Phenotypic Plasticity of Cuticular Hydrocarbon Profiles in Insects.

The insect integument is covered by cuticular hydrocarbons (CHCs) which provide protection against environmental stresses, but are also used for communication. Here we review current knowledge on environmental and insect-internal factors which shape phenotypic plasticity of solitary living insects, especially herbivorous ones. We address the dynamics of changes which may occur within minutes, but may also last weeks, depending on the species and conditions. Two different modes of changes are suggested, i.e. stepwise and gradual. A switch between two distinct environments (e.g. host plant switch by phytophagous insects) results in stepwise formation of two distinct adaptive phenotypes, while a gradual environmental change (e.g. temperature gradients) induces a gradual change of numerous adaptive CHC phenotypes. We further discuss the ecological and evolutionary consequences of phenotypic plasticity of insect CHC profiles by addressing the question at which conditions is CHC phenotypic plasticity beneficial. The high plasticity of CHC profiles might be a trade-off for insects using CHCs for communication. We discuss how insects cope with the challenge to produce and "understand" a highly plastic, environmentally dependent CHC pattern that conveys reliable and comprehensible information. Finally, we outline how phenotypic plasticity of CHC profiles may promote speciation in insects that rely on CHCs for mate recognition.

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.

Inter- and Intrapopulation Variability in the Composition of Larval Defensive Secretions of Willow-Feeding Populations of the Leaf Beetle Chrysomela lapponica.

We explored the inter- and intrapopulation variability in the larval defensive chemistry of the leaf beetle Chrysomela lapponica with respect to the salicylic glycoside (SG) content of its host species. Secretions of larvae from three populations associated in nature with SG-poor willows contained nearly twice as many components and 40-fold higher concentrations of autogenously produced isobutyrates and 2-methylbutyrates than secretions of larvae from three populations associated with SG-rich willows, which in turn had 200-fold higher concentrations of host-derived salicylaldehyde. Reciprocal transfer experiments showed that the larvae from populations associated with SG-rich willows did not produce appreciable amounts of butyrates on either SG-rich or SG-poor willows, while populations feeding on several SG-poor willow species retained the ability for efficient sequestration of SGs, along with their ability to produce high amounts of isobutyrates and 2-methylbutyrates. Only the populations associated with SG-poor willows demonstrated among-family variation in the composition of defensive secretion and differential responses of individual families to willows with alternative SG levels, which can be seen as the prerequisites for shifting to novel hosts. These non-specialized populations show a dual defensive strategy, which corresponds to the ancestral state of this species, while populations that fully depend on host-derived toxins (feeding on SG-rich willows) or have lost the ability to produce salicylaldehyde (feeding on birches) are most deviant from the ancestral state. The results indicate that defensive strategies may differ between populations within a species, and suggest that this variation reduces extinction risks and maintains the high ecological diversity and wide distribution of C. lapponica.

Cuticular extracts from Acromis sparsa (Coleoptera: Cassidinae) mediate arrestment behavior of the commensal canestriniid mite Grandiella rugosita.

Astigmatid mites in the family Canestriniidae are often closely associated with tortoise leaf beetles (Chrysomelidae: Cassidinae). For example, the survival of the commensal canestriniid mite Grandiella rugosita depends on dispersal to the cassidine beetle Acromis sparsa. Here, we tested whether the beetle cuticle provides chemical cues for host recognition for G. rugosita. In two-choice assays with cuticular extracts from A. sparsa and the co-occurring, non-host cassidine Chelymorpha alternans offered simultaneously, mites clearly preferred the area treated with extract from their host. In no-choice assays, G. rugosita spent three times longer and moved three times slower on host cuticular extracts compared to non-host extracts and the solvent control. Analyses of the chemical composition of cuticular extracts from males and females of A. sparsa and C. alternans revealed complex mixtures of mainly methyl branched hydrocarbons, which clearly separated both species in a principal component analysis. We found no qualitative difference between males and females of either species, but in C. alternans quantitative differences between males and females were detected. Our results demonstrate that G. rugosita is able to discriminate between cuticular extracts from its host A. sparsa and the non-host C. alternans. The components eliciting the observed arrestment behavior remain to be determined.

Looking for a similar partner: host plants shape mating preferences of herbivorous insects by altering their contact pheromones.

The role of phenotypical plasticity in ecological speciation and the evolution of sexual isolation remains largely unknown. We investigated whether or not divergent host plant use in an herbivorous insect causes assortative mating by phenotypically altering traits involved in mate recognition. We found that males of the mustard leaf beetle Phaedon cochleariae preferred to mate with females that were reared on the same plant species to females provided with a different plant species, based on divergent cuticular hydrocarbon profiles that serve as contact pheromones. The cuticular hydrocarbon phenotypes of the beetles were host plant specific and changed within 2 weeks after a shift to a novel host plant species. We suggest that plant-induced phenotypic divergence in mate recognition cues may act as an early barrier to gene flow between herbivorous insect populations on alternative host species, preceding genetic divergence and thus, promoting ecological speciation.

Evidence for damage-dependent hygienic behaviour towards Varroa destructor-parasitised brood in the western honey bee, Apis mellifera.

The ectoparasitic mite Varroa destructor and honey bee pathogenic viruses have been implicated in the recent demise of honey bee colonies. Several studies have shown that the combination of V. destructor and deformed wing virus (DWV) poses an especially serious threat to honey bee health. Mites transmitting virulent forms of DWV may cause fatal DWV infections in the developing bee, while pupae parasitised by mites not inducing or activating overt DWV infections may develop normally. Adult bees respond to brood diseases by removing affected brood. This hygienic behaviour is an essential part of the bees' immune response repertoire and is also shown towards mite-parasitised brood. However, it is still unclear whether the bees react towards the mite in the brood cell or rather towards the damage done to the brood. We hypothesised that the extent of mite-associated damage rather than the mere presence of parasitising mites triggers hygienic behaviour. Hygienic behaviour assays performed with mites differing in their potential to transmit overt DWV infections revealed that brood parasitised by 'virulent' mites (i.e. mites with a high potential to induce fatal DWV infections in parasitised pupae) were removed significantly more often than brood parasitised by 'less virulent' mites (i.e. mites with a very low potential to induce overt DWV infections) or non-parasitised brood. Chemical analyses of brood odour profiles suggested that the bees recognise severely affected brood by olfactory cues. Our results suggest that bees show selective, damage-dependent hygienic behaviour, which may be an economic way for colonies to cope with mite infestation.

Insect egg deposition induces indirect defense and epicuticular wax changes in Arabidopsis thaliana.

Egg deposition by the Large Cabbage White butterfly Pieris brassicae on Brussels sprouts plants induces indirect defense by changing the leaf surface, which arrests the egg parasitoid Trichogramma brassicae. Previous studies revealed that this indirect defense response is elicited by benzyl cyanide (BC), which is present in the female accessory reproductive gland (ARG) secretion and is released to the leaf during egg deposition. Here, we aimed (1) to elucidate whether P. brassicae eggs induce parasitoid-arresting leaf surface changes in another Brassicacean plant, i.e., Arabidopsis thaliana, and, if so, (2) to chemically characterize the egg-induced leaf surface changes. Egg deposition by P. brassicae on A. thaliana leaves had similar effects to egg deposition on Brussels sprouts with respect to the following: (a) Egg deposition induced leaf surface changes that arrested T. brassicae egg parasitoids. (b) Application of ARG secretion of mated female butterflies or of BC to leaves had the same inductive effects as egg deposition. Based on these results, we conducted GC-MS analysis of leaf surface compounds from egg- or ARG-induced A. thaliana leaves. We found significant quantitative differences in epicuticular waxes compared to control leaves. A discriminant analysis separated surface extracts of egg-laden, ARG-treated, untreated control and Ringer solution-treated control leaves according to their quantitative chemical composition. Quantities of the fatty acid tetratriacontanoic acid (C34) were significantly higher in extracts of leaf surfaces arresting the parasitoids (egg-laden or ARG-treated) than in respective controls. In contrast, the level of tetracosanoic acid (C24) was lower in extracts of egg-laden leaves compared to controls. Our study shows that insect egg deposition on a plant can significantly affect the quantitative leaf epicuticular wax composition. The ecological relevance of this finding is discussed with respect to its impact on the behavior of egg parasitoids.

The role of cuticular hydrocarbons in male mating behavior of the mustard leaf beetle, Phaedon cochleariae (F.).

We investigated the role that cuticular hydrocarbons (CHC) play in sexual communication by the mustard leaf beetle, Phaedon cochleariae (Coleoptera: Chrysomelidae). In laboratory bioassays, male P. cochleariae attempted to copulate with living or freeze-killed females as often as with males. However, the duration of copulation with females was longer than with males. To elucidate the impact of CHC on this behavior, cuticular compounds of adults of both sexes were extracted with dichloromethane. Male mating attempts with glass beads treated with the dichloromethane extract were nearly as frequent as with living beetles. The dichloromethane extract was fractionated by silica gel chromatography, and the biological activity of the fractions was tested by applying them to glass beads. A non-polar hexane fraction significantly elicited mating behavior, whereas the polar methanol fraction did not, likely because it contained defensive compounds from exocrine glands located in the elytra and pronota. Interestingly, a mixture of both the non-polar and polar fraction tended to elicit more mating attempts than did the non-polar hexane fraction alone. Further fractionation of the significantly active hexane fraction by silver nitrate column chromatography revealed that saturated CHC elicited mating behavior, but the olefins did not. GC-MS analyses of dichloromethane cuticular extracts showed that the male and female CHC profiles were qualitatively identical, but differed in their relative composition. Canonical discriminant analysis showed that CHC profiles of males and females formed separate clusters. Nevertheless, the results of our bioassays demonstrated that male and female CHC did not elicit sex discriminative male behavior, but induced mating by males regardless of the sex of the partner.

Phenotypic plasticity of mate recognition systems prevents sexual interference between two sympatric leaf beetle species.

Maladaptive sexual interactions among heterospecific individuals (sexual interference) can prevent the coexistence of animal species. Thus, the avoidance of sexual interference by divergence of mate recognition systems is crucial for a stable coexistence in sympatry. Mate recognition systems are thought to be under tight genetic control. However, we demonstrate that mate recognition systems of two closely related sympatric leaf beetle species show a high level of host-induced phenotypic plasticity. Mate choice in the mustard leaf beetles, Phaedon cochleariae and P. armoraciae, is mediated by cuticular hydrocarbons (CHCs). Divergent host plant use causes a divergence of CHC phenotypes, whereas similar host use leads to their convergence. Consequently, both species exhibit significant behavioral isolation when they feed on alternative host species, but mate randomly when using a common host. Thus, sexual interference between these syntopic leaf beetles is prevented by host-induced phenotypic plasticity rather than by genotypic divergence of mate recognition systems.

Divergence of cuticular hydrocarbons in two sympatric grasshopper species and the evolution of fatty acid synthases and elongases across insects.

Cuticular hydrocarbons (CHCs) play a major role in the evolution of reproductive isolation between insect species. The CHC profiles of two closely related sympatric grasshopper species, Chorthippus biguttulus and C. mollis, differ mainly in the position of the first methyl group in major methyl-branched CHCs. The position of methyl branches is determined either by a fatty acid synthase (FAS) or by elongases. Both protein families showed an expansion in insects. Interestingly, the FAS family showed several lineage-specific expansions, especially in insect orders with highly diverse methyl-branched CHC profiles. We found five putative FASs and 12 putative elongases in the reference transcriptomes for both species. A dN/dS test showed no evidence for positive selection acting on FASs and elongases in these grasshoppers. However, one candidate FAS showed species-specific transcriptional differences and may contribute to the shift of the methyl-branch position between the species. In addition, transcript levels of four elongases were expressed differentially between the sexes. Our study indicates that complex methyl-branched CHC profiles are linked to an expansion of FASs genes, but that species differences can also mediated at the transcriptional level.

Pre-exposure of Arabidopsis to the abiotic or biotic environmental stimuli "chilling" or "insect eggs" exhibits different transcriptomic responses to herbivory.

Plants can retain information about environmental stress and thus, prepare themselves for impending stress. In nature, it happens that environmental stimuli like 'cold' and 'insect egg deposition' precede insect herbivory. Both these stimuli are known to elicit transcriptomic changes in Arabidposis thaliana. It is unknown, however, whether they affect the plant's anti-herbivore defence and feeding-induced transcriptome when they end prior to herbivory. Here we investigated the transcriptomic response of Arabidopsis to feeding by Pieris brassicae larvae after prior exposure to cold or oviposition. The transcriptome of plants that experienced a five-day-chilling period (4 °C) was not fully reset to the pre-chilling state after deacclimation (20 °C) for one day and responded differently to herbivory than that of chilling-inexperienced plants. In contrast, when after a five-day-lasting oviposition period the eggs were removed, one day later the transcriptome and, consistently, also its response to herbivory resembled that of egg-free plants. Larval performance was unaffected by previous exposure of plants to cold and to eggs, thus indicating P. brassicae tolerance to cold-mediated plant transcriptomic changes. Our results show strong differences in the persistence of the plant's transcriptomic state after removal of different environmental cues, and consequently differential effects on the transcriptomic response to later herbivory.

Priming and memory of stress responses in organisms lacking a nervous system.

Experience and memory of environmental stimuli that indicate future stress can prepare (prime) organismic stress responses even in species lacking a nervous system. The process through which such organisms prepare their phenotype for an improved response to future stress has been termed 'priming'. However, other terms are also used for this phenomenon, especially when considering priming in different types of organisms and when referring to different stressors. Here we propose a conceptual framework for priming of stress responses in bacteria, fungi and plants which allows comparison of priming with other terms, e.g. adaptation, acclimation, induction, acquired resistance and cross protection. We address spatial and temporal aspects of priming and highlight current knowledge about the mechanisms necessary for information storage which range from epigenetic marks to the accumulation of (dormant) signalling molecules. Furthermore, we outline possible patterns of primed stress responses. Finally, we link the ability of organisms to become primed for stress responses (their 'primability') with evolutionary ecology aspects and discuss which properties of an organism and its environment may favour the evolution of priming of stress responses.

Egg laying of cabbage white butterfly (Pieris brassicae) on Arabidopsis thaliana affects subsequent performance of the larvae.

Plant resistance to the feeding by herbivorous insects has recently been found to be positively or negatively influenced by prior egg deposition. Here we show how crucial it is to conduct experiments on plant responses to herbivory under conditions that simulate natural insect behaviour. We used a well-studied plant--herbivore system, Arabidopsis thaliana and the cabbage white butterfly Pieris brassicae, testing the effects of naturally laid eggs (rather than egg extracts) and allowing larvae to feed gregariously as they do naturally (rather than placing single larvae on plants). Under natural conditions, newly hatched larvae start feeding on their egg shells before they consume leaf tissue, but access to egg shells had no effect on subsequent larval performance in our experiments. However, young larvae feeding gregariously on leaves previously laden with eggs caused less feeding damage, gained less weight during the first 2 days, and suffered twice as high a mortality until pupation compared to larvae feeding on plants that had never had eggs. The concentration of the major anti-herbivore defences of A. thaliana, the glucosinolates, was not significantly increased by oviposition, but the amount of the most abundant member of this class, 4-methylsulfinylbutyl glucosinolate was 1.8-fold lower in larval-damaged leaves with prior egg deposition compared to damaged leaves that had never had eggs. There were also few significant changes in the transcript levels of glucosinolate metabolic genes, except that egg deposition suppressed the feeding-induced up-regulation of FMOGS-OX2 , a gene encoding a flavin monooxygenase involved in the last step of 4-methylsulfinylbutyl glucosinolate biosynthesis. Hence, our study demonstrates that oviposition does increase A. thaliana resistance to feeding by subsequently hatching larvae, but this cannot be attributed simply to changes in glucosinolate content.

Impact of chemical manipulation of tarsal liquids on attachment in the Colorado potato beetle, Leptinotarsa decemlineata.

Insect tarsal attachment forces are thought to be influenced by the viscosity and surface tension of a thin film of adhesive liquid (wet adhesion). In beetles, this fluid has been shown to be composed mainly of lipophilic substances that are similar to the cuticular lipids. In this study we investigate whether and how the chemical composition of footprint lipids affects attachment forces in the Colorado potato beetle, Leptinotarsa decemlineata. After application of standardised mixtures of synthetic n-alkanes or alkenes, or a concentrated hydrocarbon extract to the surface of the elytra, we tested the beetles' attachment performance using a beam force transducer. The results show that only the unsaturated components, but not the straight-chained alkanes reduced friction forces, confirming that attachment performance is influenced by the chemical composition of the adhesive secretion. We estimated the volume of footprint droplets and calculated a mean thickness of the liquid layer of 0.04 microm. The measured friction exceeded the viscous and capillary force expected for a film of this thickness. Therefore, alternative mechanisms (i.e. shear-thinning and solid-like behaviour) for the generation of attachment forces and their dependence on the chemical composition of the liquid are discussed.

A sex pheromone in the desert tenebrionid beetle Parastizopus armaticeps.

Males of the desert beetle Parastizopus armaticeps (Pér.) (Coleoptera: Tenebrionidae) exhibit a characteristic calling behavior that attracts females by raising the tip of the abdomen, exposing the aedeagus, and remaining in this posture for a few seconds while emitting a pheromone. We collected the pheromone by holding a solid phase microextraction fiber (100 mum polydimethylsiloxane) close to the aedeagus for 5 s and analyzed the volatiles collected by gas chromatography/mass spectrometry. The volatiles consisted of 3-methylphenol (52%), ethyl-1,4-benzoquinone (48%), and 3-ethylphenol (2%). The pheromone originated from the aedeagal glands. In the gland reservoirs, these compounds (2.1%) were mixed with ethyl, isopropyl, and propyl esters of fatty acids (24.2%), and a mixture of hydrocarbons (69.1%). The mean amount of volatiles extracted from gland reservoirs was 0.92 +/- 0.83 microg. Chemo-orientation experiments with a servosphere show that females responded only to the ternary volatile mixture. Females stopped walking, elevated the front parts of their bodies with erected antennae, turned slowly on their own axis, and walked upwind toward the odor source. Single components or binary mixtures did not elicit responses from females. Males did not respond to the pheromone. Evolutionary aspects of this pheromone system are discussed.

1-Tridecene--male-produced sex pheromone of the tenebrionid beetle Parastizopus transgariepinus.

Males of the genus Parastizopus (Coleoptera: Tenebrionidae) exhibit a special pheromone-emitting behaviour. They do a headstand, expose the aedeagus and remain in this posture for a few seconds. The pheromone emitted by P. transgariepinus was collected by solid-phase micro-extraction (100 microm polydimethylsiloxane fibre) and identified as 1-tridecene by gas chromatography/mass spectrometry. Presumably, this compound originates from the aedeagal gland, a special feature in Parastizopus, as 1-tridecene is the main compound in the gland reservoirs (23.6+/-3.8%), accompanied by various less volatile fatty acid esters (25.2+/-2.0%) and hydrocarbons (51.2+/-5.7%). 1-Tridecene is also part of the pygidial defensive secretion of both sexes, together with other 1-alkenes, monoterpene hydrocarbons and 1,4-benzoquinones, but as none of these other compounds was detected during calling, the pygidial gland could be ruled out as pheromone source. Extracts of the aedeagal gland reservoirs and the pygidial defensive secretion contained comparable amounts of 1-tridecene, 1.24+/-0.41 and 1.88+/-0.54 microg/male, respectively. Chemo-orientation experiments using a servosphere showed that 1 microg of 1-tridecene was attractive to females but not to males.

The chemistry of the postpharyngeal gland of female European beewolves.

Females of the European beewolf, Philanthus triangulum, possess a large glove-shaped gland in the head, the postpharyngeal gland (PPG). They apply the content of the PPG to their prey, paralyzed honeybees, where it delays fungal infestation. Here, we describe the chemical composition of the gland by using combined GC-MS, GC-FTIR, and derivatization. The PPG of beewolves contains mainly long-chain unsaturated hydrocarbons (C23-C33), lower amounts of saturated hydrocarbons (C14-C33), and minor amounts of methyl-branched hydrocarbons (C17-C31). Additionally, the hexane-soluble gland content is comprised of small amounts of an unsaturated C25 alcohol, an unknown sesquiterpene, an octadecenylmethylester, and several long-chain saturated (C25, C27) and unsaturated (C23-C27) ketones, some of which have not yet been reported as natural products. Surprisingly, we found a dimorphism with regard to the major component of the PPG with some females having (Z)-9-pentacosene, whereas others have (Z)-9-heptacosene as their predominant component. The biological relevance of the compounds for the prevention of fungal growth on the prey and the significance of the chemical dimorphism are discussed.