Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs.

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa - spiders and birds - to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.

The evolution and ecology of multiple antipredator defences.

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such "defence portfolios" that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.

Sequestration of Defenses against Predators Drives Specialized Host Plant Associations in Preadapted Milkweed Bugs (Heteroptera: Lygaeinae).

AbstractHost plant specialization across herbivorous insects varies dramatically, but while the molecular mechanisms of host plant adaptations are increasingly known, we often lack a comprehensive understanding of the selective forces that favor specialization. The milkweed bugs (Heteroptera: Lygaeinae) are ancestrally associated with plants of the Apocynaceae from which they commonly sequester cardiac glycosides for defense, facilitated by resistant Na+/K+-ATPases and adaptations for transport, storage, and discharge of toxins. Here, we show that three Lygaeinae species independently colonized four novel nonapocynaceous hosts that convergently produce cardiac glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three milkweed bug species tested, feeding on seeds containing toxins did not improve growth or speed of development and even impaired growth and development in two species, but sequestration mediated protection of milkweed bugs against two natural predators: lacewing larvae and passerine birds. We conclude that physiological preadaptations and convergent phytochemistry facilitated novel specialized host associations. Since toxic seeds did not improve growth but either impaired growth or, at most, had neutral effects, selection by predators on sequestration of defenses, rather than the exploitation of additional profitable dietary resources, can lead to obligatory specialized host associations in otherwise generalist insects.

Analysis of defensive secretion of a milkweed bug Lygaeus equestris by 1D GC-MS and GC×GC-MS: sex differences and host-plant effect.

The composition of defensive secretion produced by metathoracic scent glands was analysed in males and females of the milkweed bug Lygaeus equestris (Heteroptera) using gas chromatography with mass spectrometric detection (GC-MS). The bugs were raised either on cardenolide-containing Adonis vernalis or on control sunflower seeds in order to determine whether the possibility to sequester cardenolides from their host plants would affect the composition of defensive scent-gland secretion. Profiles of the composition of defensive secretions of males and females raised on sunflower were closely similar, with predominant presence of (E)-2-octenal, (E)-2-octen-1-ol, decanal and 3-octen-1-ol acetate. The secretion of bugs raised on A. vernalis was more sexually dimorphic, and some chemicals e.g. (E,E)-2,4-hexadienyl acetate and 2-phenylethyl acetate were dominant in males, but absent in females. Compared to bugs from sunflower, the scent-gland secretion of bugs raised on A. vernalis was characterized by lower overall intensity of the peaks obtained for detected chemicals and by absence of some chemicals that have supposedly antipredatory function ((E)-2-hexenal, (E)-4-oxo-hex-2-enal, 2,4-octadienal). The results suggest that there might be a trade-off between the sequestration of defensive chemicals from host plants and their synthesis in metathoracic scent-glands.

No evidence for self-recognition in a small passerine, the great tit (Parus major) judged from the mark/mirror test.

Self-recognition is a trait presumed to be associated with high levels of cognition and something previously considered to be exclusive to humans and possibly apes. The most common test of self-recognition is the mark/mirror test of whether an animal can understand that it sees its own reflection in a mirror. The usual design is that an animal is marked with a colour spot somewhere on the body where the spot can only be seen by the animal by using a mirror. Very few species have passed this test, and among birds, only magpies have been affirmatively demonstrated to pass it. In this study, we tested great tits (Parus major), small passerines, that are known for their innovative foraging skills and good problem-solving abilities, in the mirror self-recognition test. We found no indication that they have any ability of this kind and believe that they are unlikely to be capable of this type of self-recognition.

The effect of social learning on avoidance of aposematic prey in juvenile great tits (Parus major).

Social learning plays an important role in acquiring new foraging skills and food preferences in many bird species but its potential role in learning to avoid aposematic prey has never been studied. We tested the effect of social learning on the acquisition of avoidance of aposematic insect prey (firebug Pyrrhocoris apterus; Heteroptera) in juvenile, hand-reared great tits (Parus major). Behaviour towards aposematic prey was compared between two groups of birds: (1) the observers that were, prior to encounter with firebugs, allowed to watch the experienced conspecific demonstrator repeatedly refuse to attack the prey, and (2) the control birds that lacked this opportunity. Observing an experienced demonstrator was not sufficient for learning complete avoidance, because birds from both groups attacked at least the first firebug they had encountered in avoidance training. However, the opportunity to observe the avoidance behaviour of another bird significantly increased the rate of subsequent individual learning of observers in comparison with control birds. Social learning also decreased mortality of firebugs killed by the birds during the avoidance learning. Socially enhanced learning to avoid aposematic prey might be a mechanism important especially for naive juvenile birds learning from their parents, but it could also enhance learning in adults from their more experienced flock mates. Because social learning of avoidance may also lead to decreased mortality of aposematic prey, its effect should be taken into account in scenarios considering evolution and maintenance of prey warning signals.

Comparative Analysis of Volatile Defensive Secretions of Three Species of Pyrrhocoridae (Insecta: Heteroptera) by Gas Chromatography-Mass Spectrometric Method.

The true bugs (Hemiptera: Heteroptera) have evolved a system of well-developed scent glands that produce diverse and frequently strongly odorous compounds that act mainly as chemical protection against predators. A new method of non-lethal sampling with subsequent separation using gas chromatography with mass spectrometric detection was proposed for analysis of these volatile defensive secretions. Separation was performed on Rtx-200 column containing fluorinated polysiloxane stationary phase. Various mechanical irritation methods (ultrasonics, shaking, pressing bugs with plunger of syringe) were tested for secretion sampling with a special focus on non-lethal irritation. The preconcentration step was performed by sorption on solid phase microextraction (SPME) fibers with different polarity. For optimization of sampling procedure, Pyrrhocoris apterus was selected. The entire multi-parameter optimization procedure of secretion sampling was performed using response surface methodology. The irritation of bugs by pressing them with a plunger of syringe was shown to be the most suitable. The developed method was applied to analysis of secretions produced by adult males and females of Pyrrhocoris apterus, Pyrrhocoris tibialis and Scantius aegyptius (all Heteroptera: Pyrrhocoridae). The chemical composition of secretion, particularly that of alcohols, aldehydes and esters, is species-specific in all three pyrrhocorid species studied. The sexual dimorphism in occurrence of particular compounds is largely limited to alcohols and suggests their epigamic intraspecific function. The phenetic overall similarities in composition of secretion do not reflect either relationship of species or similarities in antipredatory color pattern. The similarities of secretions may be linked with antipredatory strategies. The proposed method requires only a few individuals which remain alive after the procedure. Thus secretions of a number of species including even the rare ones can be analyzed and broadly conceived comparative studies can be carried out.

Through experience to boldness? Deactivation of neophobia towards novel and aposematic prey in three European species of tits (Paridae).

European tits (Paridae) exhibit species-specific levels of initial wariness towards aposematic prey. This wariness may be caused by neophobia, dietary conservatism or innate bias against particular prey traits. We assessed the contribution of these three mechanisms to the behaviour of juvenile tits towards novel palatable prey and novel aposematic prey. We compared levels of initial wariness in great tits (Parus major), blue tits (Cyanistes caeruleus) and coal tits (Periparus ater), and tested how the wariness can be deactivated by experience with a palatable prey. One group of birds was pre-trained to attack familiar naturally coloured mealworms the other one, novel red-painted mealworms. Then all the birds were offered a novel palatable prey of different colour and shape: cricket (Acheta domestica) with blue sticker, and then a novel aposematic firebug (Pyrrhocoris apterus). The three species of tits differed in how the experience with a novel palatable prey affected their behaviour towards another novel prey. Great tits and coal tits from experienced groups significantly decreased their neophobia towards both palatable prey and aposematic prey while blue tits did not change their strongly neophobic reactions. The interspecific differences may be explained by differences in body size, geographic range, and habitat specialisation.

Capillary electrophoresis of pterin derivatives responsible for the warning coloration of Heteroptera.

A new capillary electrophoretic (CE) method has been developed for analysis of 10 selected derivatives of pterin that can occur in the integument (cuticle) of true bugs (Insecta: Hemiptera: Heteroptera), specifically L-sepiapterin, 7,8-dihydroxanthopterin, 6-biopterin, D-neopterin, pterin, isoxanthopterin, leucopterin, xanthopterin, erythropterin and pterin-6-carboxylic acid. Pterin derivatives are responsible for the characteristic warning coloration of some Heteroptera and other insects, signaling noxiousness or unpalatability and are used to discourage potential predators from attacking. Regression analysis defining the parameters significantly affecting CE separation was used to optimize the system (the background electrolyte (BGE) composition, pH value and applied voltage). The optimized separation conditions were as follows: BGE with composition 2 mmol L(-1) the disodium salt of ethylendiamintetraacetic acid, 100 mmol L(-1) tris(hydroxymethyl)aminomethane and 100 mmol L(-1) boric acid, pH 9.0, applied voltage 20 kV and UV detection at 250 nm. Under these conditions, all the 10 studied derivatives of pterin were baseline separated within 22 min. The optimized method was validated from the viewpoint of linearity (R(2)≥0.9980), accuracy (relative error ≤7.90%), precision (for repeatability RSD≤6.65%), detection limit (LOD in the range 0.04-0.99 µg mL(-1)) and limit of quantitation (LOQ in the range 0.13-3.30 µg mL(-1)). The developed method was used for identification and determination of the contents of pterin derivatives in adults of four species of Heteroptera (Eurydema ornata cream color morph, Scantius aegyptius, Pyrrhocoris apterus and Corizus hyoscyami) and their distribution in the individual species was determined.

Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection applied for analysis of pteridines in two Graphosoma species (Insecta: Heteroptera).

A new separation method involving hydrophilic interaction chromatography with tandem mass spectrometric detection has been developed for the analysis of pteridines, namely biopterin, isoxanthopterin, leucopterin, neopterin, xanthopterin and erythropterin in the cuticle of heteropteran insect species. Two columns, Atlantis HILIC Silica and ZIC(®)-HILIC were tested for the separation of these pteridines. The effect of organic modifier content, buffer type, concentration and pH in mobile phase on retention and separation behavior of the selected pteridines was studied and the separation mechanism was also investigated. The optimized conditions for the separation of pteridines consisted of ZIC(®)-HILIC column, mobile phase composed of acetonitrile/5mM ammonium acetate, pH 6.80, 85/15 (v/v), flow rate 0.5mL/min and column temperature 30°C. Detection was performed by tandem mass spectrometry operating in electrospray ionization with Agilent Jet Stream technology using the selected reaction monitoring mode. The optimized method provided a linearity range from 0.3 to 5000ng/mL (r>0.9975) and repeatability with relative standard deviation<8.09% for all the studied pteridines. The method was applied to the analysis of pteridines in the cuticle of larvae and three adult color forms of Graphosoma lineatum and one form of Graphosoma semipunctatum (Insecta: Hemiptera: Heteroptera: Pentatomidae). The analysis shows that different forms of Graphosoma species can be characterized by different distribution of individual pteridines, which affects the coloration of various forms. Only isoxanthopterin was found in all the five forms tested.

Personality matters: individual variation in reactions of naive bird predators to aposematic prey.

Variation in reactions to aposematic prey is common among conspecific individuals of bird predators. It may result from different individual experience but it also exists among naive birds. This variation may possibly be explained by the effect of personality--a complex of correlated, heritable behavioural traits consistent across contexts. In the great tit (Parus major), two extreme personality types have been defined. 'Fast' explorers are bold, aggressive and routine-forming; 'slow' explorers are shy, non-aggressive and innovative. Influence of personality type on unlearned reaction to aposematic prey, rate of avoidance learning and memory were tested in naive, hand-reared great tits from two opposite lines selected for exploration (slow against fast). The birds were subjected to a sequence of trials in which they were offered aposematic adult firebugs (Pyrrhocoris apterus). Slow birds showed a greater degree of unlearned wariness and learned to avoid the firebugs faster than fast birds. Although birds of both personality types remembered their experience, slow birds were more cautious in the memory test. We conclude that not only different species but also populations of predators that differ in proportions of personality types may have different impacts on survival of aposematic insects under natural conditions.