of the jewel wasps Nasonia giraulti and Nasonia vitripennis. We analyzed the CHC profiles of pure strain and of F(2) hybrid males using gas chromatography coupled with mass spectrometry and distinguished 54 peaks, of which we identified 52 as straight-chain, monounsaturated, or methyl-branched CHCs. The latter compound class proved to be particularly abundant and diverse in Nasonia. Quantitative trait locus (QTL) analysis suggests fixed genetic differences between the two strains in 42 of the 54 studied traits, making Nasonia a promising genetic model for identifying genes involved in CHC biosynthesis. QTL for methyl-branched CHCs partly clustered in genomic regions with high recombination rate: a possible indication for pleiotropic genes that control their biosynthesis, which is largely unexplored so far. Finally, we identified and mapped genes in the Nasonia genome with high similarity to genes that have been implicated in alkene biosynthesis in Drosophila and discuss those that match in their position with predicted QTL for alkenes.

Identification of quantitative trait loci function through analysis of multiple cuticular hydrocarbons differing between Drosophila simulans and Drosophila sechellia females

The genetics of sexual isolation, behavioral differences between species that prevent mating, is understood poorly. Pheromonal differences between species can influence sexual isolation in many animals and in some cases a single locus can cause large functional changes in pheromonal mating signals. Drosophila cuticular hydrocarbons (CHCs) can function as pheromones and consequently affect mate recognition. In a previous study of the two major CHCs in females that affect mating discrimination between Drosophila simulans and D. sechellia, quantitative trait loci (QTL) were identified on the X and third chromosome, and a few candidate genes were potentially implicated. Here we specifically test candidate genes for CHC biosynthesis and determine the genetic architecture of four additional CHCs that differ in abundance between D. simulans and D. sechellia females. The same QTL, and new ones, were found for additional CHCs. By examining all these CHCs and exploring their covariance, we were able to ascribe putative function to the major QTL. Although desaturases have received considerable attention for their role in CHC biosynthesis, evidence here implies that elongases may be just as important. Sex determination genes do not seem to have a role in this species difference although D. sechellia is sexually dimorphic in CHCs, whereas D. simulans is not. Epistatic interactions, only detected for CHCs limited to D. sechellia, imply that complex interactions among loci may also be having a role in these compounds that affect mating isolation.

Evolution of a desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis and courtship behavior in Drosophila

Drosophila species exhibit polymorphism in female pheromonal cuticular hydrocarbons, with 7-monoenes produced in Drosophila simulans and 7,11-dienes in most populations of Drosophila melanogaster (5,9-dienes in several African populations). A female-biased desaturase, desatF, expressed only in D. melanogaster is involved in the synthesis of 7,11-dienes. We investigated the role of desatF in 5,9-diene flies. We constructed a 5,9-diene strain knock-down for desatF and showed that desatF is involved in 5,9-diene formation. We also studied D. melanogaster/D. simulans hybrids. These hybrid females produced dienes and received normal courtship from D. melanogaster males, but copulation success was reduced. With D. simulans males, courtship was decreased and no copulation occurred. Hybrids with a chromosomal deletion of the D. melanogaster desatF gene had no dienes and received normal courtship from D. simulans males; depending on the D. simulans parental strain, 7–19% of them succeeded in mating. D. simulans desatF promoter region shows 21–23% gaps and 86–89% identity with D. melanogaster promoter region, the coding region 93–94% identity, depending on the strain. These differences could explain the functional polymorphism of desatF observed between both species, contributing to different cuticular hydrocarbon profiles, that constitute an effective barrier between species.

Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster

The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults.

Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster

The biosynthesis of cuticular hydrocarbons was investigated in male and female Drosophila melanogaster (Canton-S strain), especially in those with a pheromonal role i.e. male 7-tricosene and female 7, 11- heptacosadiene. The incorporation of radioactivity was followed after topical application of 14C-labelled myristic, palmitic and stearic acid and 3H-labelled cis-vaccenic acid on one to ten day old flies. The incorporation levels into unsaturated hydrocarbons are similar in both sexes, depending markedly on the chain length of the saturated precursor, with a maximum level from myristic acid. Cis-vaccenic acid leads only to unsaturated compounds. With this precursor, there is an enhanced incorporation into female monoenes and dienes, maximum in two to three day old females. The total fatty acid composition shows the highest abundance of fatty acids with 16 carbon atoms and the presence of a major position for double bond, Δ9. Moreover, cis-vaccenic acid and 17-tetracosenoic acid are identified by GC–MS analysis. These data support an elongation–decarboxylation mechanism for the biosynthesis of D. melanogaster cuticular hydrocarbons. Its early steps for male monoenes and female monoenes and dienes might involve a Δ9 desaturase transforming palmitic acid into palmitoleic acid which would then be elongated into vaccenic acid, an important common precursor for all pheromones.

Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly Sarcophaga bullata

Ecdysterone has been shown to stimulate hydrocarbon biosynthesis in Sarcophaga bullata at pupariation. When post-feeding larva were treated with 3H-acetate 10·5 hr after hormone administration, a 1·3 times greater quantity of 3H-acetate was incorporated into hydrocarbon in the ecdysterone injected insects than controls. A similar experiment with a 24 hr delay of 3H-acetate administration following hormone treatment resulted in 3·3 times greater incorporation into hydrocarbon of treated animals. Isolated integuments synthesize hydrocarbon from acetate better than internal tissues, and the integuments of ecdysterone-treated insects incorporate acetate into hydrocarbon 9·8 times better than integuments of control insects. This indicates that cuticular hydrocarbon biosynthesis not only occurs in the integument, but that a locus of regulation is present in the integument.